WO2005077099A2 - Reduction de la replication du vih-1 a l'aide de proteines mutantes apogec3g - Google Patents

Reduction de la replication du vih-1 a l'aide de proteines mutantes apogec3g Download PDF

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WO2005077099A2
WO2005077099A2 PCT/US2005/004371 US2005004371W WO2005077099A2 WO 2005077099 A2 WO2005077099 A2 WO 2005077099A2 US 2005004371 W US2005004371 W US 2005004371W WO 2005077099 A2 WO2005077099 A2 WO 2005077099A2
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hiv
apobec3g
nucleic acid
polypeptide
mutant
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WO2005077099A3 (fr
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Vinay K. Pathak
Hongzhan Xu
Evguenia S. Svarovskaia
Rebekah Barr
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Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • FIELD This application relates to mutant apolipoprotein B mRNA-editing enzyme- catalytic polypeptide-like-3G (APOBEC3G) molecules, and methods of their use to decrease human immunodeficiency virus- 1 (HIV-1) infection, as well as to methods for identifying other agents that reduce HIV-1 infection.
  • APOBEC3G mutant apolipoprotein B mRNA-editing enzyme- catalytic polypeptide-like-3G
  • the human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G (APOBEC3G), first identified as CEM15 (Sheehy et al. Nature 418:646-50, 2002), is packaged into retroviral virions (Mariani et al. Cell 114:21-31, 2003; Harris et al. Cell 113:803-9, 2003; and Kao et al. J. Virol.
  • HIV-1 Vif interacts with human APOBEC3G and promotes its rapid degradation through the proteosomal pathway by recruiting cellular factors Cul5, elongins B and C, and Rbxl to form an Skpl-cullin-F-box (SCF)-like complex (Yu et al. Science 302:1056-60, 2003).
  • SCF Skpl-cullin-F-box
  • HIV-1 Vif can overcome the inhibitory effects of human and chimpanzee (CPZ) APOBEC3Gs but not the homologous Afiican green monkey (AGM) or macaque (MAC) APOBEC3Gs (Mariani et al. Cell 114:21-31, 2003). The explanation for this observation has not yet been determined. The rate of HIV infection is increasing. HIV and its associated acquired immune deficiency syndrome (AIDS) accounted for approximately 5% of all deaths in the United States in 2000, and over 313,000 persons were reported to be living with AIDS in that same year (Centers for Disease Control and Prevention, HIV/AIDS Surveillance Supplemental Report, 8(1): 1 -22, 2002).
  • AIDS acquired immune deficiency syndrome
  • APOBEC3G is a protem that can protect cells from HIV-1 infection by deaminating deoxycytidine to deoxuridine, thereby inducing G-to-A hypermutations in the replicated HIV-1.
  • this protective action is thwarted by HIV- Vif, a protein that binds to APOBEC3G and induces ubiquitination and proteosomal degradation of APOBEC3G.
  • mutant APOBEC3G is resistant to proteosomal degradation induced by HIV-1 Vif, while retaining the ability to deaminate deoxycytidine to deoxuridine and interact with HIV-1 Vif.
  • the disclosed mutant APOBEC3G molecules can be used to treat a subject having an HIV-1 infection, such as a subject with AIDS. It is disclosed herein that amino acid substitutions in human APOBEC3G, at position D128 (numbering is relative to a human APOBEC3G sequence, such as SEQ ID NO: 2), allows APOBEC3G to interact with HIV-1 Vif and to deaminate deoxycytidine to deoxuridine. Particular examples of substitutions that can be made include, but are not limited to, D128K, D128E, D128A, and D128V. However, because mutant APOBEC3G is not depleted from cells, it can decrease HIV-1 replication.
  • mutant APOBEC3G containing a substitution at D128, and abrogated its antiviral activity, indicating that it can be a substrate for the proteosomal pathway.
  • HIV- 1 Vif interaction triggers a conformational change in wild type APOBEC3G protein that initiates degradation.
  • mutant APOBEC3G proteins suppress the conformational change and thereby escapes degradation. Based on these observations, methods are provided for using mutant APOBEC3G molecules to reduce HIV-1 infection, for example to treat a subject having an HIV-1 infection. Examples of particular mutant APOBEC3G sequences are disclosed herein.
  • the disclosure provides purified peptides that include amino acids 1-384 of SEQ ED NO: 10, 22, 32, 34 and 40 as well as variants, fragments, and fusions thereof that retain mutant APOBEC3G activity, such as the ability to reduce HIV-1 infection.
  • Nucleic acids encoding the disclosed peptides are also encompassed by this disclosure, as well as host cells expressing the peptides.
  • mutant APOBEC3G nucleic acids include, but are not limited to, nucleotide sequences that include nucleotides 5-1156 of SEQ ID NOS: 9 and 21, or nucleotides 1-1152 of SEQ ID NOS: 31, 33, and 39, as well as variants, fragments, and fusions thereof that encode proteins having mutant APOBEC3G activity.
  • Binding agents that specifically bind to a mutant APOBEC3G peptide, such as antibodies, are encompassed by this disclosure.
  • Methods are also disclosed for reducing HIV-1 infection, for example to treat a subject having an HIV-1 infection, such as a subject having AIDS, using the disclosed mutant APOBEC3G proteins, or a nucleic acid encoding such peptides.
  • Subjects having an HIV-1 infection can receive the disclosed agents alone, or in the presence of other therapeutically effective molecules, such as other anti-viral agents.
  • Methods are provided for determining whether a subject (or population) is more resistant to HIV-1 infection.
  • the method includes determining the amino acid sequence at position 128 of human APOBEC3G (or the nucleic acid sequence encoding this codon). If the subject has a wild-type sequence, D128, this indicates that the subject is more susceptible to HIV-1 infection.
  • FIG. la is a schematic drawing showing mutant APOBEC3G sequences.
  • the ten mutants containing clusters of amino acid substitutions, starting at the N-terminal end of APOBEC3G, are labeled Apo4, Apol4, Apol5, Apol, Apol2, Apol2.1, Apol 1, Apo2, Apo9, and Apo3.
  • the clusters of substitution mutations are shown above the sequence in brackets; rounded brackets indicate a discontinuous cluster of substitutions.
  • the HIV-1 Vif resistant Apo 12 mutant is shown in bold and underlined letters.
  • FIG. lb is a bar graph showing the effect of APOBEC3G mutants on reducing
  • HIV-1 replication in the absence (Vif-) or presence (Vif+) of HIV-1 Vif was set to 100% (on average, approximately 24% of infected cells). Error bars represent the standard error of the mean of two experiments.
  • FIG. 2a is a digital image of a western blot showing the analysis of 293T cell lysates without transfection (293T control), or transfected with wild-type APOBEC3G (APO-WT), wild-type APOBEC3G and pC-Help (APO-WT + Vif), Apol2 mutant (APO 12), Apo 12 mutant and pC-Help (APO 12 + Vif), and pC-Help alone (Vif).
  • FIG. 2b is a digital image of a western blot showing the quantitation of wild- type APOBEC3G degradation in the presence of Vif.
  • FIG. 2c is a digital image of a western blot showing the results of a co- immunoprecipitation of wild-type or mutant Apo 12 APOBEC3G with HIV-1 Vif.
  • FIG. 2d is a bar graph showing flow cytometry analysis of the effect of wild type APOBEC3G on inhibition of HIV-1 replication by the Apo 12 mutant. Equimolar concentrations of the wild-type and Apo 12 mutants were cotransfected.
  • FIG. 3 a is a bar graph showing the effect of single amino acid substitutions (D128K, E133Q, and S137I) in APOBEC3G on reducing HIV-1 replication in the absence (Vif-) or presence (Vif ) of HIV-1 Vif. Error bars represent the standard error of the mean of 2 to 7 experiments.
  • FIG. 3b is a bar graph showing the effect of wild type and D128K mutant
  • FIG. 3c is a digital image of a western blot analysis of the steady state levels of wild type APOBEC3G (APO-WT) and D128K mutant of APOBEC3G (D128K) in the absence of any Vif or presence of HIV-1, SIVmac239 (SlVmac), and HIV-2 Vif proteins.
  • APO-WT wild type APOBEC3G
  • D128K D128K mutant of APOBEC3G
  • FIG. 4 is a bar graph showing the effects of amino acid substitutions on position 128 of APOBEC3G on sensitivity to HIV-1 Vif. Error bars represent the standard error of the mean of 4 experiments.
  • SEQ ID NO: 1 is a cDNA nucleic acid sequence of a wild-type human APOBEC3G (Genbank Accession No. BC024268).
  • SEQ ID NO: 2 is the corresponding protein sequence of SEQ ED NO: 1.
  • SEQ ID NO: 3 is a cDNA nucleic acid sequence of a variant human
  • SEQ ID NO: 4 is the corresponding protein sequence of SEQ ID NO: 3.
  • SEQ ID NO: 5 is a cDNA nucleic acid sequence of a variant human APOBEC3G (Apo 14) including a C-myc tag (nucleotides 1157-1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 6 is the corresponding protein sequence of SEQ ID NO: 5.
  • SEQ ID NO: 7 is a cDNA nucleic acid sequence of a variant human APOBEC3G (Apo 1) including a C-myc tag (nucleotides 1157-1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 8 is the corresponding protein sequence of SEQ ID NO: 7.
  • SEQ ID NO: 9 is a cDNA nucleic acid sequence of a mutant human APOBEC3G (Apo 12) including a C-myc tag (nucleotides 1157-1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 10 is the corresponding protein sequence of SEQ ID NO: 9.
  • SEQ ID NO: 11 is a cDNA nucleic acid sequence of a variant human
  • SEQ ID NO: 12 is the corresponding protein sequence of SEQ ID NO: 11.
  • SEQ ED NO: 13 is a cDNA nucleic acid sequence of a variant human APOBEC3G (Apo 2) including a C-myc tag (nucleotides 1157-1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 14 is the corresponding protein sequence of SEQ ID NO: 13.
  • SEQ ID NO: 15 is a cDNA nucleic acid sequence of a variant human APOBEC3G (Apo 9).
  • SEQ ID NO : 16 is the corresponding protein sequence of SEQ ID NO : 15.
  • SEQ ID NO: 17 is a cDNA nucleic acid sequence of a variant human APOBEC3G (Apo 15) including a C-myc tag (nucleotides 1157-1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 18 is the corresponding protein sequence of SEQ ID NO: 17.
  • SEQ ID NO: 19 is a cDNA nucleic acid sequence of a variant human
  • APOBEC3G (Apo 3) including a C-myc tag (nucleotides 1157-1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 20 is the corresponding protein sequence of SEQ ID NO: 19.
  • SEQ ID NO: 21 is a cDNA nucleic acid sequence of a mutant human APOBEC3G that encodes a D128K mutation with a C-myc tag (nucleotides 1157- 1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 22 is the corresponding protein sequence of SEQ ID NO: 21.
  • SEQ ID NO: 23 is a cDNA nucleic acid sequence of a wild-type human APOBEC3G sequence that includes a C-myc tag (nucleotides 1157-1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 24 is the corresponding protein sequence of SEQ ID NO: 23.
  • SEQ ID NO: 25 is a cDNA nucleic acid sequence of a variant human APOBEC3G (E133Q) including a C-myc tag (nucleotides 1157-1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 26 is the corresponding protein sequence of SEQ ID NO: 25.
  • SEQ ID NO: 27 is a cDNA nucleic acid sequence of a variant human
  • SEQ ID NO: 28 is the corresponding protein sequence of SEQ ID NO: 27.
  • SEQ ID NO: 29 is a cDNA nucleic acid sequence of a variant human APOBEC3G (Apo 12.1) including a C-myc tag (nucleotides 1157-1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 30 is the corresponding protem sequence of SEQ ID NO: 29.
  • SEQ ID NO: 31 is a cDNA nucleic acid sequence of a mutant human APOBEC3G that encodes a D128V mutation and a C-myc tag (nucleotides 1157- 1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 32 is the corresponding protein sequence of SEQ ID NO: 31.
  • SEQ ED NO: 33 is a cDNA nucleic acid sequence of a mutant human APOBEC3G that encodes a D128E mutation and a C-myc tag (nucleotides 1157- 1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 34 is the corresponding protein sequence of SEQ ID NO: 33.
  • SEQ ID NO: 35 is a cDNA nucleic acid sequence of a mutant human APOBEC3G that encodes a D128G mutation and a C-myc tag (nucleotides 1157- 1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 36 is the corresponding protein sequence of SEQ ED NO: 35.
  • SEQ ID NO: 37 is a cDNA nucleic acid sequence of a mutant human
  • SEQ ID NO: 38 is the corresponding protein sequence of SEQ ED NO: 37.
  • SEQ ED NO: 39 is a cDNA nucleic acid sequence of a mutant human APOBEC3G that encodes a D128A mutation and a C-myc tag (nucleotides 1157- 1213) and a His tag (nucleotides 1214-1231).
  • SEQ ID NO: 40 is the corresponding protein sequence of SEQ ID NO: 39.
  • AGM African green monkey APOBEC3G: Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G HIV: Human Immunodeficiency Virus MAC: Macaque VIF: Viral infectivity factor
  • Agent Any protein, nucleic acid molecule, compound, small molecule, organic compound, inorganic compound, or other molecule of interest.
  • Analog An agent (such as a protein) that is structurally similar to another, but differs slightly in composition, for example the replacement of one atom by an atom of a different element or functional group.
  • Antiviral agent An agent that reduces the ability of a virus to infect a cell. Apolipoprotein B mRNA-editing enzyme-catalytic polyp eptide-like-3G
  • APOBEC3G An enzyme that can deaminate deoxycytidine to deoxuridine, for example in a retroviral minus (first-)-strand DNA during reverse transcription, resulting in G-to-A substitutions in the viral plus (genomic) strand, and is susceptible to ubiquitination and proteosomal degradation by HIV-1 viral infectivity factor (Vif).
  • Vif HIV-1 viral infectivity factor
  • An example of an APOBEC3G protein includes the sequence provided in GenBank Accession No. AAH24268.
  • an APOBEC3G nucleic acid sequence includes the sequence shown in SEQ ID NO: 1, or fragments, variants, or fusions thereof that retain the ability to encode a peptide or protein having APOBEC3G activity.
  • an APOBEC3G protein includes the amino acid sequence shown in SEQ ED NO: 2, or fragments, fusions, or variants thereof that retain APOBEC3G activity.
  • This description includes APOBEC3G allelic variants, as well as any variant, fragment, or fusion sequence that retains the ability to deaminate deoxycytidine to deoxuridine and that is susceptible to ubiquitination and proteosomal degradation by HIV-1 Vif.
