WO2007130646A2 - Hcv coreceptor and methods of use thereof - Google Patents
Hcv coreceptor and methods of use thereof Download PDFInfo
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- WO2007130646A2 WO2007130646A2 PCT/US2007/010958 US2007010958W WO2007130646A2 WO 2007130646 A2 WO2007130646 A2 WO 2007130646A2 US 2007010958 W US2007010958 W US 2007010958W WO 2007130646 A2 WO2007130646 A2 WO 2007130646A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Definitions
- HCV hepatitis C virus
- HCV hepatitis C virus
- HCV tetraspanin CD 81 and scavenger receptor BI/II (SR-BI/II) are cell surface molecules that bind the HCV E2 glycoprotein and participate in HCV entry.
- SR-BI/II scavenger receptor BI/II
- HCV is a member of the family Flaviviridae, which also includes Pestiviruses and Flaviviruses.
- the HCV virion consists of an enveloped nucleocapsid containing the viral genome, a single, positive stranded RNA of approximately 9,600 nucleotides. Viral entry into the host cell is thought to require a tightly regulated interaction between the viral envelope proteins, El and E2, and host proteins at the cell surface. Moreover, it has been shown that host cell infection requires endosomal acidification suggesting that fusion of the viral envelope with cellular membranes is a pH triggered event. After cell entry the nucleocapsid is released into the cytosol and the viral RNA is translated through action of an internal ribosome entry site (IRES) present in the 5' untranslated region (5'UTR).
- IRS internal ribosome entry site
- the HCV genome encodes a single long open reading frame giving rise to a viral polyprotein of over 3000 amino acids that then undergoes co- and post-translational proteolytic processing to generate the mature viral proteins: C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (Fig.l).
- the viral structural proteins, including core, the capsid protein El, and E2 are encoded by the first third of the polyprotein.
- p7 (a putative ion channel) and nonstructural (NS) proteins encoded by the C-terminal two-thirds of the polyprotein, are components of the HCV RNA replication complex. The replication of the viral genome through a negative strand RNA intermediate occurs.
- HCVpp systems take advantage of the ability of retroviruses to incorporate heterologous glycoproteins into their envelope. HCVpp can infect a number of liver derived human cell lines and their reporter genes allow convenient quantification of target cell infection, making HCVpp the first robust assay for the study of HCV glycoprotein mediated cell entry.
- HCVcc More has been elucidated in the life cycle of HCV via the development of HCVcc (Fig. 2). This was made possible through the discovery of a genotype 2a HCV isolate from a Japanese patient with acute fulminant hepatitis (JFH)-I, that is capable of efficient subgenomic RNA replication in multiple cell types without the need for adaptive mutations 39,86,87. Surprisingly, when full length JFH-I genome RNA was transfected into Huh-7 cells, viral particles, termed HCVcc, were released that were capable of infecting na ⁇ ve cells. Efficient in vitro systems based on the JFH genome recapitulating the entire HCV life cycle have been developed.
- JFH acute fulminant hepatitis
- Virus produced from full length JFH-I RNA exhibits characteristics predicted for an HCV virion: the infectivity of these particles is blocked by antibodies against E2 or CD81 and by purified soluble CD81; moreover, virion density is similar to that found in sera of infected individuals.
- JFH-I initially yielded low titers, higher viral titers were obtained by using Huh-7.5 cells and derived sublines 99,168, which are highly permissive for HCV replication 22 due to a defect in the RIG-I intrinsic immune response pathway 146.
- Virion production was further enhanced through the use of a chimeric genotype 2a full length genome, expressing the core through NS2 region of the HC 36 HCV isolate cloned into the JFH-I genome (J6/JFH) (Fig. IB), which, unexpectedly, produced higher initial titers post transfection than the full length JFH- 1 genome 99.
- cell culture grown HCVcc has been used to infect both chimpanzees and uP A-SCID mice transplanted with human hepatocytes 100. In both cases rising viral loads and an infection sustained for several weeks ensued, validating the usefulness of the HCVcc system.