  • APOBEC3G activity The ability of an APOBEC3G molecule to deaminate deoxycytidine to deoxuridine, and to be susceptible to ubiquitination and proteosomal degradation induced by HIV-1 Vif. In the presence of an APOBEC3G molecule and HIV-1 Vif, HIV-1 infection (such as HIV-1 replication), is not significantly decreased. These activities can be measured using any assay known in the art, for example the HlV-replication assays described in Example 2 and the immunoprecipitation/Western blotting assays described in Example 3.
  • Antibody A molecule including an antigen-binding site which specifically binds (immunoreacts with) an antigen.
  • Examples include polyclonal antibodies, monoclonal antibodies, humanized monoclonal antibodies, or immunologically effective portions thereof. Includes immunoglobulin molecules and immunologically active portions thereof.
  • Naturally occurring antibodies for example IgG
  • Naturally occurring antibodies include four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody.
  • Immunologically effective portions of monoclonal antibodies include, but are not limited to: Fab, Fab', F(ab') 2 , Fabc and Fv portions (for a review, see Better and Horowitz, Methods. Enzymol. 178:476-96, 1989).
  • antigen-binding fragments include, but are not limited to: (i) an Fab fragment consisting of the VL, VH, CL and CHI domains; (ii) an Fd fragment consisting of the VH and CHI domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment which consists of a VH domain; (v) an isolated complimentarity determining region (CDR); and (vi) an F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • a synthetic linker can be made that enables them to be made as a single protein chain (known as single chain Fv (scFv) by recombinant methods.
  • single chain Fv single chain Fv
  • Single chain antibodies are also included.
  • Specifically binds refers to the ability of a particular agent (a "specific binding agent") to specifically react with a particular analyte, for example to specifically immunoreact with an antibody, or to specifically bind to a particular peptide sequence. The binding is a non-random binding reaction, for example between an antibody molecule and an antigenic determinant.
  • Binding specificity of an antibody is typically determined from the reference point of the ability of the antibody to differentially bind the specific antigen and an unrelated antigen, and therefore distinguish between two different antigens, particularly where the two antigens have unique epitopes.
  • An antibody that specifically binds to a particular epitope is referred to as a "specific antibody”.
  • Monoclonal or polyclonal antibodies can be produced to a mutant
  • APOBEC3G peptide such as SEQ ID NO: 22
  • fragments of a wild-type APOBEC3G peptide such as those that include amino acid 128 of SEQ ED NO: 22
  • variants, fusions, or fragments thereof such as SEQ ID NO: 10
  • antibodies raised against one or more epitopes on a polypeptide antigen will specifically detect that polypeptide. That is, antibodies raised against one particular polypeptide would recognize and bind that particular polypeptide, and would not substantially recognize or bind to other polypeptides.
  • an antibody that is specific for a mutant APOBEC3G peptide (such as amino acids 1-384 of SEQ ID NOS: 10, 22, 32, 34, or 40), will not substantially bind to a wild-type APOBEC3G peptide (such as SEQ ED NO: 1), and vice-versa.
  • the determination that an antibody specifically binds to a particular polypeptide is made by any one of a number of standard immunoassay methods; for instance, Western blotting.
  • Antibody fragments can be used in place of whole antibodies and can be readily expressed in prokaryotic host cells. Methods of making and using immunologically effective portions of monoclonal antibodies, also referred to as "antibody fragments," are well known and include those described in Better &
  • Antigen A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are administered, such as injected or absorbed, to an animal.
  • cDNA complementary DNA
  • a piece of DNA lacking internal, non-coding segments (introns) and transcriptional regulatory sequences.
  • a cDNA also can contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule.
  • cDNA can be produced using various methods, such as synthesis in the laboratory by reverse transcription from messenger RNA extracted from cells. Conservative substitution: One or more amino acid substitutions (for example 1, 2, 5 or 10 residues) for amino acid residues having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide.
  • a conservative substitution is an amino acid substitution in a mutant APOBEC3G peptide that does not substantially affect the ability of the peptide to decrease HIV-1 replication in a cell.
  • a conservative substitution is an amino acid substitution in a mutant APOBEC3G peptide, such as a conservative substitution in SEQ ED NO: 10 or 22, which does not significantly alter the mutant APOBEC3G activity of the peptide, such as its ability to decrease HIV-1 replication.
  • Methods that can be used to determine mutant APOBEC3G activity are disclosed herein (for example see Examples 2 and 3 below).
  • An alanine scan can be used to identify which amino acid residues in a mutant APOBEC3G peptide can tolerate an amino acid substitution (see Example 6).
  • mutant APOBEC3G activity is not altered by more than 25%, for example not more than 20%, for example not more than 10%, when an alanine, or other conservative amino acid (such as those listed below), is substituted for one or more native amino acids. In one example, at least one conservative substitution is included in a mutant
  • APOBEC3G peptide such as a conservative substitution in amino acids 1-384 of SEQ ED NO: 10, 22, 32, 34, or 40.
  • at least 2, 3, 4, 5 or 10 conservative substitutions are included in the peptide.
  • the peptide includes no more than 10, such as no more than 5, 4, 3, or 2, conservative substitutions.
  • a polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR.
  • a polypeptide can be produced to contain one or more conservative substitutions by using standard peptide synthesis methods.
  • Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place.
  • amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gin or His for Asn; Glu for Asp; Ser for Cys; Asn for Gin; Asp for Glu; Pro for Gly; Asn or Gin for His; Leu or Val for He; lie or Val for Leu; Arg or Gin for Lys; Leu or He for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and He or Leu for Val.
  • DNA is a long chain polymer which includes the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid, RNA).
  • the repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.
  • Triplets of nucleotides, referred to as codons in DNA molecules code for amino acid in a polypeptide.
  • codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
  • Exogenous refers to any nucleic acid that does not originate from that particular cell as found in nature. Thus, a non-naturally-occurring nucleic acid is considered to be exogenous to a cell once introduced into the cell.
  • a nucleic acid that is naturally-occurring also can be exogenous to a particular cell. For example, an entire chromosome isolated from a cell of person X is an exogenous nucleic acid with respect to a cell of person Y once that chromosome is introduced into Y's cell.
  • functionally equivalent molecules include different molecules that retain the function of a mutant APOBEC3G molecule.
  • functional equivalents can be provided by sequence alterations in a mutant APOBEC3G peptide, wherein the peptide with one or more sequence alterations retains a function of the unaltered peptide, such that it retains its ability to reduce HIV-1 replication in a cell, as compared to an amount of HIV-1 replication in the presence of a wild-type APOBEC3G protein.
  • sequence alterations include, but are not limited to, conservative substitutions, deletions, mutations, frameshifts, and insertions.
  • a given polypeptide binds an antibody
  • a functional equivalent is a polypeptide that binds the same antibody.
  • a functional equivalent includes peptides that have the same binding specificity as a polypeptide, and that can be used as a reagent in place of the polypeptide (such as in the reduction of HIV-1 replication).
  • a functional equivalent includes a polypeptide wherein the binding sequence is discontinuous, wherein the antibody binds a linear epitope.
  • the polypeptide is functionally equivalent to amino acids 124-133 of SEQ ED NO: 22 if the three dimensional structure of the polypeptide is such that it can bind a monoclonal antibody that binds amino acids 124-133 of SEQ ED NO: 22.
  • HIV Human Immunodeficiency Virus
  • AIDS acquired immunodeficiency syndrome
  • This immunosuppression results from a progressive depletion and functional impairment of T lymphocytes expressing the CD4 cell surface glycoprotein.
  • the loss of CD4 helper/inducer T cell function may underlie the loss of cellular and humoral immunity leading to the opportunistic infections and malignancies seen in AIDS.
  • Depletion of CD4 T cells results from the ability of HIV to selectively infect, replicate in, and ultimately destroy these T cells (for example see Klatzmann et al., Science 225:59, 1984).
  • CD4 itself is an important component, and in some examples an essential component, of the cellular receptor for HTV.
  • HIV subtypes can be identified by particular number, such as HIV-1 and HIV- 2.
  • the virus enters a host cell in at least three stages: receptor docking, viral-cell membrane fusion, and particle uptake (D'Souza et ah, JAMA
  • Receptor docking begins with a gpl20 component of a virion spike binding to the CD4 receptor on the host cell. Conformational changes in gpl20 induced by gpl20-CD4 interaction promote an interaction between gpl20 and either CCR5 or CXCR4 cellular co-receptors. The gp41 protein then mediates fusion of the viral and target cell membranes. More detailed information about HIV can be found in Coffin et al, Retroviruses (Cold Spring Harbor Laboratory Press, 1997).
  • HIV-1 viral infectivity factor A protein that allows replication of HIV-1, by inducing APOBEC3G ubiquitination and proteosomal degradation, and thus preventing incorporation of APOBEC3G into virions. Includes any HIV-1 Vif gene, cDNA, RNA, or protein.
  • An example of an HIV-1 Vif protein includes the sequence provided in GenBank Accession No. NP_05785, as well as fragments, variants, or fusions thereof that retain the ability to induce APOBEC3G ubiquitination and proteosomal degradation.
  • An example of an HIV-1 Vif RNA includes the sequence provided in GenBank Accession No.
  • HIV-1 Vif mediated degradation The ability of a HIV-1 Vif sequence to induce proteosomal degradation of APOBEC3G. Reduction or inhibition of such activity can refer to full, partial, or enhanced induction of degradation, for example reducing an amount of wild-type HIV- Vif activity by at least 25%, such as at least 50%, at least 50%, at least 90%, or even at least 100% of such activity.
  • Hybridization of a nucleic acid occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other.
  • the stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acids used. For example, temperature and ionic strength (such as Na + concentration) can affect the stringency of hybridization.
  • T m is the temperature at which 50% of a given strand of nucleic acid is hybridized to its complementary strand.
  • the T m of a particular nucleic acid can be determined by various methods, such as observing the transition state between a single-stranded and double-stranded state during a temperature change, such as heating a dsDNA from about 30 C to about 100 C, and detecting when the dsDNA denatures to ssDNA. This can be accomplished by determining a melting profile for the nucleic acid. For longer nucleic acid fragments, such as PCR products, the nearest-neighbor method can be used to determine T m (Breslauer et al., Proc. Natl. Acad. Sci. USA 83:3746-50, 1986).
  • stringent conditions encompass conditions under which hybridization only will occur if there is less than 25% mismatch between the hybridization molecule and the target sequence.
  • “Moderate stringency” conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of "medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of "high stringency” are those under which sequences with more than 10% mismatch will not hybridize.
  • Conditions of "very high stringency” are those under which sequences with more than 5% mismatch will not hybridize.
  • Moderately stringent hybridization conditions are when the hybridization is performed at about 42°C in a hybridization solution containing 25 mM KPO 4 (pH 7.4), 5X SSC, 5X Denhart's solution, 50 ⁇ g/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15 ng/mL probe (about 5xl0 7 cpm/ g), while the washes are performed at about 50°C with a wash solution containing 2X SSC and 0.1 % sodium dodecyl sulfate.
  • Highly stringent hybridization conditions are when the hybridization is performed at about 42°C in a hybridization solution containing 25 mM KPO 4 (pH 7.4), 5X SSC, 5X Denhart's solution, 50 j-tg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15 ng/mL probe (about 5xl0 7 cpm/ ⁇ g), while the washes are performed at about 65°C with a wash solution containing 0.2X SSC and 0.1% sodium dodecyl sulfate.
  • Induction of HIV-1 mutation The ability of APOBEC3G sequences
  • mutant or wild-type to cause G-to-A substitutions in a viral plus (genomic) strand as a result of deaminating deoxycytidine to deoxuridine in a retroviral minus (firsts- strand.
  • Preservation of such activity can refer to full, partial, or enhanced induction, for example retaining at least 50% of such activity as compared to a wild-type APOBEC3G sequence, such as at least 70%, at least 90%, or even at least 100% of such activity.
  • Infection The entry, replication, insertion, lysis or other event or process involved with the pathogensis of a virus into a host cell.
  • decreasing infection includes decreasing entry, replication, insertion, lysis, or other pathogensis of a virus into a cell or subject, or combinations thereof.
  • Infection includes the introduction of an infectious agent, such as a non-recombinant virus, recombinant virus, plasmid, or other agent capable of infecting a host, such as the cell of a subject.
  • infection is the introduction of a recombinant vector into a host cell via transduction, transformation, transfection, or other method.
  • Vectors include, but are not limited to, viral, plasmid, cosmid, and artificial chromosome vectors.
  • a recombinant vector can include a mutated APOBEC3G molecule, such as variants, fragments or fusions of nucleotides 5-1156 of SEQ ED NOS: 9 or 21, or nucleotides 1-1152 of SEQ ID NOS: 31, 33, or 39.
  • Isolated An isolated biological component (such as a nucleic acid or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods.
  • isolated refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally-occurring genome of the organism from which it is derived.
  • an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (such as a cDNA or a genomic DNA fragment produced by PCR or restriction endonudease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (such as a retrovirus, adenovirus, or he ⁇ es virus), or into the genomic DNA of a prokaryote or eukaryote.
  • an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.
  • the term "isolated" as used with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.
  • non-naturally- occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid.
  • Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques.
  • Mimetic A molecule (such as an organic chemical compound) that mimics the activity of an agent, such as the activity of a mutant APOBEC3G protein on HIV-1 infectivity.
  • Peptidomimetic and organomimetic embodiments are within the scope of this term, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains in the peptide, resulting in such peptido- and organomimetics of the peptides having substantial specific activity.
  • a pharmacophore is an idealized, three- dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with computer modeling software (using computer assisted drug design or CADD).
  • Mutant APOBEC3G An APOBEC3G sequence that includes at least one amino acid substitution or deletion compared to a wild-type sequence, which results in resistance to proteosomal degradation by HIV-1 Vif.
  • Mutant APOBEC3G retains the ability to deaminate deoxycytidine to deoxuridine, for example in a newly synthesized viral minus-strand DNA, thereby inducing G-to-A hypermutation.
  • a mutant APOBEC3G protein includes a D128K, D128V, D128E, or D128A substitution (wherein the numbering refers to the wild-type human sequence, such as in SEQ ED NO: 2).
  • mutant APOBEC3G peptides include, but are not limited to, amino acids 1-384 of SEQ ID NOS: 10, 22, 32, 34, and 40, as well as variants, fragments, and fusions thereof that retain mutant APOBEC3G activity.