- virus recovered from HCVcc inoculated animals (ex vivo HCVcc; chHCVcc and muHCVcc for virus recovered from chimps and mice, respectively) was infectious in cell culture establishing the first robust ex vivo culture system.
- Virus recovered from infected animals displayed both altered biophysical properties and increased specific infectivity (ratio of infectious units to RNA copies) indicating that ex vivo HCVcc is of great use in elucidating the role of host factors in modulating HCV infection. lOOlO] Using HCVpp, much has been learned about the mechanism of HCV cell entry.
- HCVpp Even with HCVcc available, HCVpp still offer certain advantages, most notably (1) the ability to investigate HCV glycoprotein dependent entry in cells non-permissive to HCV replication and (2) the availability of stringent controls in the form of pseudoparticles bearing glycoproteins from viruses other than HCV 5 such as VSV or MLV, and pseudoparticles devoid of glycoproteins (no envelope). Nonetheless, future studies are needed to elucidate possible mechanistic differences between the cell entry properties of HCVpp, HCVcc and ex vivo HCVcc.
- HCVpp infectivity requires both El and E2 with their intact transmembrane domains 12, 83.
- the structure of the infectious unit in vivo may be more complex through the above mentioned association between the virus and host serum factors including different lipoprotein species (VLDL 5 LDL, HDL) and immunoglobulins 92, 113, 149, 150.
- Such associations may explain the heterogeneous buoyant density observed for both plasma-derived HCV (1.03-1.2g/ml) 23, 82, 150 and HCVcc (1.04- 1.18g/ml) 99, 156, 168.
- the highest infectivity seems to be associated with fractions of low to medium density (1.1 lg/ml and below) 23, 82, 99 indicating that an interaction with plasma lipids may enhance virion infectivity.
- HCVpp entry has been shown to be sensitive to endosomal acidification inhibitors such as ammonium chloride, bafilomycin and concanamycin, as is HCVcc entry, as shown herein.
- endosomal acidification inhibitors such as ammonium chloride, bafilomycin and concanamycin
- HCVcc entry As shown herein.
- This pH dependence indicates that virus interaction with putative cell surface receptors is followed by endocytotic uptake of the particle rather than fusion at the plasma membrane and that endosomal low pH is required, in some embodiments, to initiate virus-cell membrane fusion.
- An endosomal route of entry has also been described for the related flaviviruses 68, 69 as well as more evolutionarily distant alphaviruses 79.
- HCV minimal host cell factor requirement for HCV cell entry
- Numerous molecules have been proposed to function as HCV (co-)receptors (i.e., cell surface molecules required for entry that bind virus).
- HCV co-receptors
- none of these have had a precise function in the entry process conclusively defined, nor has the temporal sequence of interactions required for entry been determined.
- additional molecules that perform other functions in the entry process e.g., endosomal proteases
- CD81 a member of the tetraspanin superfamily with four transmembrane domains and short cytosolic N- and C-terminal tails, was initially identified as a candidate HCV receptor based on its ability to bind sE2124.
- the HCV-CD81 interaction is thought to take place between the CD81 large-extracellular loop (LEL) between transmembrane domains 3 and 4 (Drummer et al., 2005, Biochem Biophys Res Commun 328:251-7; Drummer et al., 2002, J Virol 76: 11143-7; Higginbottom et al., 2000, J Virol 74:3642-9) and a conformational epitope on E252.
- LEL large-extracellular loop
- CD81's role as an essential (co-)receptor for HCV (1) The human hepatoma cell line HepG2 does not express CD81 and cannot be infected with HCVpp or HCVcc, but becomes infectable with both upon transduction with CD 81 (Bartosch et al., 2003, J Biol Chem 278:41624-30; Lindenbach et al., 2005, Science 309:623-6; Zhang et al., 2004, J Virol 78:1448-55).
- CD81 expression alone is insufficient to allow HCVpp entry 12
- 83, 166 and the expression of CD81 in all human cell types except erythrocytes and platelets 97 does not explain HCVs apparent liver tropism.