  • mutant APOBEC3G nucleic acid sequences are shown in nucleotides 5-1156 of SEQ ID NOS: 9 and 21, or nucleotides 1-1152 of SEQ ID NOS: 31, 33, and 39, or fragments, variants, or fusions thereof that retain the ability to encode a peptide or protein having mutant APOBEC3G activity.
  • Mutant APOBEC3G activity The ability of a mutant APOBEC3G molecule to deaminate deoxycytidine to deoxuridine, and to be resistant to proteosomal degradation induced by HIV-1 Vif, thereby decreasing HIV-1 infection of a cell (such as decreasing HIV-1 replication in a cell).
  • HIV-1 infection (such as HIV-1 replication), is significantly decreased, such as at least 50%, such as at least 80%, compared to an amount of HIV-1 infection in the presence of a wild-type or non-mutant APOBEC3G molecule.
  • mutant APOBEC3G protein is not significantly degraded, such as no more than about 20%, compared to an amount of degradation that would occur to a wild-type or non- mutant APOBEC3G molecule.
  • Nucleic acid Encompasses both RNA and DNA including, without limitation, cDNA, genomic DNA, and synthetic (such as chemically synthesized) DNA.
  • the nucleic acid can be double-stranded or single-stranded. Where single- stranded, the nucleic acid can be the sense strand or the antisense strand.
  • nucleic acid can be circular or linear. Unless otherwise specified, any reference to a nucleic acid molecule includes the reverse complement of nucleic acid.
  • any nucleic acid written to depict only a single strand encompasses both strands of a corresponding double-stranded nucleic acid.
  • depiction of a plus-strand of a dsDNA also encompasses the complementary minus-strand of that dsDNA.
  • reference to the nucleic acid molecule that encodes a specific protein, or a fragment thereof encompasses both the sense strand and its reverse complement.
  • a nucleic acid includes a mutant APOBEC3G nucleotide sequence shown in nucleotides 5-1156 of SEQ ED NO: 21 or nucleotides 1- 1152 of SEQ ID NOS: 31, 33, and 39, or a variant, fragment, or fusion thereof, such as SEQ ED NO: 9.
  • a fragment can be any portion of the nucleic acid corresponding to at least 5 contiguous bases from a nucleic acid sequence, such as nucleotides 5-1156 of SEQ ID NOS: 9 and 21, or nucleotides 1-1152 of SEQ ID NOS: 31, 33, and 39, for example at least 20 contiguous bases, at least 50 contiguous bases, at least 100 contiguous bases, at least 250 contiguous bases, or even at least 500 or more contiguous bases.
  • a fragment can be chosen from a particular portion of any of the target sequences associated with nucleotides 5-1156 of SEQ ID NOS: 9 or 21, or nucleotides 1-1152 of SEQ ED NOS: 31, 33, or 39, such as a particular half, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or smaller portion of any of the target sequences associated with nucleotides 5-1156 of SEQ ID NOS: 9 or 21, or nucleotides 1-1152 of SEQ ID NOS: 31, 33, or 39. Fragments of the nucleic acids described herein can be used as probes and primers.
  • Oligonucleotide A linear polynucleotide (such as DNA or RNA) sequence of at least 9 nucleotides, for example at least 15, 18, 24, 25, 30, 50, 100, 200 or even 500 nucleotides long.
  • an oligonucleotide is about 6-50 bases, for example about 10-25 bases, such as 12-20 bases.
  • An oligonucleotide analog refers to moieties that function similarly to oligonucleotides, but have non-naturally occurring portions.
  • oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.
  • Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules.
  • Open reading frame A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.
  • Operably linked A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Peptide Modifications The present disclosure includes mutant APOBEC3G peptides, as well as synthetic embodiments.
  • analogues non-peptide organic molecules
  • derivatives chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences
  • variants homologs having mutant APOBEC3G activity
  • the peptides disclosed herein include a sequence of amino acids that can be either L- and/or D- amino acids, naturally occurring and otherwise.
  • Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties.
  • carboxylic acid groups of the protein may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a Ci-C. ⁇ ester, or converted to an amide of formula NR ⁇ R 2 wherein Ri and R 2 are each independently H or C 1 -C 16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring.
  • Amino groups of the peptide may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to Ci-Ci ⁇ alkyl or dialkyl amino or further converted to an amide.
  • Hydroxyl groups of the peptide side chains can be converted to C ⁇ -C 16 alkoxy or to a C 1 -C 16 ester using well-recognized techniques.
  • Phenyl and phenolic rings of the peptide side chains can be substituted with one or more halogen atoms, such as F, CI, Br or I, or with C ⁇ -C 16 alkyl, Ci-Ci ⁇ alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
  • Methylene groups of the peptide side chains can be extended to homologous C 2 -C alkylenes.
  • Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • a C- or N-terminal cysteine can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, generating a cyclic peptide.
  • Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.
  • Peptidomimetic and organomimetic embodiments are also within the scope of the present disclosure, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of the proteins of this invention having detectable mutant APOBEC3G activity.
  • a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer- Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, EL, pp. 165-174 and Principles of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CADD. Also included within the scope of the disclosure are mimetics prepared using such techniques.
  • a mimetic mimics the mutant APOBEC3G activity generated by a mutant APOBEC3G or a variant, fragment, or fusion thereof.
  • Pharmaceutical agent or drug A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when administered to a subject, alone or in combination with another therapeutic agent(s) or pharmaceutically acceptable carriers.
  • a pharmaceutical agent decreases or even inhibits HIV-1 infection of a cell, such as the cell of a subject.
  • Preventing or treating a disease "Preventing" a disease refers to inhibiting the full development of a disease, for example preventing development of a viral infection.
  • Probes and primers refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to a HIV-1 infection, such as inhibiting or decreasing HIV-1 infection.
  • a probe includes an isolated nucleic acid attached to a detectable label or other reporter molecule.
  • Typical labels include, but are not limited to radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al.
  • Primers are short nucleic acid molecules, such as DNA oligonucleotides ten nucleotides or more in length. Longer DNA oligonucleotides can be about 15, 20, 25, 30 or 50 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example by the polymerase chain reaction (PCR) or other nucleic- acid amplification methods.
  • PCR polymerase chain reaction
  • probes and primers can be selected that include at least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of a nucleic acid disclosed herein.
  • Promoter An array of nucleic acid control sequences which direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase ⁇ type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements that can be located as much as several thousand base pairs from the start site of transcription.
  • Purified The term purified does not require absolute purity; rather, it is a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its environment within a cell, such that the peptide is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that may accompany it.
  • a purified peptide preparation is one in which the peptide is substantially-free from contaminants, such as those that might be present following chemical synthesis of the peptide.
  • a peptide is purified when at least 60% by weight of a sample is composed of the peptide, for example when 75%, 95%, or 99% or more of a sample is composed of the peptide, such as a mutant APOBEC3G peptide. Examples of methods that can be used to purify proteins, include, but are not limited to the methods disclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Ch. 17).
  • Protein purity can be determined by, for example, polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band upon staining the polyacrylamide gel; high- pressure liquid chromatography; sequencing; or other conventional methods.
  • Recombinant A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids or proteins, for example, by genetic engineering techniques.
  • Sequence identity The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences.
  • Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.
  • Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.
  • Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species that are more closely related (such as human and mouse sequences), compared to species more distantly related (such as human and C. elegans sequences). Methods of alignment of sequences for comparison are well known in the art.
  • Biol. 215:403-10, 1990 is available from several sources, including the National Center for Biological Information (NCBI, National Library ofMedicine, Building 38 A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
  • NCBI National Center for Biological Information
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • the options can be set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (such as C: ⁇ seql.txt); -j is set to a file containing the second nucleic acid sequence to be compared (such as C: ⁇ seq2.txt); -p is set to blastn; -o is set to any desired file name (such as C: ⁇ output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting.
  • the following command can be used to generate an output file containing a comparison between two sequences: C: ⁇ B12seq -i c: ⁇ seql .txt -j c: ⁇ seq2.txt -p blastn -o c: ⁇ outputtxt -q -1 -r 2.
  • the options of B12seq can be set as follows: -i is set to a file containing the first amino acid sequence to be compared (such as C: ⁇ seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C: ⁇ seq2.txt); -p is set to blastp; -o is set to any desired file name (such as C: ⁇ outputtxt); and all other options are left at their default setting.
  • -i is set to a file containing the first amino acid sequence to be compared (such as C: ⁇ seql.txt)
  • -j is set to a file containing the second amino acid sequence to be compared (e.g., C: ⁇ seq2.txt)
  • -p is set to blastp
  • -o is set to any desired file name (such as C: ⁇ outputtxt); and all other options are left at their default setting.
  • the following command can be used to generate an output file containing a comparison between two amino acid sequences: C: ⁇ B12seq — i c: ⁇ seql.txt -j c: ⁇ seq2.txt -p blastp -o c: ⁇ output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
  • the percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 1O0 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.
  • the percent sequence identity value is rounded to the nearest tenth.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • Homologs are typically characterized by possession of at least 70% sequence identity counted over the full-length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs use SEG. In addition, a manual alignment can be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site.
  • nucleic acid molecules that hybridize under stringent conditions to a mutated APOBEC3G gene sequence typically hybridize to a probe based on either an entire mutated APOBEC3G gene or selected portions of the gene, respectively, under conditions described above.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
  • homologous nucleic acid sequences can, for example, possess at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity determined by this method.
  • sequence identity ranges are provided for guidance only; it is possible that strongly significant homologs could be obtained that fall outside the ranges provided.
  • An alternative (and not necessarily cumulative) indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • Subject Living multi-cellular vertebrate organisms, including human and veterinary subjects, such as cows, pigs, horses, dogs, cats, birds, reptiles, and fish.
  • Therapeutically Effective Amount A_n amount of a pharmaceutical preparation that alone, or together with an additional therapeutic agent(s) (for example other anti-viral agents), induces the desired response.
  • the preparations disclosed herein are administered in therapeutically effective amounts.
  • a desired response is to decrease or inhibit HIV-1 infection of a cell, such as a cell of a subject. HIV-1 infection does not need to be completely inhibited for the pharmaceutical preparation to be effective.
  • a pharmaceutical preparation can decrease viral infection by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to an amount of HIV-1 infection in the absence of the pharmaceutical preparation, such as a preparation including a mutant APOBEC3G nucleic acid or protein.
  • This decrease or inhibition can result in halting or slowing the progression of, or inducing a regression of a pathological condition caused by HIV-1 infection (such as AEDS or opportunistic infections associated with HIV infection), or which is capable of relieving signs or symptoms caused by the condition.
  • a desired response is to increase the number of CD4 T-cells in the subject, for example to an amount greater than 200 cells per microliter ( ⁇ L).
  • a CD4+ cell count of fewer than 2O0 cells/ ⁇ L indicates acquired immunodeficiency syndrome (AEDS) and a high risk for opportunistic infections.
  • AEDS acquired immunodeficiency syndrome
  • a therapeutically effective amount can increase the number of CD4 T-cells in the subject to at least 350 cells/ ⁇ L, such as at least 400 cells/ ⁇ L, such as at least 500 cells/ ⁇ L, such as at least 600 cells/ ⁇ L, such as at least 800 cells/ ⁇ L, such as at least 1000 cells/ ⁇ L, such as at least 1200 cells/ ⁇ L.
  • it is an amount sufficient to partially or completely alleviate symptoms of HIV-1 infection within a host subject.
  • Treatment can involve only slowing the progression of the infection temporarily, but can also include halting or reversing the progression of the infection permanently.
  • Effective amounts of the therapeutic agents described herein can be determined in many different ways, such as assaying for a reduction in the rate of infection of cells or subjects, a reduction in the viral load within a host, improvement of physiological condition of an infected subject, or increased resistance to infection following exposure to the virus. Effective amounts also can be determined through various in vitro, in vivo or in situ assays, including the assays described herein.
  • a virus or vector "transduces” or “transfects” a cell when it transfers nucleic acid into the cell.
  • a cell is “transformed” by a nucleic acid transduced into the cell when the DNA becomes stably replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication.
  • transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • transfected cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • the term transfection encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • Transgene An exogenous nucleic acid sequence supplied by a vector.
  • a transgene includes any mutant APOBEC3G sequence, such as nucleotides 5-1156 of SEQ ID NOS: 9 or 21, or nucleotides 1-1152 of SEQ ID NOS: 31, 33, or 39, as well as or variants, fragments, or fusions thereof that retain mutant APOBEC3G activity.
  • variants, fragments or fusions include variants, fragments, and fusions thereof that retain the mutant APOBEC3G biological activity (such as decreasing HFV-l infection).
  • DNA sequences which encode for a protein or fusion thereof, or a fragment or variant of thereof can be engineered to allow the protein to be expressed in eukaryotic or prokaryotic cells, such as mammalian cells, bacterial cells, insect cells, and plant cells.
  • the DNA sequence can be altered and operably linked to other regulatory sequences.
  • the final product which contains the regulatory sequences and the therapeutic protein, is referred to as a vector.
  • This vector can be introduced into the desired cell. Once inside the cell the vector allows the protein to be produced.
  • PCR can be used to produce variations in the DNA sequence that encodes a protein.
  • Such variants can be variants optimized for codon preference in a host cell used to express the protein, or other sequence changes that facilitate expression.
  • Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication, and can also include one or more selectable marker genes and other genetic elements.
  • An insertional vector is capable of inserting itself into a host nucleic acid. Wild-type. A naturally occurring, non-mutated version of a nucleic acid or protein sequence. Among multiple alleles, the allele with the greatest frequency within the population is usually (but not necessarily) the wild-type. The term “native" can be used as a synonym for "wild-type.”
  • Mutant APOBEC3G Nucleic Acids and Proteins Provided herein are mutant APOBEC3G nucleic acid and protein sequences, which in some examples are used to reduce HIV-1 infection.
  • a mutant APOBEC3G protein includes a D128K, D128V, D128E, or D128A, amino acid substitution (wherein the numbering refers to a full-length wild-type human amino acid sequence, such as SEQ ED NO: 1).
  • mutant APOBEC3G proteins including a D128K, D128V, D128E, or Dl 28A amino acid substitution retain the ability to deaminate deoxycytidine to deoxuridine, but because mutated APOBEC3G is resistant to HIV-1 Vif-induced degradation, HIV-1 infection is decreased.
  • Polypeptides having mutant APOBEC3G activity are disclosed herein.