- the precise role of CD81 in the entry process is unclear; some evidence suggests it may function as a co -receptor, interacting with the virus only after binding of the virus to another receptor molecule has occurred 34.
- scavenger receptor class B member I was first proposed as an HCV entry factor because of its ability to bind sE2136.
- SR-BI scavenger receptor class B member I
- SR-BI is expressed at high levels in the liver and steroidogenic tissues with lower levels detectable in placenta, small intestine, monocytes/macrophages and other tissues. It mediates selective uptake of cholesterol esters from HDL into the cellular membrane 1, 130 and possibly also endocytosis of entire HDL particles 140.
- the role of SR-BI in HCV cell entry is less clear than that of CD81. No SR-BI negative cell line that becomes permissive to HCV infection when transfected with SR-BI has been reported.
- Antibodies and siRNA directed against SR-BI inhibit HCVpp infection (Bartosch et al., 2003, J Biol Chem 278:41624-30; Lavillette et al., 2005, Hepatology 41:265-74), but both effects are less striking than those obtained for CD81 and vary considerably between HCV genotypes (Lavillette et al., 2005, Hepatology 41:265-74) (and unpublished data).
- HCVpp infectivity was found to be enhanced significantly in the presence of HDL (Bartosch et al., 2005, J Virol 79:8217-29; Meunier et al., 2005, Proc Natl Acad Sci USA 102:4560-5; Voisset et al., 2005, J Biol Chem 280:7793-9).
- the enhancement depends on functional SR-BI on the target cell since both SR-BI siRNA and BLT-4, a drug that inhibits flux of cholesteryl esters from SR-BI bound HDL into the target cell membrane 116, completely abrogate the enhancing effect of HDL.
- HCVpp The interaction between HCVpp and a cell expressing DC- or L-SIGN does not result in infection; however, bound HCVpp can be transmitted to permissive cells in co-culture (Cormier et al., 2004, Proc Natl Acad Sci USA 101 : 14067-7; Lozach et al., 2004, J Biol Chem 279:32035- 45), as is the case for HIV (Geijtenbeeket al., 2000, Cell 100:587-97).
- L-SIGN and DC-SIGN are expressed on liver sinusoidal endothelial cells and DCs, respectively, a model where they capture and transmit HCV particles to susceptible hepatocytes is feasible but unproven.
- LDL-R low density lipoprotein receptor
- HCV entry factors heparan sulfates (HS)IO and asialoglycoprotein receptor (ASGP-R)135 have been suggested as HCV entry factors, but their roles have not been rigorously validated in an infection assay.
- HCVpp became available it was quickly noted that only a select group of cell lines, all of which were derived from human liver, could be infected (Bartosch et al., 2003, J Exp Med 197:633-642; Hsu et al., 2003, Proc. Natl. Acad. Sci. USA 100:7271-76; Zhang et al., 2004, J Virol 78:1448-55). What precisely defines this narrow tropism is as yet unclear.
- Claudin 2 Claudin 2
- TJ de novo tight junction
- Claudins are small (20-27kD) molecules with a short cytoplasmic N- and C-terminal tails.
- Four membrane-spanning helices are separated by a larger ( ⁇ 53aa) first and a smaller ( ⁇ 24aa) second extracellular loop (ELl and EL2, respectively) and a very short intracellular loop (Fig. 3).
- Claudins are thought to be the major structural component of the TJ in epithelia where claudin family members and other TJ associated membrane proteins such as occludin, the junction-adhesion-molecule (JAM) and the coxsackie- adenovirus-receptor (CAR) associate laterally to form the TJ strand 64, 70.
- TJ strands in the membrane of neighboring cells then interact across the intercellular space to form the actual TJ.
- the extracellular loops of the claudins are thought to be central to these intercellular contacts that narrow and largely obliterate the intercellular space, thus forming the barrier between the apical and the basolateral side of the epithelium64.
- TJs do not completely seal off the paracellular pathway but allow selective flux of solutes based on size and charge.
- Paracellular pathway selectivity seems to be determined largely by the extracellular domains of the claudins present in a given tight junction 31, 61.
- the modular claudin composition of the tight junction may determine both the transepithelial electrical resistance and the paracellular pathway selectivity for certain solutes in epithelial tissues.
- claudins interact with PDZ- domain containing adaptor proteins, such as the TJ associated proteins zonula occludens (ZO)-I, -2 and -384.
- CLDNl is expressed in a number of epithelia, with the highest levels detected in the liver followed by kidney, skin and other tissues 60, 145. Claudin- 1 knockout mice die in the neonatal period due to water loss through the skin 62. However, loss of CLDNl is tolerated in humans; individuals homozygous for a two nucleotide deletion in the Claudin- 1 gene resulting in frame shift and a premature stop codon have been found in two inbred families ofixie descent 72. Affected patients exhibit scaling skin (ichthyosis) and liver disease due to neonatal sclerosing cholangitis.
- Claudin-14 mutations cause recessive non-syndromic deafness 159 and this phenotype was replicated in Claudin-14 knockout mice 15.
- Defects in Claudin-16 result in renal magnesium loss in humans 141.
- CPE Clostridium perfringens enterotoxin
- Claudin-1 (CLDNl) was identified as an HCV entry factor.
- CLDNl transduction of 293T cells expressing CD81 and SR-BI/II renders these normally non-permissive cells fully permissive for HCVpp entry and HCVcc infection.
- CLDNl a tight junction (TJ) constituent, is highly expressed in human liver and its expression correlates with HCV entry permissiveness in a variety of tissue culture cells.
- CLDNl contains two extracellular loops, four membrane-spanning segments, short cytoplasmic N- and C- termini, and a small intracellular loop.
- This invention first provides methods of inhibiting, mitigating or preventing infection of a subject with Hepatitis C Virus (HCV) that comprise contacting a cell in said subject with an agent which inhibits HCV interaction with a Claudin-1 protein.
- HCV Hepatitis C Virus
- the contacted cell can be a hepatocyte.
- the subject can be a mouse, a rat, a monkey, or a human.
- the agent binds to extracellular loop 1 of the Claudin-1 protein.
- the agent is selected from the group consisting of an antibody, an aptamer, or a recombinant protein.
- the antibody can be a monoclonal or a single chain antibody.
- the antibody can be a synthetic antibody.
- the variable region of the synthetic antibody that binds to extracellular loop 1 of the Claudin-1 protein can be obtained from a phage display, bacterial expression, or yeast expression library.
- the agent can also be a peptide or peptidomimetic compound that is structurally related to amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1.
- the peptide or peptidomimetic compound used in the method can comprise amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1, conservative amino acid substitutions thereof, or chemically derivatized variants thereof, wherein residue 32 is isoleucine and residue 48 is glutamic acid.
- residue 41 of said peptide or peptidomimetic compound is isoleucine.
- residue 31 of said peptide or peptidomimetic compound is isoleucine and/or residue 33 is serine.
- transgenic animal models for the study of Hepatitis C Virus (HCV) infection, replication and pathogenesis that comprise expression of a human Claudin-1 transgene in the animal.
- the animal is selected from the group consisting of a mouse, a monkey, and a rat.
- Methods of screening for an inhibitor of Hepatitis C Virus (HCV) infection that comprise screening a library for a compound which prevents or mitigates interaction of a region of a Hepatitis C Virus with a region of a Claudin-1 protein are also contemplated by this method.
- HCV Hepatitis C Virus
- a method of identifying a compound or agent that prevents or mitigates interaction of a region of a Hepatitis C Virus with a region of a Claudin-1 protein can comprise the steps of: a) providing either i) a recombinant protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, or ii) a cell comprising a recombinant vector that provides for expression of a membrane bound protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, wherein said amino acid residues of said Claudin-1 protein are located extracellularly to said cell; b) contacting the protein or the cell from step (a) with an agent or a compound and an HCV envelope protein, a cell expressing HCV envelope proteins El and E2, an HCV pseudotyped retroviral particle, an HCV cell culture particle, an ex vivo H
- the recombinant protein or the cell in step (a) can comprise amino acid residues 28 to 81 of a Claudin 1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof.