  • mutant APOBEC3G activity is characterized by the ability of the mutant protein to retain the ability to deaminate deoxycytidine to deoxuridine and bind HIV-1 Vif, but unlike wild-type APOBEC3G, is more resistant to proteosomal degradation induced by HIV-1 Vif.
  • mutant APOBEC3G sequences reduce HPV-1 infection (in the presence of HTV-1 Vif) by at least 10%, at least 20%, at least 30%, at least 50%, at least 80%, at least 90%, at least 95%, or even at least 99%, as compared to an amount of HIV-1 infection in the presence of wild-type human APOBEC3G, such as SEQ ID NO: 1 or 2.
  • mutant APOBEC3G proteins are more resistant to HIV-1 Vif-induced degradation than wild-type APOBEC3G proteins.
  • a mutated APOBEC3G amino acid sequence includes an amino acid substitution at D128, such as a substitution with a non-polar amino acid, such as Gly, Ala, or Val.
  • a mutated APOBEC3G amino acid sequence includes a D128K, D128V, D128E, or D128A amino acid substitution, such as amino acids 1-384 of SEQ ID NO: 22, 32, 34 or 40.
  • the disclosure also encompasses variants, fusions, and fragments of amino acids 1-384 of SEQ ED NO: 22, 32, 34 or 40 that retain mutant APOBEC3G activity, such as amino acids 1-384 of SEQ ID NO: 10.
  • a particular example of a variant sequence is one that includes one or more amino acid substitutions, such as at least 2, 3, 4, 5, 6, 10, 12, 15, or even more substitutions.
  • substitutions that can be made to a mutant APOBEC3G amino acid sequence include, but are not limited to, one or more of the following: S18V, Y22N, G43D, R46G, R55Q, Y124A, Y125A, F126A, W127A, P129A, D130A, Y131A, Q132A, Q57K, L62A, K79Q, T101A, R102N, D103S; E133Q, S137I, D143G, R146H, D155N, S162N, Y166D, S167G, Q168R, R169G, E170K, L171P, E173K, W175R, Y181H, E209K R238H, C243R, L253P, E254K, V265L, A329H, A33 ID, S18V, Y22N, G43D, R46G, R55Q, Y124A, Y125A, F126A,
  • a variant mutant APOBEC3G protein sequence is one that includes a fragment of a mutant APOBEC3G protein sequence, such as fragments that include a D128K, D128A, D128V, or D128E substitution.
  • the disclosure provides mutant APOBEC3G polypeptides that include at least 15 contiguous amino acids of a disclosed mutant APOBEC3G peptide, such as at least 15 contiguous amino acids of amino acids 1-384 of SEQ ID NO: 10, 22, 32, 34, and 40 that includes a D128K, D128A, D128V, or D128E substitution, for example amino acids 120-140 of SEQ ED NO: 10, 22, 32, 34, or 40.
  • mutant APOBEC3G polypeptides that contain an amino acid sequence that is greater than at least 15 amino acid residues of a disclosed mutant APOBEC3G peptide (such as at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 150, 200, 250, 300, 350 or more amino acid residues that include a D128K, D128A, D128V, or D128E substitution).
  • the disclosure also provides variant mutant APOBEC3G polypeptides that include at least one amino acid insertion, deletion, or substitution, such as 1, 2, 3, 4, 5, or 10 amino acid insertions, deletions, or substitutions, or any combination thereof (such as a single deletion together with 1-10 insertions).
  • polypeptides share at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99% sequence identity with a mutant APOBEC3G amino acid sequence (such as amino acids 1-384 of SEQ ID NOS: 10, 22, 32, 34 or 40), as long as the peptide encoded by the amino acid sequence retains mutant APOBEC3G activity.
  • a mutant APOBEC3G amino acid sequence such as amino acids 1-384 of SEQ ID NOS: 10, 22, 32, 34 or 40
  • One type of variation includes the substitution of one or more amino acid residues, and in some examples no more than 10 amino acids, for amino acid residues having a similar biochemical property, that is, a conservative amino acid substitution. Accordingly, mutant APOBEC3G polypeptides having at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 conservative substitutions are provided herein.
  • substitutions that are less conservative for example by selecting residues that differ more significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the polypeptide at the target site; or (c) the bulk of the side chain.
  • substitutions that in general are expected to produce the greatest changes in polypeptide function are those in which: (a) a hydrophilic residue, such as serine or threonine, is substituted for (or by) a hydrophobic residue, such as leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, such as lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, such as glutamic acid or aspartic acid; or (d) a residue having a bulky side chain, such as phenylalanine, is substituted for (or by) one not having a side chain, such as glycine.
  • a hydrophilic residue such as serine or threonine
  • a hydrophobic residue such as leucine, isoleucine, phenylalanine
  • polypeptides having mutant APOBEC3G activity can be assessed for polypeptides having mutant APOBEC3G activity by analyzing the ability of the polypeptide to decrease HIV-1 infection, for example as described in Example 2.
  • isolated nucleic acid molecules that encode polypeptides having mutant APOBEC3G activity for example a nucleic acid sequence that includes nucleotides 5-1156 of SEQ ID NOS: 9 or 21, or nucleotides 1-1152 of SEQ ED NOS: 31, 33, or 39.
  • an isolated nucleic acid molecule encoding a polypeptide having mutant APOBEC3G activity is operably linked to a promoter sequence, and can be part of a vector.
  • the nucleic acid molecule can be a recombinant nucleic acid molecule that can be used to transform cells and make transformed cells or transgenic primates.
  • Transformed cells including at least one exogenous nucleic acid molecule encoding a polypeptide having mutant APOBEC3G activity (such as a sequence that includes nucleotides 5-1156 ofSEQ ID NOS: 9 or 21, or nucleotides l-1152 ofSEQ ID NOS: 31, 33, or 39, or fragments, fusions, or variants thereof that retain mutant APOBEC3G activity), are disclosed.
  • a transformed cell is more resistant to HIV-1 infection, has decreased HIV-1 replication, or both, than a comparable non-transformed cell.
  • nucleic acid sequences encoding mutant APOBEC3G proteins disclosed herein can contain an entire nucleic acid sequence encoding the protein, as well as a portions thereof that retain the desired mutant APOBEC3G activity.
  • a mutant APOBEC3G nucleic acid can include at least 15 contiguous nucleotides of a mutant APOBEC3G nucleic acid sequence, wherein the at least 15 contiguous nucleotides include a sequence encoding for a D128K, D128V, D128A, or D128E substitution (such as nucleotides 380-394 of SEQ ED NO: 21, 31, 33, or 39).
  • the disclosure also provides isolated nucleic acids that contain a nucleotide sequence that is greater than 15 nucleotides (such as at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 75, 10, 200, 500, 750 or more nucleotides) in length of a mutant APOBEC3G sequence shown in nucleotides 5-1156 of SEQ ED NOS : 9 or 21 , or nucleotides 1 - 1152 of SEQ ID NOS: 31, 33, or 39, wherein the fragment includes a codon encoding for a D128K, D128E, D128V, or D128A substitution.
  • a nucleotide sequence that is greater than 15 nucleotides (such as at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 75, 10, 200, 500, 750 or more nucleotides) in length of a mutant APOBEC3G sequence shown in nucleotides 5-1156 of S
  • variants include at least one insertion, deletion, or substitution, such as 1, 2, 3, 4, 5, or 10 insertions, deletions, or substitutions, or any combination thereof (such as a single deletion together with 1-10 insertions) as long as the peptide encoded thereby retains mutant APOBEC3G activity.
  • the disclosed isolated nucleic acid molecules share at least 60, 70, 75, 80, 85, 90, 92, 95, 97, 98, or 99% sequence identity with a mutant APOBEC3G sequence (such as a sequence including nucleotides 5-1156 of SEQ ID NOS: 9 or 21, or nucleotides 1- 1152 of SEQ ID NOS: 31, 33, or 39), as long as the peptide encoded by the nucleic acid sequence retains mutant APOBEC3G activity.
  • a mutant APOBEC3G sequence such as a sequence including nucleotides 5-1156 of SEQ ID NOS: 9 or 21, or nucleotides 1- 1152 of SEQ ID NOS: 31, 33, or 39
  • the disclosure also provides isolated nucleic acid sequences that encode for a mutant APOBEC3G peptide that includes a D128K, D128V, D128E, or D128A substitution, wherein the nucleic acid sequence is at least 12 bases in length (such as at least 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 100, 250, 500, 750, 1000, 1500, 2000, 3000, 4000, or 5000 bases in length) and hybridizes, under hybridization conditions, to the sense or antisense strand of a nucleic acid encoding the protein. Particular examples of hybridization conditions are provided herein.
  • nucleic acid substitutions can be assessed for sequences that encode for peptides having mutant APOBEC3G activity, for example by determining the ability of the nucleic acid sequence to encode a protein that decreases HIV-1 infection, for example using the methods described in Example 2.
  • Mutant APOBEC3G peptides and nucleic acid sequences encoding a mutant APOBEC3G peptide are in some examples produced by standard DNA mutagenesis techniques, for example, Ml 3 primer mutagenesis or PCR. Details of these techniques are provided in Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual 2nd ed., vol.
  • Nucleic acid molecules can include changes of a coding region to fit the codon usage bias of the particular organism into which the molecule is to be introduced.
  • the disclosed peptides are in some examples produced using chemical synthesis.
  • Various automatic peptide synthesizers are commercially available and can be used in accordance with known protocols. Chemical synthesis of peptides is described in: S. B. H. Kent, Biomedical Polymers, eds. Goldberg and Nakajima, Academic Press, New York, pp. 213-242, 1980; Mitchell et al, J. Org.
  • the length of the amino acid sequence produced can depend on the method of producing the sequence. If the sequence is made by assembling amino acids by chemical means, the sequence ideally does not exceed, for example, about 50, about 40, or about 30 amino acids. If the synthetic peptide is made by translating a nucleic acid, the peptide can be any length, including, for example, about 100 amino acids or more.
  • the coding region is altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence in such a way that, while the nucleic acid sequence is substantially altered, it nevertheless encodes a polypeptide having an amino acid sequence identical or substantially similar to the native amino acid sequence.
  • nucleic acid variants can be derived from a nucleic acid sequence, for example by using standard DNA mutagenesis techniques or by synthesis of nucleic acid sequences.
  • this disclosure also encompasses nucleic acid molecules that encode the same polypeptide but vary in nucleic acid sequence by virtue of the degeneracy of the genetic code.
  • specific binding agents such as antibodies, which can distinguish between mutant and wild-type APOBEC3G proteins.
  • Such antibodies can be generated in an experimental animal, such as a mouse or rabbit, using methods well known in the art.
  • Methods of Using Mutant APOBEC3G Sequences to Decrease HIV-1 Infection It is disclosed in the Examples below that human APOBEC3G sequences that include (or encode for) a D128K, D128V, D128E, or D128A substitution can decrease HIV-1 infection.
  • methods for decreasing HTV-1 infection, such as HIV-1 replication, by contacting a cell with a therapeutically effective amount of a mutant APOBEC3G nucleic acid sequence (including derivatives, analogs, or mimetics thereof), for a time long enough to allow expression of the mutant APOBEC3G protein in the cell, and for a time long enough for the encoded protein to induce mutation in HIV-1 and decrease HIV-1 infection.
  • a mutant APOBEC3G nucleic acid sequence including derivatives, analogs, or mimetics thereof
  • methods for decreasing HIN-1 infection includes contacting a cell with a therapeutically effective amount of a mutant APOBEC3G protein (including derivatives, analogs, or mimetics thereof), or contacting the cell with agents identified using the screening methods described herein, or combinations thereof, for a time long enough t decrease HIV-1 infection, for example by inducing G-to-A mutations in HIV-1.
  • a mutant APOBEC3G protein including derivatives, analogs, or mimetics thereof
  • Decreasing HIV-1 infection does not require a 100% reduction in infection, and in some examples includes decreasing HIV-1 replication by at least 25% (such as at least 50%, at least 75%, or even at least 99%), or increased resistance of mutant APOBEC3G to HIV-1 Vif-induced degradation by at least 25% (such as at least 50%, at least 75%, or even at least 99%), or combinations thereof, as compared to an amount in the absence of the therapeutic agent.
  • the therapeutic agents such as mutant APOBEC3G proteins and nucleic acids, can be part of an in vitro solution, an in vivo expression system, or in situ with a host tissue or subject.
  • mutant APOBEC3G proteins are part of a larger molecule or complex, such as a peptide expressed as part of a fusion protein or contained as one subunit of a larger protein.
  • a mutant APOBEC3G nucleic acid can be part of a larger molecule, complex, organism or microorganism such as a mutant APOBEC3G nucleic acid contained within its genome, a recombinant vector, or a transgenic organism or microorganism (including both extrachromosomal molecules or genomic insertions).
  • the method includes contacting a cell with a therapeutically effective amount of an agent that decreases HIV-1 infection, such as a mutant APOBEC3G protein.
  • the cell is incubated with the protein for a time sufficient to allow the mutant APOBEC3G protein to be taken up by the cell, for example by endocytosis.
  • the protein can be present in a liposomal vesicle, which fuses with the cell membrane, thereby allowing the protein to enter the cell.
  • the present disclosure is not limited to particular means of administration.
  • the method includes contacting a cell with a therapeutically effective amount of a mutant APOBEC3G nucleic acid sequence, hi particular examples, the cell is incubated with the nucleic acid molecule for a time sufficient to allow the mutant APOBEC3G nucleic acid sequence to be taken up by the cell, and the protein encoded by the nucleic acid molecule expressed in the cell.
  • the nucleic acid molecule is part of a vector, which is used to transform the cell.
  • expression vectors including mutant APOBEC3G nucleic acid molecules can be introduced into the bone manow of a subject.
  • the vector, or other nucleic acid carrying the mutant APOBEC3G nucleic acid is introduced into a subject by any standard molecular biology method and can be included in a composition containing a pharmaceutically acceptable carrier.
  • the cell is present in a subject, and the method includes administering a therapeutically effective amount of the therapeutic agent (such as a mutant APOBEC3G nucleic acid or protein sequence, including derivatives, analogs, or mimetics thereof, agents identified using the screening methods described herein, or combinations thereof) to the subject, thereby reducing HIV-1 infection in the subject.
  • the HIV-1 status of the subject such as a human subject, is determined prior to administering a therapeutically effective amount of the desired agent. This allows one, such as a physician, to determine whether the subject is HIN- 1 positive and thus could benefit from the disclosed therapies.
  • the subject can be administered a mutant APOBEC3G nucleic acid or protein sequence (including derivatives, analogs, or mimetics thereof), in order to reduce HIN-1 infection of the subject.
  • a mutant APOBEC3G proteins or nucleic acid molecules that encode such proteins
  • the therapeutic agent can be administered in a pharmaceutically acceptable carrier.
  • the therapeutic agent can be administered with additional therapeutic agents (such as before, during of after administration of a mutant APOBEC3G protein or nucleic acid molecule), such as an anti-viral agent, for example AZT.
  • Mutant APOBEC3G proteins variants, fragments, and fusions can be employed in the pharmaceutical compositions, and can include one or more amino acid additions, amino acid deletions, amino acid replacements, or by isostereomer (a modified amino acid that bears close structural and spatial similarity to the original amino acid) substitutions, and isostereomer additions, so long as the resulting mutant APOBEC3G proteins can reduce HIV-1 infection.
  • such variants, fragments, and fusions provide an advantage, such as increasing the solubility of the protein, or easing linking or coupling of the protein.
  • the disclosed mutant APOBEC3G proteins can also be engineered to include other amino acids (to generate a fusion protein), such as residues of various moieties, such as additional amino acid segments or polysaccharides. Examples include, but are not limited to, moieties that augment protein stability, manufacture, or delivery within the body to sites appropriate for reducing HIV-1 infection.
  • additional amino acid sequences can be of varying length, such as at least about 5 amino acids, at least abut 10 amino acids, at least about 25 amino acids, at least about 50 amino acids, at least about 100 amino acids, or no more than about 500 amino acids, such as no more than about 250 amino acids, no more than about 100 amino acids, no more than about 75 amino acids, no more than about 50 amino acids, no more than about 25 amino acids, no more than about 15 amino acids, or no more than about 10 amino acids.
  • the subject has an HIV-1 infection, AEDS, or both, hi some examples, expression of a mutant APOBEC3G protein decreases HIV-1 infection, decreases symptoms associated with HIV-1 infection, or both.
  • expression of a mutant APOBEC3G protein can decrease, inhibit, or even prevent infection of a cell by HIV-1, or otherwise inhibit the progression or clinical manifestation of the HIV-1 infection.
  • expression of a mutant APOBEC3G protem reduces or alleviate one or more symptoms associated with HIV- 1 infection or AIDS, such as fever, headaches, aching muscles, sore throat, swollen lymph glands, diarrhea, nausea, vomiting, weight loss, or reduction in CD4 T-cell count (for example 200 cells per microliter or less).
  • the mutant APOBEC3G proteins described herein can be used to screen samples for the presence or absence of an antibody that specifically recognizes mutant APOBEC3G.
  • a mutant APOBEC3G protein can be used in an ELISA to screen a sample obtained from an individual for the presence of anti mutant APOBEC3G antibodies generated by that individual, such as a blood sample.
  • protein binding agents such as agents that specifically bind mutant APOBEC3G proteins, for example antibodies
  • Screening for Agents that Decrease HIV-1 Infection Provided herein are methods that can be used to screen agents for their ability to decrease HIV-1 infection, for example decreasing HIV-1 replication.
  • the agents are mutant APOBEC3G proteins, nucleic acid molecules, or antibodies, as well as mimetics, analogs, or derivatives thereof that may have mutant APOBEC3G activity.
  • the agents to be screened are variants, fragments or fusions .of amino acids 1-384 of SEQ ID NOS: 10, 22, 32, 34, or 40, of nucleotides 5-1156 of SEQ ED NOS: 9 or 21, or nucleotides 1-1152 of SEQ ED NOS: 31, 33, or 39.
  • the variant, fragments, or fusions of the disclosed amino acid and nucleic acid sequences include a D128K, D128A, D128E, or D128V substitution (or encode for such a substitution).
  • the method includes screening an agent for an ability to reduce HIV-1 infection. The method includes contacting the agent with a cell that included HIV-1 and HIV-1 Vif, under conditions that allow the agent to enter the cell and under conditions that reduce HIV-1 infection. In some examples, HIV-1 Vif and HIV-1 are expressed from a vector. Similarly, if the test agent is a protein, it can be expressed recombinantly in a cell.
  • HIV-1 infection was reduced, for example by comparing an amount of HIV-1 infection in the presence and absence of the test agent.
  • a determination is made as to whether the agent binds to HIV-1 Vif.
  • Agents that decrease HIV-1 infection, for example by at least 25%, and that bind HIV-1 Vif, can be selected for further study. Several types of assays can be used (alone or in combination) to determine whether HIV-1 infection was decreased.
  • the method includes determining an amount of HIV-1 replication in the cell, wherein a decrease in HIV-1 replication as compared to an amount of HIV-1 replication in the absence of the test agent indicates that the agent decreased HIV-1 infection, hi particular examples, HTV- 1 replication decreases by at least 10%, such as at least 20%, at least 30%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, or even at least 99% in the presence of the agent, as compared to an amount of HIV-1 replication in the absence of the agent. Any method used by those skilled in the art can be used to measure HIV-1 replication, hi one example, HIV-1 replication is measured as follows.
  • Cells such as human 293 T cells, are transfected with vectors that permit HIV-1 replication, such as an HFV-l based vector and a HIV-1 Vif based vector. Particular examples of vectors are described in Example 2.
  • the HIV-based vector can include a label that permits detection of HIV-1 as it is replicated, such as green fluorescent protein (GFP) or EGFP.
  • GFP green fluorescent protein
  • the HIV-1 and HIV-1 Vif based vectors can be co-transfected with a test nucleic acid sequence, such as a nucleic acid sequence encoding a mutant APOBEC3G. If the agent is a protein or non-nucleic acid based agent, the agent is incubated with the transfected cells for a time sufficient to allow uptake of the agent by the cell.
  • the amount of HIV-1 produced is determined, for example by detecting a label present on HIV-1.
  • Methods of detecting a label include, but are not limited to, flow cytometry, microscopy, and spectroscopy.
  • a negative control cells not expressing an HIV-1 Vif can be used, since in the absence of Vif, HTV-1 replication will be decreased even in the presence of wild-type APOBEC3G.
  • Such a control will also allow one to determine if the agent has deoxycytidine deaminase activity, resulting in G-to-A hypermutation of HIV-1.
  • Another assay that can be used to determine if HIV-1 infection is decreased in the presence of a test agent includes determining an amount of an HFV-l protein expressed by the cell, such as present on the cell surface, wherein a decrease in the amount of HIV-1 proteins compared to an amount of HIV-1 proteins in the absence of the agent indicates that the agent decreased HEV-1 infection.
  • the amount of HEV-1 protein present in or on the cell decreases by at least 20%, such as at least 25%, at least 40%, at least 50%, at least 75%, at least 90%, at least 95%, or even at least 99% in the presence of the agent, as compared to an amount of HTV-1 protein in the absence of the agent. Any method used by those skilled in the art can be used to measure an amount of HIV-1 protein.
  • cells can be transfected and incubated with a test agent as described above. Following the incubation, the amount of HTV-1 protein present can be determined by using a specific binding agent, such as an antibody, that specifically recognizes and binds to an HIV-1 protein.
  • a specific binding agent such as an antibody
  • the HIV-1 protein is p24.
  • the presence of this binding is then detected, hi some examples, the antibody itself includes a label.
  • the antibody is detected by a secondary antibody containing a label.
  • the presence of bound antibody (and the label) can then be detected, for example by using microscopy, flow cytometry, or ELISA.
  • the method includes determining if the agent has deoxycytidine deaminase activity, for example determining if a mutant APOBEC3G variant, fragment or fusion has deoxycytidine deaminase activity.
  • Methods for measuring deoxycytidine deaminase activity are known in the art.
  • the method is a DNA deamination assay, for example as described by Harris et al. (Cell, 113:803-9, 2003). Briefly, tagged agent, such as a His 6 -tagged mutant APOBEC3G protein, is expressed in bacteria (such as inE. coli) and purified (for example using Ni-ATA-Sepharose).
  • DNA deamination can be monitored using a UDG-based assay with biotinylated oligonucleotides SPM167 and SPM168 (see Petersen-Mahrt and Neuberger, J. Biol. Chem. 278:19583-6, 2003).
  • Isolated tagged- proteins are subjected to SDS-PAGE, transferred to a membrane, and detected, for example by using an anti-His antibody and chemiluminescence.
  • deoxycytidine deaminase activity is measured indirectly, for example by sequencing retroviral DNA following incubation with the test agent. Sequencing nucleic acids is routine in the art.
  • Therapeutic agents identified with the disclosed approaches can be used as lead compounds to identify other agents having even greater antiviral activity.
  • chemical analogs of identified chemical entities, or variant, fragments of fusions of peptide agents are screened for their ability to decrease HIV-1 infection using the disclosed assays.
  • Candidate agents are also tested for safety in animals and then used for clinical trials in animals or humans.
  • Screening Subjects for Resistance to HIV-1 Infection Also provided herein are methods of screening subjects for resistance to HIV-1 infection by characterizing an APOBEC3G nucleotide or amino acid sequence of a subject, for example by detecting an APOBEC3G mutation that is resistant to HIV-1 degradation.
  • the APOBEC3G nucleic acid molecule or protein of a subject is isolated, sequenced, and compared to a wild-type APOBEC3G sequence (such as SEQ ED NO: 1 or 2, respectively), a mutant APOBEC3G sequence (such as amino acids 1-384 of SEQ ED NO: 21 or nucleotides 5-1156 of SEQ ED NO: 22, respectively), or both, to determine if the subject has a substitution at amino acid 128 of a human APOBEC3G sequence.
  • a wild-type APOBEC3G sequence such as SEQ ED NO: 1 or 2, respectively
  • a mutant APOBEC3G sequence such as amino acids 1-384 of SEQ ED NO: 21 or nucleotides 5-1156 of SEQ ED NO: 22, respectively
  • the presence of an aspartic acid at position 128 that is, greater the similarity between the subject's APOBEC3G sequence and a wild- type human APOBEC3G sequence), the more susceptible that person
  • a substitution at amino acid 128 indicates that the subject is more resistant to HFV-l infection.
  • Assessing the genetic characteristics of a population can provide information about the susceptibility or resistance of that population to HIV-1 infection. For example, polymorphic analysis of APOBEC3G alleles in a particular human population, such as the population of a particular city or geographic area, can indicate how susceptible that population is to HIV-1 infection.
  • a large number of human APOBEC3G alleles that that does not result in an aspartic acid at amino acid 128 indicates that a population is more resistant to HEV-1 infection.
  • Such information can be used, for example, in making public health decisions about vaccinating or screening susceptible populations.
  • Example 1 Generation of human APOBEC3G Mutants This example describes methods used to generate mutant APOBEC3G sequences. Similar methods can be used to introduce other or additional mutations into APOBEC3G.
  • Ten human APOBEC3G mutants were generated in which clusters of amino acids were substituted with equivalent residues in AGM and MAC APOBEC3G (FIG. la). Thirty-eight amino acid residues that were the same in the human and the CPZ APOBEC3Gs, but were substituted with identical residues in both AGM and MAC APOBEC3Gs, were targeted. Most amino acid substitution mutations were introduced into pcDNA-APO3G (Kao et al. J. Virol.
  • the Apo4 mutant contained substitutions S18V, Y22N, G43D, R55G, Q57K, and L62A; the Apol4 mutant contained substitutions G43D, R46G, and K79Q; The Apol mutant contained substitutions T101A, R102N, and D103S; the Apol2 mutant contained substitutions D128K, E133Q, S137I, D143G and R146H; the Apol 1 mutant contained substitutions D143G and D155N; the Apo2 mutant contained substitutions S162N, Y166D, S167G, Q168R, R169G, E170K, L171P, E173K, W175R, and Y181H; the Apo9 mutant contained substitutions E209K and V265L; the Apol5 mutant contained substitutions K79Q, R238H, C243R, L253P, and E254K; the Apo
  • Example 2 Mutations in human APOBEC3G decrease HIV-1 replication The example describes the methods used to demonstrate the effects of mutations in human APOBEC3G on HEV-1 replication.
  • One skilled in the art will appreciate that other methods can be used to measure HIV-1 replication, such as those disclosed in the Examples below.
  • Cells were co-transfected with pHDV-EGFP, pC-Help- ⁇ Vif, pHCMV-G, and wild-type or mutated APOBEC3G plasmids were performed.
  • pHDV-EGFP an HIV- 1 -based vector, Unutmaz et al. J. Exp. Med. 189:1735-46, 1999
  • pC-Help- ⁇ Vif (Kao et al. J. Virol. 77:11398-407, 2003) does not express Vif, Env or packagable viral RNA, but expresses all other viral proteins needed to complete one cycle of replication.
  • pC-Help is an HIV-1 helper construct that lacks several cis-acting elements needed for viral replication, including the packaging signal and primer-binding site; it expresses all of the viral proteins except Nef and Env.
  • the vector pC-Help ⁇ Vif is identical to pC-Help except that the Vif open reading frame has been disrupted with a deletion. pHCMV-G (Yee et al. Methods Cell. Biol.
  • VSV vesicular stomatitis virus
  • Cells were infected with virus that was equivalent to 100 ng of p24 capsid.
  • the infected cells were analyzed by flow cytometry (FACScan; Becton-Dickinson) for green fluorescence 36-48 hours after infection and the results analyzed using CellQuest software (Becton-Dickinson).
  • the mutated APOBEC3G plasmids express an enzymatically active deoxycytidine deaminase that, in the absence of Vif, is incorporated into the HDV-EGFP virion, resulting in G-to-A hypermutation and diminished GFP expression.
  • FIG. lb bar graphs labeled Vif+.
  • expression of wild type APOBEC3G did not diminish GFP expression in infected cells. Therefore, HIV-1 Vif expressed from pC- Help protected HDV-EGFP replication from wild-type APOBEC3G.
  • pC- Help cotransfection and the resulting HIV-1 Vif expression protected HDV-EGFP replication from most of the mutated APOBEC3G plasmids, indicating that these mutations in APOBEC3G did not influence the ability of HIV-1 Vif to counteract their inhibitory effects on HIV-1 replication.
  • the Apo 12 mutant inhibited the replication of HDN-EGFP even in the presence of pC-Help, indicating that its antiviral activity was resistant to HIV-1 Vif.
  • Example 3 HIV-1 Vif does not reduce intracellular steady-state levels of the Apol2 mutant
  • Intracellular steady-state levels of wild-type and Apol2 mutant APOBEC3G proteins were determined by Western blotting detection of C-terminal myc-tagged APOBEC3G proteins in the presence and absence of HIV-1 Vif.
  • an anti-c-Myc antibody Sigma- Aldrich was coupled to paramagnetic beads according to manufacturer's instructions (Dynal Biotech).