- the recombinant protein or cell in step (a) can comprise amino acid residues 1 to 102 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, a Claudin-1 protein, or a Claudin-1 protein that has an amino acid sequence as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof.
- the recombinant protein in step (a) can be provided in a soluble form, in a liposome, or attached to a solid support.
- the recombinant protein in step (a) further comprises a detectable label.
- This detectable label can be selected from the group consisting of a radiolabel, a spectrophotometrically detectable label, a fluorescent label, a time- resolved fluorescence label, a hapten and an epitope.
- the cell in step (a) can be a CD81 positive cell.
- the cell is an SR-BI positive cell.
- the cell in step (a) is a 293T or an SWl 3 cell.
- the agent in step (b) can be provided by an antibody library, an aptamer library, a peptide library, a recombinant protein library or a peptidomimetic library.
- the agent is an antibody
- the antibody library can be a phage display, bacterial expression, or yeast expression library.
- Compounds can be from a compound library.
- Various recombinant vectors can be used in these methods of identifying a compound or agent that prevents or mitigates interaction of a region of a Hepatitis C Virus with a region of a Claudin-1 protein.
- the recombinant vector can be a DNA vector or an RNA vector.
- the recombinant vector comprises at least one sequence encoding a transmembrane domain that is operably linked to a sequence encoding at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof.
- the recombinant vector comprises a first sequence encoding a signal peptide, wherein said sequence is operably linked to a sequence encoding at least amino acid residues 28 to 49 the amino acid residues of the Claudin-1 protein or conservative amino acid substitutions thereof.
- Recombinant vectors that comprise a sequence encoding a signal peptide can further comprise a second sequence encoding a domain that binds to a membrane bound protein, wherein this second sequence is operably linked to the first sequence encoding the signal peptide and to a sequence encoding at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof.
- Recombinant vectors that comprise a sequence encoding a signal peptide can further comprise a second sequence encoding a domain that provides for interaction to a membrane, wherein this second sequence is operably linked to the first sequence encoding the signal peptide and to a sequence encoding at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO: 1.
- step (c) inhibition of binding or interaction of the recombinant protein of step (a) is determined in step (c) by assaying for retention of the recombinant protein by a HCV envelope protein, by a cell expressing HCV envelope proteins El and E2, by a HCV pseudotyped retroviral particle, by a HCV cell culture particle, by an ex vivo HCV cell culture particle, or by an HCV provided in step (b), wherein decreased retention of the recombinant protein is indicative of binding inhibition.
- step (c) determination of interaction or fusion in step (c) is effected by assaying a reporter protein.
- This reporter protein can be selected from the group consisting of a fluorescent protein, an immunologically detectable protein or an enzyme.
- Fluorescent proteins can be selected from the group consisting of a red fluorescent protein, a green fluorescent protein, or a yellow fluorescent protein.
- Reporter enzymes can be selected from the group consisting of a chloramphenicol acetyl transferase, a beta galactosidase, a beta glucuronidase, and an alkaline phosphatase.
- Other embodiments where a selectable maker gene is used are also contemplated. This selectable marker gene can be suitable for either dominant or negative selections.
- Selectable marker genes include, but are not limited to, zeocin resistance, neomycin, G418, DHFR, TK, or hygromycin resistance genes.
- Compounds that prevent or mitigate interaction of a region of HCV with Claudin-1 are also provided.
- Compounds that prevent or mitigate interaction of a region of a Hepatitis C Virus with extracellular loop 1 of a Claudin-1 protein, wherein said compound is identified by the methods of the invention are provided by this invention.
- Agents that prevent or mitigate interaction of a region of a Hepatitis C Virus with extracellular loop 1 of a Claudin-1 protein are also provided by this invention.
- These agents can be identified by the methods of the invention.
- the identified agent can be an antibody, an aptamer, a peptide, a peptidomimetic compound, or a recombinant protein. When the agent is antibody, it can be a synthetic antibody.
- the antibody can be a monoclonal or a single chain antibody.