  • 293T cells were cotransfected with APOBEC3G expressing plasmids and either pC-Help or pC-Help ⁇ Vif. Approximately 36 hours after transfection, 2 x 10 6 cells were harvested, washed twice with ice-cold PBS, and lysed in 1 ml of cell extraction buffer (20 mM Tris-Cl, pH 8.0, 137 mM ⁇ aCl, 1 mM EDTA, 1 mM ⁇ aVO 3 , 10% glycerol, 1% Triton X-100 and protease inhibitor cocktail [Roche]).
  • cell extraction buffer (20 mM Tris-Cl, pH 8.0, 137 mM ⁇ aCl, 1 mM EDTA, 1 mM ⁇ aVO 3 , 10% glycerol, 1% Triton X-100 and protease inhibitor cocktail [Roche]).
  • Cell extracts were adjusted to equivalent protein concentration by using Bradford reagent (BioRad Labotories), and equal aliquots were then used for co-immunoprecipitation and Western blotting analysis.
  • Cell extracts were centrifuged at 1,500 x g for four minutes, and the supe atants incubated with anti-c-Myc antibody conjugated paramagnectic beads for three hours in slow rotation on RKDynal rotor (Dynal Biotech) at 4°C.
  • the beads were washed three times with 50 mM Tris-HCI, pH 7.5, 500 mM LiCl, 1 mM NaVO 3 and 0.5% Triton X-100, 3 times with 50 mM Tris-HCI, pH 7.5, 500 mM LiCl, 1 mM NaVO 3 , and once with 1 mM NaVO 3 .
  • the bound proteins were eluted from the beads by heating to 90°C for five minutes in SDS-PAGE loading buffer.
  • 2 x 10 6 cells were harvested, washed with ice-cold PBS 36 hours after transfection, lysed in 1 x SDS-PAGE loading buffer, and heated to 90°C for five minutes.
  • the myc epitope-tagged APOBEC3G proteins were detected by using the anti-c-Myc antibody
  • the tubulin protein was detected by using the anti-tubulin antibody (Sigma- Aldrich) to insure that equivalent aliquots were loaded on to gels
  • the HIV-1 Vif protein was detected by using anti-HIV-lHXB2 Vif antiserum (Dana Gabuzda, Dana- Farber Cancer Institute) obtained through the AEDS Reagents and Reference Program, Division of AIDS, MAID, NEEL As shown in FIG.
  • Wild type APOBEC3G and the Apo 12 mutant proteins were immunoprecipitaed from cell lysates.
  • the cellular proteins that were co-immunoprecipitated were analyzed by Western blotting for the presence of Vif using an anti- Vif antibody.
  • FIG. 2c a trace amount of HIV-1 Vif was non-specifically co-precipitated in the absence of APOBEC3G; in contrast, significantly greater amounts of HIV- 1 Vif co-immunoprecipitated with both the wild- type and the Apol2 mutant APOBEC3G proteins. Therefore, the Apol2 APOBEC3G mutant can interact with HIV-1 Vif.
  • the proportion of GFP+ cells after infection with HDV-EGFP in the presence of wild-type APOBEC3G and HIV-1 Vif was set to 100%.
  • the Apol2 mutant dominantly inhibited replication of HDV-EGFP in the presence of HTV-1 Vif and wild-type APOBEC3G. Therefore, a single amino acid substitution (D128K) renders human APOBEC3G resistant to HIV-1 Vif. This mutant provides a tool for decreasing HIV- 1 replication in vivo.
  • Example 4 D128K Mutation responsible for Vif-resistant phenotype This example describes methods used to identify the amino acid substitution in Apo 12 responsible for the HIV-1 Vif-resistant phenotype described in the Examples above.
  • the HIV-1 Vif-resistant Apo 12 mutant contains five single amino acid substitutions (D128K, E133Q, S137I, D143G, and R146H). To determine which of these mutations resulted in the Vif-resistant phenotype, mutants containing single substitutions D128K (SEQ ED NOS: 21 and 22), E133Q (SEQ ID NOS: 25 and 26), and S137I (SEQ ID NOS: 27 and 28) were generated. Because the Vif-sensitive
  • Apol2.1 mutant contained the D143G and R146H substitutions, they were unlikely to be responsible for the Vif-resistant phenotype.
  • the effects of these single amino acid substitution mutations in APOBEC3G on antiviral activity were determined in the absence and presence of HIV-1 Vif, using the methods described in Examples 2 and 3.
  • the proportion of GFP positive cells generated in the presence of wild type APOBEC3G in the presence of HIV-1 Vif was set to 100%, and the relative proportion of GFP positive cells generated by infection with HDV-EGFP virion in the presence of wild type or mutant APOBEC3G are shown in FIG. 3a. As shown in FIG.
  • Vif expression constructs were generated by PCR amplification of the Vif open reading frames from the respective proviral constructs and cloning the PCR products into pCR 3.1 (Invitrogen). The structure of the Vif expression constructs was verified by DNA sequencing (SAIC, Core Facility). The proportion of GFP positive cells generated in the presence of wild type APOBEC3G in the presence of HIV-1 Vif was set to 100%. As shown in FIG. 3b, in the absence of Vif, both the wild type and the D128K mutant APOBEC3G inhibited HDV-EGFP replication.
  • D128K mutant is resistant to HIV-1 Vif, but not to SIVmac239 or HIV-2 Vif, indicating that it can be a substrate for the proteosomal pathway.
  • Example 5 Vertical Mutagenesis of D128K APOBEC3G Mutation This example describes methods used to identify additional substitutions that can be made at amino acid 128 of human APOBEC3G, and retain the ability to reduce HIV-1 viral infection in the presence of HIV-1 Vif. The aspartic acid at position 128 was substituted with V, E, G, R and A
  • the relative proportion of GFPH- cells generated by infection with HDV-EGFP virion in the presence of wild type or mutant/variant APOBEC3G are shown in FIG. 4.
  • the proportion of GFP+- cells generated in the presence of wild type APOBEC3G in the presence of HIV-1 Vif was set to 100%.
  • D128E, D128A, and D128V substitutions exhibit an HEV- 1 Vif-resistant phenotype and are at least as resistant to HIV-1 Vif as the D128K mutant described in the above examples.
  • the D128R and D128G variants were not measurably Vif-resistant.
  • Example 6 Alanine Scan This example describes methods used to identify additional amino acid substitutions that can be made to mutant APOBEC3G. Such substitutions, in combination with a D128K, D128A, D128V, or D128E substitution, retain the ability to reduce HIV-1 viral infection in the presence of HIV-1 Vif. Amino acid substitution mutations at positions adjacent to D128 (Y124A,
  • Y125A, F126A, W127A, P129A, D130A, Y131A and Q132A were introduced into pcDNA-APO3G (Kao et al. J. Virol. 77:11398-407, 2003) using the Multi-Site Mutagenesis Kit as per the manufacturer's instructions (Stratagene).
  • the presence of the desired mutations and the absence of undesired mutations were verified by DNA sequencing (Core Facility, SAIC-Frederick). The variant sequences were then tested for their ability to decrease HIV-1 replication in the presence of HIV-1 Vif, as described in Example 2.
  • mutant APOBEC3G molecules such as those including a D128K, D128A, D128V, or D128E substitution, decrease HIV-1 infection, even in the presence of HIV-1 Vif.
  • this example provides exemplary methods that can be used to reduce HIV-1 infection, for example of a cell, such as a cell of a subject having an HFV-l infection. Such methods can also be used to or reduce the symptoms of HIV-1 infection, AEDS, or both.
  • mutant APOBEC3G molecules can be administered to a subject at a therapeutically effective dose, thereby relieving the symptoms associated with HFV-l AEDS, or both.
  • the disclosed agents can also be administered with other therapeutic agents, such as other anti- viral compounds.
  • the additional anti-viral compounds can be administered before, during, or subsequent to administration of the disclosed therapeutic molecules. Because mutant APOBEC3G is resistant to Vif-degradation, but retains deoxycytidine deaminase activity, HIV-1 infection is decreased in the presence of mutant APOBEC3G molecules.
  • the mutant APOBEC3G is administered in combination with other drugs (such as anti-viral and anti-bacterial agents) that are used to treat opportunistic infections in HIV infected patients.
  • drugs such as anti-viral and anti-bacterial agents
  • such agents include, but are not limited to interleukin 2, antibiotics, protease inhibitors, non-nucleoside reverse-transcriptase inhibitors, for example Ziagen (abacavir), tenofovir, Pentafuside (enfuvirtide), Sustiva (efavirenz), Crixivan (indinavir), Viracept (nelfinavir), AZT (zidovudine) and 3TC (amivudine).
  • a subject susceptible to or having an HIV-1 infection, having AIDS, or both, wherein decreased amounts of infection by the vims is desired, can be treated with a therapeutically effective amount of a mutant APOBEC3G molecule (including derivatives, analogs, or mimetics thereof).
  • a mutant APOBEC3G molecule including derivatives, analogs, or mimetics thereof.
  • the mutant APOBEC3G molecule has produced an effect (a decreased level of HIV-1 infection is observed, or symptoms associated with HIV-1 infection decrease), for example after 24-48 hours, the subject can be monitored for diseases associated with HTV-1 infection.
  • other agents having mutant APOBEC3G activity can also be used to decrease or inhibit HIV-1 infection.
  • Other exemplary agents include those identified using the methods described in Example 10 below.
  • agents such as antibodies, peptides, nucleic acids, organic or inorganic compounds
  • the agent After the agent has produced an effect (a decreased level of HIV-1 infection is observed, or symptoms associated with HIV-1 infection decrease), for example after 24-48 hours, the subject can be monitored for diseases and symptoms associated with HIV-1 infection.
  • the treatments disclosed herein can also be used prophylactically, for example to inhibit or prevent a HIV-1 infection. Such adminisfration is indicated where the treatment is shown to have utility for treatment or prevention of the disorder.
  • the prophylactic use is indicated in conditions known or suspected of progressing to disorders associated with a HFV-l infection, such as AIDS.
  • Example 8 Recombinant Expression With the disclosed mutant APOBEC3G sequences that reduce HIV-1 infection, native and variant sequences can be generated. Expression and purification by standard laboratory techniques of any variant, such as a polymorphism, mutant, fragment or fusion of a mutant APOBEC3G sequence, which can reduce HIV-1 infection, is enabled.
  • mutant APOBEC3G sequences that reduce HIV-1 infection, as well as variants thereof that retain this biological activity can be produced recombinantly in any cell or organism of interest, and purified prior to use. Methods for producing recombinant proteins are well known in the art. Therefore, the scope of this disclosure includes recombinant expression of any host protein or variant or fragment thereof involved in viral infection.
  • partial, full-length, or variant mutant APOBEC3G cDNA sequences that encode for a protein that reduces HIV-1 infection can be ligated into an expression vector, such as a bacterial expression vector.
  • Proteins or peptides can be produced by placing a promoter upstream of the cDNA sequence.
  • promoters include, but are not limited to lac, trp, tac, trc, major operator and promoter regions of phage lambda, the control region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma, adenovirus, refrovirus, baculovirus and simian vims, the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors and combinations thereof.
  • Vectors suitable for the production of intact proteins include pKC30 (Shimatake and Rosenberg, 1981, Nature 292:128), pKK177-3 (Amann and Brosius, 1985, Gene 40:183) and pET-3 (Studiar and Moffatt, 1986, J. Mol. Biol. 189:113).
  • a DNA sequence can be transferred to other cloning vehicles, such as other plasmids, bacteriophages, cosmids, animal viruses and yeast artificial chromosomes (YACs) (Burke et al, 1987, Science 236:806-12).
  • vectors can be introduced into a variety of hosts including somatic cells, and simple or complex organisms, such as bacteria, fungi (Timberlake and Marshall, 1989, Science 244:1313-7), invertebrates, plants (Gasser and Fraley, 1989, Science 244:1293), and mammals (Pursel et al, 1989, Science 244:1281-8), that are rendered transgenic by the introduction of the heterologous cDNA.
  • somatic cells such as bacteria, fungi (Timberlake and Marshall, 1989, Science 244:1313-7), invertebrates, plants (Gasser and Fraley, 1989, Science 244:1293), and mammals (Pursel et al, 1989, Science 244:1281-8), that are rendered transgenic by the introduction of the heterologous cDNA.
  • a mutant APOBEC3G cDNA sequence for expression in mammalian cells, can be ligated to heterologous promoters, such as the simian virus SV40, promoter in the pSV2 vector (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6), and introduced into cells, such as monkey COS-1 cells (Gluzman, 1981, Cell 23:175-82), to achieve transient or long-term expression.
  • heterologous promoters such as the simian virus SV40, promoter in the pSV2 vector (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6)
  • cells such as monkey COS-1 cells (Gluzman, 1981, Cell 23:175-82), to achieve transient or long-term expression.
  • the stable integration of the chimeric gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin (Southern and Berg, 1982, J. Mol. Appl. Genet. 1:327-41) and mycophoenohc acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6).
  • biochemical selection such as neomycin (Southern and Berg, 1982, J. Mol. Appl. Genet. 1:327-41) and mycophoenohc acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6).
  • the transfer of DNA into eukaryotic, such as human or other mammalian cells is a conventional technique.
  • the vectors are introduced into the recipient cells as pure DNA (transfection) by, for example, precipitation with calcium phosphate (Graham and vander Eb, 1973, Virology 52:466) strontium phosphate
  • the cDNA can be introduced by infection with vims vectors, for example retroviruses (Bernstein et al, 1985, Gen. Engrg. 7:235) such as adeno iruses (Ahmad et al, J. Virol. 57:267, 1986) or He ⁇ es (Spaete et ⁇ /., Cell 30:295, 1982).
  • vims vectors for example retroviruses (Bernstein et al, 1985, Gen. Engrg. 7:235) such as adeno iruses (Ahmad et al, J. Virol. 57:267, 1986) or He ⁇ es (Spaete et ⁇ /., Cell 30:295, 1982).
  • mutant AP BEC3G molecules can be used to reduce HIV-1 infection.
  • the subject can be monitored for HFV-l infection, symptoms associated with HIV-1 infection, or both.
  • This example describes several methods that can be used to administer the therapeutic molecules disclosed herein, such as mutant APOBEC3G molecules that reduce HTV-1 infection.
  • Administering the therapeutic molecules the present disclosure can be accomplished by any means known to the skilled artisan.
  • the disclosed therapeutic molecules can be administered to a subject directly, or can be administered to a cell ex vivo, and then the cell introduced into the subject.