- Other agents of the invention are peptide or peptidomimetic compounds comprising amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO: 1, conservative amino acid substitutions thereof, or a chemically derivatized variant thereof, wherein residue 32 is isoleucine and residue 48 is glutamic acid.
- the peptide or peptidomimetic compound agents can comprise any one or all of a residue 41 isoleucine, a residue 31 isoleucine, and a residue 33 serine of a Claudin-1 peptide sequence as shown in SEQ ID NO: 1.
- kits for identifying a compound or agent that prevents or mitigates interaction of a region of a Hepatitis C Virus with a region of a Claudin-1 protein comprise either i) a recombinant protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof; or ii) a recombinant vector that provides for expression of a membrane bound protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, wherein said amino acid residues of said Claudin-1 protein are located extracellularly to a cell expressing said membrane bound protein, or a cell comprising said recombinant vector and instructions for using the kit to identify a compound or agent that prevents or mitigates interaction of a region of a Hepatitis C Virus with a region of a Claudin-1 protein.
- kits can further comprise an HCV envelope protein, a recombinant vector encoding HCV envelope proteins El and E2, a recombinant vector encoding an HCV pseudotyped retroviral particle, a recombinant vector encoding an HCV cell culture particle, or a recombinant vector encoding an infectious HCV particle.
- the kit can contain a recombinant protein, a membrane bound protein provided by a vector or a membrane bound protein of a cell that comprises amino acid residues 28 to 81 or amino acid residues 1 to 102 of a Claudin 1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof.
- the kit can contain a recombinant protein, a membrane bound protein provided by a vector or a membrane bound protein of a cell that comprises a Claudin 1 protein or a Claudin 1 protein as shown in SEQ ID NO: 1.
- the invention further provides cell culture compositions that are useful for identifying compounds or agents that prevents or mitigates interaction of a region of a Hepatitis C Virus with a region of a Claudin- 1 protein.
- Cell culture compositions of the invention comprise: i) a cell comprising a recombinant vector that provides for expression of a membrane bound protein comprising at least amino acid residues 28 to 49 of a Claudin- 1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, wherein said amino acid residues of said Claudin- 1 protein are located extracellularly to said cell, and either ii) a cell comprising a recombinant vector that encodes HCV envelope proteins El and E2 or iii) any one of an HCV pseudotyped retroviral particle, an HCV cell culture particle, an ex vivo HCV cell culture particle, or an HCV particle.
- the recombinant vector of (i) provides for expression of a membrane bound protein comprising at least amino acids 28 to 81 or at least amino acids 1 to 102 of a Claudin- 1 protein as shown in SEQ ID NO: 1 or conservative amino acid substitutions thereof.
- the recombinant vector of (i) can provide for expression of a membrane bound protein comprising a Claudin-1 protein or for expression of a Claudin- 1 protein as shown in SEQ NO.l or conservative amino acid substitutions thereof.
- the HCV pseudotyped retroviral vector can comprise an HCV El protein, an HCV E2 protein and a packaging competent retroviral genome containing a reporter gene.
- the packaging competent retroviral genome can be an HIV or an MLV packaging competent retroviral genome.
- the packaging competent retroviral genome is an envelope deficient retroviral genome.
- the recombinant vector of the cell culture composition can comprise at least one sequence encoding a transmembrane domain that is operably linked to a sequence encoding at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof.
- the recombinant vector comprises a first sequence encoding a signal peptide, wherein said sequence is operably linked to a sequence encoding at least amino acid residues 28 to 49 the amino acid residues of the Claudin-1 protein or conservative amino acid substitutions thereof.
- Recombinant vectors that comprise a sequence encoding a signal peptide can further comprise a second sequence encoding a domain that binds to a membrane bound protein, wherein this second sequence is operably linked to the first sequence encoding the signal peptide and to a sequence encoding at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof.
- Recombinant vectors that comprise a sequence encoding a signal peptide can further comprise a second sequence encoding a domain that provides for binding to a membrane, wherein this second sequence is operably linked to the first sequence encoding the signal peptide and to a sequence encoding at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof.