  • the disclosed therapeutic agents can be administered alone, or in combination with a pharmaceutical carrier, other therapeutic agents (such as other anti-viral therapeutics and other agents that alleviate symptoms associated with HIV-1 infection), or both. If the disclosed therapeutic agents are administered with one or more other therapeutic agents, administration of the disclosed therapeutic agents can before, during, or subsequent to administration of other therapeutic agents.
  • Pharmaceutical compositions are disclosed that include a therapeutically effective amount of a disclosed therapeutic nucleic acid molecule, protein, antibody, or other therapeutic agent, alone or with a pharmaceutically acceptable carrier. In one example, a composition that includes a disclosed therapeutic agent is formulated and administered with an additional antiviral compound as a single dose.
  • compositions administered to a subject can begin whenever treatment of signs, symptoms, or laboratory results associated with HIN-1 infection or progression is desired, or when it is desired to reduce viral load in asymptomatic HIV- infected subjects. While the disclosed compositions can be used to treat human subjects, they can also be used to treat similar diseases in other vertebrates such as other primates, farm animals such as swine, cattle and poultry, and sport animals and pets such as horses, dogs and cats.
  • the pharmaceutical compositions that include a mutant APOBEC3G molecule, or agent identified using the methods described in Example 10 (or combinations thereof), can be formulated in unit dosage form, suitable for individual administration of precise dosages.
  • a therapeutically effective amount of an agent can be administered in a single dose, or in multiple doses, for example daily, during a course of freatment.
  • Compositions that include a therapeutic agent can be administered whenever the effect (such as decreased signs, symptom, or laboratory results of HIV-1 infection) is desired.
  • a time-release formulation can also be utilized.
  • a therapeutically effective amount of a composition that includes a disclosed therapeutic molecule can be administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time. In pulse doses, a bolus administration of a composition that includes a disclosed therapeutic molecule is provided, followed by a time-period wherein no disclosed therapeutic molecule is administered to the subject, followed by a second bolus administration.
  • pulse doses of compositions that include a disclosed therapeutic molecule are administered during the course of a day, during the course of a week, or during the course of a month.
  • Amounts effective for therapeutic use can depend on the severity of the disease and the age, weight, general state of the patient, and other clinical factors. Thus, the final determination of the appropriate treatment regimen will be made by the attending clinician.
  • dosages used in vitro can provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders.
  • APOBEC3G protein is from about 0.1 ⁇ g/kg body weight to about 100 mg/kg body weight.
  • Other suitable ranges include doses of from about 1 ⁇ g/kg to 10 mg/kg body weight.
  • the dose is about 1.0 ⁇ g to about 50 mg, for example, 1 ⁇ g to 1 mg, such as 1 mg peptide per subject.
  • the dosing schedule can vary from daily to as seldom as once a year, depending on clinical factors, such as the subject's sensitivity to the peptide and tempo of their disease. Therefore, a subject can receive a first dose of a disclosed therapeutic molecule, and then receive a second dose (or even more doses) at some later time(s), such as at least one day later, such as at least one week later.
  • compositions disclosed herein can be prepared and administered in dose units.
  • Solid dose units include tablets, capsules, transdermal delivery systems, and suppositories.
  • the administration of a therapeutic amount can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • Suitable single or divided doses include, but are not limited to about 0.01, 0.1, 0.5, 1, 3, 5, 10, 15, 30, or 50 ⁇ g protein/kg/day
  • the disclosed therapeutic agents and pharmaceutical compositions can be administered by any method used in the art, for example locally or systeroically, such as topically, intravenously, orally, parenterally, nasally, vaginally, rectally, intradermally, subcutaneously, sublingually, transdermally, transmucosally, or as implants.
  • parenteral refers to non-oral modes of administration, such as intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug deli-very systems.
  • compositions can be provided as parenteral compositions, such as for injection or infusion.
  • parenteral compositions such as for injection or infusion.
  • Such compositions are formulated generally by mixing a disclosed therapeutic agent at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, for example one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • a disclosed therapeutic agent can be suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0.
  • useful buffers include sodium citrate- citric acid and sodium phosphate-phosphoric acid, and sodium acetate/acetic acid buffers.
  • the active ingredient, optionally together with excipients, can also be in the form of a lyophilisate and can be made into a solution prior to parenteral administration by the addition of suitable solvents. Solutions such as those that are used, for example, for parenteral administration can also be used as infusion solutions.
  • a form of repository or "depot” slow release preparation can be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.
  • Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Pharmaceutical compositions that include a disclosed therapeutic agent as an active ingredient can be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. The product can be shaped into the desired formulation.
  • the carrier is a parenteral canier, such as a solution that is isotonic with the blood of the recipient.
  • parenteral canier such as a solution that is isotonic with the blood of the recipient.
  • carrier vehicles are known in the art (for example see Remington's Pharmaceutical Sciences by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975; and Wang and Hanson, J. Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2S, 1988).
  • the disclosed pharmaceutical compositions can also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Excipients that can be included in the disclosed compositions include flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
  • Various delivery systems for administering the therapies disclosed herein are known, and include encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (Wu and Wu, J. Biol. Chem. 1987, 262:4429-32), and construction of therapeutic nucleic acids as part of a retroviral or other vector.
  • compositions including a disclosed therapeutic agent are administered by sustained-release systems.
  • sustained-release systems include suitable polymeric materials (such as, semi-permeable polymer matrices in the form of shaped articles, for example films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
  • suitable polymeric materials such as, semi-permeable polymer matrices in the form of shaped articles, for example films, or mirocapsules
  • suitable hydrophobic materials for example as an emulsion in an acceptable oil
  • ion exchange resins for example as an emulsion in an acceptable oil
  • sparingly soluble derivatives such as, for example, a sparingly soluble salt
  • Sustained-release compositions can be administered orally, parenterally, intracistemally, infraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), or as
  • a disclosed therapeutic agent is administered to a subject in a colloidal dispersion system.
  • Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo.
  • Large uni-lamellar vesicles (LUV) which range in size from 0.2-4.0 ⁇ m, can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al, 1981, Trends Biochem. Sci.
  • liposomes containing a disclosed therapeutic agent are administered to a subject (see generally, Langer, Science 249:1527-1533, 1990; Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-65, 1989).
  • Liposomes containing a disclosed therapeutic agent can be prepared by known methods: DE 3,218,121; Epstein et al, Proc. Natl. Acad. Sci. U.S.A. 82:3688-92, 1985; Hwang et al, Proc. Natl. Acad. Sci. U.S.A.
  • the composition and methods of preparations of these liposomes are disclosed in these references.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active.
  • Passive targeting utilizes the natural tendency of liposomes to distribute to' cells of the reticulo-endothelial system (RES) in organs that contain sinusoidal capillaries.
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • the surface of the targeted delivery system can be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • lipid chains can be joined to the targeting ligand.
  • a liposome includes a mutant APOBEC3G peptide and is directed to a cell, where the liposomes then deliver the selected therapeutic epitope.
  • a variety of methods are available for preparing liposomes and are described, for example, in Szoka et al., 1980, Ann. Rev. Biophys. Bioeng 9:467 and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
  • Particular lipid residues such as palmitic acid or other uncharged fatty acid residues of different chain lengths and degrees of unsaturation, ranging from acetic to stearic acid as well as to negatively charged succinyl residues can be attached to the peptide via the appropriate carboxylic acid anhydrides.
  • the lipids can be directly attached to the peptide or indirectly through a linkage as described above.
  • a lipid can be attached directly to the amino terminus of the peptide or via a linkage such as Ser-Ser, Gly, Gly-Gly, or Ser.
  • Preparations for administration can be suitably formulated to give controlled release of a disclosed therapeutic agent.
  • the pharmaceutical compositions can be in the form of particles comprising a biodegradable polymer, a polysaccharide jellifying or bioadhesive polymer, an amphiphilic polymer, an agent modifying the interface properties of the particles, (or combinations thereof) and a pharmacologically active substance.
  • these compositions exhibit certain biocompatibility features that allow a controlled release of the active substance. See U.S. Patent No. 5,700,486.
  • Compositions that include a disclosed therapeutic agent can be delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201, 1987; Buchwald et al, Surgery 88:507, 1980; Saudek et al, N.
  • An intravenous bag solution can also be employed.
  • One factor in selecting an appropriate dose is the result obtained, as measured by the methods disclosed here, as are deemed appropriate by the practitioner.
  • Other controlled release systems are discussed in Langer (Science 249:1527-33, 1990).
  • the pump is implanted (for example see U.S. Patent Nos. 6,436,091; 5,939,380; and 5,993,414).
  • hnplantable dmg infusion devices are used to provide patients with a constant and long-term dosage or infusion of a therapeutic agent. Such device can be categorized as either active or passive.
  • Active dmg or programmable infusion devices feature a pump or a metering system to deliver the agent into the patient's system.
  • An example of such an active infusion device currently available is the Medtronic SynchroMedTM programmable pump.
  • Passive infusion devices in contrast, do not feature a pump, but rather rely upon a pressurized dmg reservoir to deliver the agent of interest.
  • An example of such a device includes the Medtronic IsoMedTM.
  • the pharmaceutical compositions can take the form of, for example, powders, pills, tablets, or capsules, prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (such as pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc or silica); disintegrants (such as potato starch or sodium starch glycolate); or wetting agents (such as sodium lauryl sulphate).
  • binding agents such as pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers such as lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants such as magnesium stearate, talc or silica
  • disintegrants such as potato starch or sodium starch glycolate
  • wetting agents such as sodium lauryl sulphate
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any normally employed excipient, and generally about 10-95% of active ingredient (such as a mutant APOBEC3G protein), for example, at a concentration of about 25%-75%.
  • active ingredient such as a mutant APOBEC3G protein
  • protein-based pharmaceuticals may be only inefficiently delivered through ingestion.
  • pill-based forms of pharmaceutical proteins can be administered subcutaneously, particularly if formulated in a slow-release composition.
  • Slow-release formulations can be produced by combining the mutant APOBEC3G protein with a biocompatible matrix, such as cholesterol.
  • the disclosed mutant APOBEC3G peptide compositions can be supplied in finely divided form along with a surfactant and propellant.
  • Typical percentages of peptides are about 0.01% - about 20% by weight, for example, about 1% - about 10%.
  • the surfactant ideally is nontoxic and soluble in the propellant, as is known in the art.
  • Exemplary propellants include dichlorodifluoromethane, trichlorofluoromethane, dichlorotefrafluoroethane, carbon dioxide or other suitable gas.
  • a carrier can also be included, as desired, for example a hydrocarbon, such as n-butane, propane, isopentane.
  • lecithin may be used for intranasal delivery.
  • a stabilizer or porous particles for deep lung delivery are included (for example, see U.S. Patent No.
  • the nucleic acid can be delivered intracellularly (for example by expression from a nucleic acid vector or by receptor-mediated mechanisms), or by an appropriate nucleic acid expression vector which is administered so that it becomes intracellular, for example by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (such as a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (for example Joliot et al, Proc.
  • a retroviral vector see U.S. Patent No. 4,980,286
  • microparticle bombardment such as a gene gun; Biolistic, Dupont
  • coating with lipids or cell-surface receptors or transfecting agents or by administering it in linkage to a homeobox-like peptide which is known to enter
  • the present disclosure includes all forms of nucleic acid delivery, including synthetic oligos, naked DNA, plasmid and viral, integrated into the genome or not.
  • a viral vector is utilized. These vectors include, but are not limited to, adenovims, herpes vims, vaccinia, or an RNA vims such as a retrovirus.
  • the retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia vims (MoMuLV), Harvey murine sarcoma vims (HaMuSV), murine mammary tumor vims (MuMTV), and Rous Sarcoma Vims (RSV).
  • MoMuLV Moloney murine leukemia vims
  • HaMuSV Harvey murine sarcoma vims
  • MuMTV murine mammary tumor vims
  • RSV Rous Sarcoma Vims
  • GaLV gibbon ape leukemia vims
  • retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • helper cell lines that have deletions of the packaging signal include, but are not limited to Q2, PA317, and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • the disclosure provides compositions that include a mutant APOBEC3G protein, for example a composition that includes at least 50%, for example at least 90%, of a mutant APOBEC3G protein in the composition.
  • compositions are useful as therapeutic agents when constituted as pharmaceutical compositions with the appropriate carriers or diluents.
  • the disclosure also provides a pharmaceutical pack or kit including one or more containers filled with one or more of the ingredients of the pharmaceutical compositions.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Instructions for use of the composition can also be included.
  • EXAMPLE 10 Screening Assays This example describes methods that can be used to screen agents for their ability to decrease HIV-1 infection.
  • mutant APOBEC3G molecules that included a D128K, D128A, D128V, or D128E substitution retained deoxycytidine deaminase activity and the ability to interact with HTV-1 Vif, but decreased HIV-1 replication because the mutant molecules were resistant to HIV-1 Vif-induced degradation. Therefore, screening assays can be used to identify and analyze other agents, such as variants, fragments, or fusions of mutant APOBEC3G nucleic acid and protein sequences, that can also decrease HIV-1 infection.
  • the present disclosure is not limited to the particular methods disclosed herein.
  • Agents identified via the disclosed assays can be useful, for example, in decreasing HFV-l infection (including HIV-1 replication), for example in treating a subject having an HIV-1 infection, such as a subject having AIDS. Assays for testing the effectiveness of the identified agents, are disclosed herein.
  • agents that can be screened include, but are not limited to, any peptide or non-peptide composition in a purified or non-purified form, such as peptides made of D-and/or L-configuration amino acids (in, for example, the form of random peptide libraries; see Lam et al, Nature 354:82-4, 1991), phosphopeptides (such as in the form of random or partially degenerate, directed phosphopeptide libraries; see, for example, Songyang et al, Cell 12:161-18, 1993), antibodies, and small or large organic or inorganic molecules.
  • a test agent can also include a complex mixture or "cocktail" of molecules.
  • test agents include variants, fragments, or fusions of the sequences shown in amino acids 1-384 of SEQ ED NOS: 10, 22, 32, 34, or 40, and nucleotides 5-1156 of SEQ ED NOS: 9 or 21, or nucleotides 1-1152 of SEQ ED NOS: 31, 33, or 39.
  • the basic principle of the assay systems used to identify agents that decrease HTV-1 infection involves preparing a reaction mixture containing HIV-1 Vif, HIV-1, and the agent to be tested under conditions and for a time sufficient to allow the agent to decrease HIV-1 infection.
  • HTV-1 Vif and HTV-1 are expressed recombinantly in a cell, and the cell is contacted with the agent.
  • controls can be included, such as reaction mixtures containing no test agent or a placebo.
  • the control includes a wild-type APOBEC3G nucleic acid or protein (such as SEQ ED NOS: 1 and 2, respectively).
  • Such controls should permit HIV-1 replication.
  • Another exemplary control includes agents known to reduce HIV-1 replication, such as mutant APOBEC3G nucleic acid or protein (such as amino acids 1-384 of SEQ ED NOS: 10, 22, 32, 34, and 40, or nucleotides 5-1156 of SEQ ED NOS: 9 or 21, or nucleotides 1- 1152 of SEQ ID NOS: 31, 33, or 39, respectively).
  • the ability of the agent to decrease HIV-1 infection is then determined.
  • the ability of the agent to bind HFV-l Vif is also determined.
  • Agents that decrease HIV-1 infection and bind HIV- 1 Vif can be selected for further investigation.
  • the ability of the agent to decrease HIV-1 infection for example decrease HIV-1 replication, decrease expression of one or more HIV-1 proteins, or combinations thereof, by a desired amount, such as a decrease of at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 95%, or even at least 99% in the presence of the agent as compared to an amount of infection/replication or protein expression in the absence of the agent, as well as an ability of the agent to bind HIV-1 Vif, indicates that the agent can be used to decrease HFV-l replication or decrease HIV-1 protein expression, and is therefore possibly an agent that can be used to treat subjects having an HIV-1 infection, AFDS, or both.
  • the ability of the agent to decrease HIV-1 infection is established by determining the number of CD4 T-cells that are present in the subject.
  • the ability of the agent to increase the number of CD4 T-cells present in the subject by a desired amount such as an increase of at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 95%, or even at least 99% in the presence of the agent as compared to an amount of CD4 T-cells present in the subject in the absence of the agent, indicates that the agent can be used to increase the number CD4 T-cells present in the subject, and is therefore possibly an agent that can be used to treat subjects having an HIV-1 infection, AIDS, or both.
  • a desired amount of CD4 T cells is an amount greater than 350 cells/ ⁇ L, such as at least 400 cells/ ⁇ L, such as at least 500 cells/ ⁇ L, such as at least 600 cells/ ⁇ L, such as at least 800 cells/ ⁇ L, such as at least 1000 cells/ ⁇ L, such as at least 1200 cells/ ⁇ L.
  • Methods of measuring the number of CD4 T-cells in a subject are known.
  • a blood sample from the subject is analyzed for the presence of CD4 T-cells, for example by incubating the sample with a CD4 antibody.
  • the CD4 antibody can include a detectable label, such as a fluorophore, or can be detected with a secondary antibody that includes a label.
  • the presence of the label can be detected, for example by flow cytometry.
  • Methods that can be used to assess a relative amount of HIV-1 infection are described in the Examples above. Briefly, cells expressing HFV-l Vif and HTV-1 are contacted with the agent. Fn examples where the agent is a nucleic acid molecule, the nucleic acid molecule can be expressed recombinantly in the cell.
  • the amount of agent administered can be determined by skilled practitioners. Fn some examples, several different doses of the potential therapeutic agent can be administered, to identify optimal dose ranges.
  • HTV-1 infection such as an amount of HFV-l replication, an amount of HFV-l protein expression, or an amount of CD4 T-cells, or combinations thereof, using the methods described herein.
  • the ability of an agent to bind HIV-1 Vif can be measured using any type of binding assay, for example by using the immunoprecipitation and Western blotting methods described in the Examples above.
  • the agent can be incubated with HTV-1 Vif for a time sufficient to allow binding to occur.
  • agent- Vif complexes can then be detected, for example by immunoprecipitating the complexes with an anti-HTV-1 -Vif antibody, and then determining whether the agent is bound to HTV-1 Vif, for example by Western blotting the complexes using an antibody that recognizes the agent.
  • Example 11 Assays for Measuring Inhibition of HIV-1 Infection Any of the agents identified in the foregoing assay systems can be tested for their ability to decrease or inhibit infection by HIV.
  • Cell-based assays Cells (such as about 20,000 to 250,000 cells) are infected with the desired pathogen, such as HIV-1, for example using the methods described in Example 2, and the incubation continued for 3-7 days.
  • the test agent can be applied to the cells before, during, or after infection with the vims.
  • the amount of vims and agent administered can be determined by skilled practitioners. Fn some examples, several different doses of the potential therapeutic agent can be administered, to identify optimal dose ranges.
  • assays are conducted to determine the resistance of the cells to infection by various agents. For example, the presence of an HTV-1 antigen can be determined by using antibody specific for an HIV-1 protein then detecting the antibody.
  • HTV- 1 antibodies include, but are not limited to, antibody against p24 of HTV in the ELISA kit (Perkin-Elmer) and anti-HTV-1 HXB2 Vif antisemm against HIV-1 Vif protein (Dana Gabuzda, Dana-Farber Cancer Institute) obtained through the AEDS Reagents and Reference Program, Division of AIDS, NIAED, NEH.
  • the antibody that specifically binds to an HIV-1 protein is labeled, for example with a detectable marker such as a flurophore.
  • the antibody is detected by using a secondary antibody containing a label. The presence of bound antibody is then detected, for example using microscopy, flow cytometry, and ELISA.
  • p24 levels are quantitated using the Coulter HTV-1 p24 Antigen Neutralization Kit according to the manufacturer's recommendation.
  • the ability of the cells to survive viral infection is determined, for example by performing a cell viability assay, such as trypan blue exclusion.
  • Animal model assays The ability of an agent, such as those identified using the methods provide above, to prevent or decrease infection by HFV-l can be assessed in animal models.
  • Several animal models for HTV-1 infection are known in the art. For example, mouse HTV models are disclosed in Sutton et al. (Res. Initiat Treat. Action, 8:22-4, 2003) and Pincus et al. (AIDS Res. Hum. Retroviruses 19:901-8, 2003) and U.S. Patent No. 6,248,721 (all herein incorporated by reference). Such animal models can also be used to screen agents for an ability to ameliorate symptoms associated with HIV-1 infection.
  • animal models can be used to determine the LD50 and the ED50 in animal subjects, and such data can be used to determine the in vivo efficacy of potential agents.
  • Animals of any species including, but not limited to, chimpanzees, can be used to generate an animal model of viral infection if needed.
  • the appropriate animal is inoculated with HIV-l, in the presence or absence of the agents identified in the examples above.
  • the amount of vims and agent administered can be determined by skilled practitioners.
  • several different doses of the potential therapeutic agent can be administered to different test subjects, to identify optimal dose ranges.
  • the therapeutic agent can be administered before, during, or after infection with HTV-1.
  • test agent is a therapeutic agent that can be used to decrease or even inhibit HIV-1 infection in a subject.
  • Example 13 Production and Use of Antibodies
  • This example describes methods that can be used to generate antibodies that are specific for mutant APOBEC3G, such as antibodies that can distinguish between wild-type human APOBEC3G and mutant human APOBEC3G proteins.
  • Monoclonal antibodies, polyclonal antibodies, or both can be produced to any mutant APOBEC3G protein herein disclosed, including variants, fragments, and fusions thereof.
  • antibodies raised against the protein will- specifically detect the protein.
  • antibodies raised against a mutant APOBEC3G protein (such as amino acids 1-384 of SEQ ID NO: 10, 22, 32, 34 or 40) recognize and bind the protein but will not substantially recognize or bind to other proteins found in human cells, such as wild-type APOBEC3G proteins (such as SEQ ED NO: 2).
  • the determination that an antibody specifically detects a mutant APOBEC3G protein is made using any standard immunoassay methods; for instance, Western blotting (Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • total cellular protein is extracted from human cells (for example, lymphocytes) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel.
  • the proteins are transfened to a membrane (for example, nitrocellulose) and the antibody preparation incubated with the membrane.
  • an appropriate secondary antibody such as an anti-mouse or anti-rabbit antibody conjugated to an enzyme such as alkaline phosphatase since application of 5-bromo-4-chloro-3-indolyl phosphate/nifro blue tetrazolium results in the production of a densely blue-colored compound by immuno-localized alkaline phosphatase.
  • Antibodies that specifically detect mutant APOBEC3G protein will, by this technique, be shown to bind to the protein band (such as the mutant APOBEC3G protein band, which localizes at a given position on the gel determined by its molecular weight and phosphorylation).
  • Non-specific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot.
  • the non-specific nature of this binding is recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody mutant APOBEC3G protein binding.
  • Substantially pure mutant APOBEC3G proteins suitable for use as an immunogen can be obtained from any suitable source, such as transfected cells, transformed cells, or wild-type cells.
  • mutant APOBEC3G protein is at least 50% pure, for example at least 75% pure. Concentration of protein in the final preparation can be adjusted, for example, by concentration on an Amicon filter device, to the level of a few ⁇ g/ml.
  • mutant APOBEC3G polypeptides ranging in size from full-length polypeptides to polypeptides having as few as nine amino acid residues can be utilized as immunogens.
  • such polypeptides are produced in cell culture, are chemically synthesized using standard methods, or obtained by cleaving large polypeptides into smaller polypeptides that can be purified.
  • Polypeptides having as few as nine amino acid residues in length can be immunogenic when presented to an immune system in the context of a Major Histocompatibility Complex (MHC) molecule such as an MHC class I or MHC class IF molecule.
  • MHC Major Histocompatibility Complex
  • mutant APOBEC3G polypeptides having at least 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, or 384, or more consecutive amino acid residues of a mutant APOBEC3G peptide (such as those that include a D128K, D128V, D128E, or D128A substitution) can be used as immunogens for producing antibodies.
  • Monoclonal antibody to epitopes of a mutant APOBEC3G protein can be identified, isolated and prepared from murine hybridomas using the method of Kohler an Milstein (Nature 256:495, 1975) or methods derivative thereof.
  • a mouse is repetitively inoculated with a few ⁇ g of the selected protein over a period of a few weeks.
  • the mouse is sacrificed and antibody-producing cells of the spleen isolated.
  • the spleen cells are fused with mouse myeloma cells using polyethylene glycol, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).
  • HAT media aminopterin
  • the fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
  • Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as described by
  • Engvall Enzymol. 70:419, 1980
  • Selected positive clones can be expanded and their monoclonal antibody product harvested for use.
  • Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies: A Laboratory Manual. 1988, Cold Spring Harbor Laboratory, New York).
  • protocols for producing humanized forms of monoclonal antibodies (for therapeutic applications) and fragments of monoclonal antibodies are known in the art.
  • Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single mutant APOBEC3G protein can be prepared by immunizing suitable animals with the expressed mutant APOBEC3G protein, which can be unmodified or modified to enhance immunogenicity.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple infradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis et al. (J. Clin. Endocrinol Metab. 33:988-91, 1971).
  • Booster injections can be given at regular intervals, and antisemm harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concenfrations of the antigen, begins to fall. See, for example, Ouchterlony et al. (hi: Handbook of Experimental Immunology, Wier, D. (ed.). Chapter 19. Blackwell. 1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of semm (about 12 ⁇ M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (Manual of Clinical Immunology, Chapter 42. 1980).
  • Another approach to raising antibodies against a mutant APOBEC3G protein is to use synthetic peptides synthesized on a commercially available peptide synthesizer based upon amino acid sequences of a mutant APOBEC3G protein, for example amino acids 1-384 of SEQ ED NOS: 10, 22, 32, 34 and 40.
  • Antibodies can be raised against a mutant APOBEC3G protein by subcutaneous injection of a DNA vector expressing a mutant APOBEC3G protein into an animal, such as mice. Delivery of the recombinant vector into the animal can be achieved using a hand-held form of the Biolistic system (Sanford et al, P articulate Sci. Technol 5:27-37, 1987) described by Tang et al.
  • Expression vectors include recombinant vectors expressing mutant APOBEC3G cDNA under transcriptional control of the human ⁇ -actin promoter or cytomegalovirus (CMV) promoter.
  • Antibody preparations prepared according to these protocols are useful in quantitative immunoassays to determine concentrations of antigen-bearing substances in samples; or semi-quantitatively or qualitatively to identify the presence of antigen in a sample.
  • Antibodies can be used to determine the susceptibility of an individual or a population to HFV-l infection. Briefly, a sample from a subject is contacted with an antibody that specifically binds to a mutant APOBEC3G protein.
  • the sample is also contacted with an antibody that specifically binds to a wild-type APOBEC3G protein. Binding of the antibody to the protein is then detected.
  • the antibody includes a detectable label.
  • the presence of the antibody is detected by using a labeled secondary antibody. Methods for detecting labeled antibodies are known in the art, and include flow cytometry and microscopy. The presence of antibodies that recognize a mutant APOBEC3G protein, indicates that the subject or population is more resistant to HFV-l infection. In contrast, the presence of antibodies that recognize a wild-type APOBEC3G protein, indicates that the subject or population is less resistant to HTV-1 infection.

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Abstract

L'invention concerne des séquences de la protéine mutante APOBEC3G permettant de réduire l'infection par le VIH-1 en présence du facteur de virulence du VIH. Dans certains exemples, les molécules de la protéine mutante APOBEC3G comprennent (ou codent pour) une substitution D128A, D128E, D128K ou D128V. L'invention concerne également des méthodes d'utilisation de ces molécules pour réduire une infection par le VIH-1, pour traiter un sujet atteint d'une infection par le VIH-1.
PCT/US2005/004371 2004-02-11 2005-02-11 Reduction de la replication du vih-1 a l'aide de proteines mutantes apogec3g WO2005077099A2 (fr)

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WO2008025564A3 (fr) * 2006-08-31 2008-04-24 Caesar Stiftung Moyens et procédés de détection d'interactions protéine-peptide
JP2011115151A (ja) * 2009-11-05 2011-06-16 National Institute Of Advanced Industrial Science & Technology Ａｐｏｂｅｃ３ｇの活性測定方法
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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2008025564A3 (fr) * 2006-08-31 2008-04-24 Caesar Stiftung Moyens et procédés de détection d'interactions protéine-peptide
AU2007291448B2 (en) * 2006-08-31 2014-02-20 Nexigen Gmbh Methods for detecting protein-peptide interactions
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JP2011115151A (ja) * 2009-11-05 2011-06-16 National Institute Of Advanced Industrial Science & Technology Ａｐｏｂｅｃ３ｇの活性測定方法
WO2019222537A1 (fr) * 2018-05-18 2019-11-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Variants catalytiquement hyperactifs de la protéine apobec3 humaine

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