- the invention further provides for compositions that are useful for preventing entry of HCV into cells in a subject or in vitro.
- composition of the invention comprises a non-naturally occurring agent or compound and a Claudin-1 protein, wherein the non-naturally occurring agent or compound is bound to extracellular loop 1 of said Claudin 1 protein and inhibits HCV entry.
- the composition can be obtained by administering the non-naturally occurring agent or compound to a subject selected from the group consisting of a mouse, a rat, a monkey, and a human.
- the composition can be obtained by contacting a cell comprising a recombinant vector that provides for expression of a membrane bound protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, wherein the amino acid residues of said Claudin-1 protein are located extracellularly to a cell expressing said membrane bound protein, with the non-naturally occurring agent or compound.
- the composition is obtained by contacting a recombinant protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO: 1 with the non-naturally occurring agent or compound.
- the non-naturally occurring agent can be an antibody, an aptamer, or a recombinant protein.
- the compound is a non-naturally occurring compound.
- FIGURE. HCV genome and HCVcc organization.
- A Schematic representation of HCV genomic RNA. The amino-terminal one third of the polyprotein, translated through the action of an IRES, encodes the HCV structural proteins, including the capsid protein, core, and the envelope glycoproteins El and E2. The remainder of the polyprotein encodes the p7 protein and the viral nonstructural proteins NS2, -3, -4A, -4B, -5A, and -5B.
- B Schematic of the infectious J6/JFH-1 full length chimeric RNA. The C-NS2 region, represented in dark boxes, is derived from the HC J6 isolate, while the remainder of the genome is from JFH-I.
- FIG. 2 Systems to study HCV entry.
- HCV pseudotypes are generated in 293T cells by expression of retroviral Gag-Pol, HCV E1-E2, and a packagable RNA encoding a provirus with incorporated reporter, which can be used to monitor infection of transduced cells.
- infectious viral particles are released from Huh-7.5 cells that stably replicate a J6/JFH RNA. These are infectious in na ⁇ ve Huh7.5 cells and animal models. Serum from infected animals contains virus that is also infectious in culture.
- FIG. 3 Diagram of screen for HCV entry factors. A single round of screening of a cDNA library is shown in the dashed box, where a cDNA library constructed in the Vl HIV proviral vector and packaged into VSVG pseudoparticles is transduced into 293T cells. Transduced cells are then challenged with drug selectable HCVpp. For CPR surviving populations were transfected with the HIV gag-pol and VSVG expression plasmids to repackage the Vl-cDNAs into pseudovirus used to infect na ⁇ ve 293T cells, allowing recycling and reselection of cDNA clones exhibiting the desired specific activity.
- FIGURE 4 Increasing susceptibility of 293T cells to HCVpp over the course of the cDNA library screen. Each row corresponds to a population of 293T cells during the screen, beginning with an untransduced population at the top and ending with a population that has been subjected to a total of 2 repackaging steps (CPR) and 5 selection steps (Puro, Zeo). At each stage the population was infected with GFP-reporter (CSGW) pseudoparticles bearing no glycoproteins ("no envelope"), HCVE1E2 (HCV) and VSV G protein (VSV-G). A gradual increase in susceptibility to HCVpp was observed (middle column) while susceptibilty to "no envelope" and VSV-G remained unchanged.
- CSGW GFP-reporter
- FIGURE 6 HCVcc infection in 293T/CLDN1 cells. Mock or pTRIP- CLDNl infected 293T cells were challenged with J6/JFH HCVcc 48 hour prior to irnrnunostaining for NS5A (in brown). Nuclei were counterstained with hematoxylin (blue). J0044] FIGURE 7. HC Vpp infection of 293 T/CLDN1 cells. A "no envelope" ⁇ p (top) and HCVpp (bottom) infection of diverse human cell lines; "endog. CLDNl” and “pTRIP CLDNl” indicate endogenous expression and lentiviral mediated overexpression, respectively. B Infection of 293T cells expressing human claudins 1, 3 and 7 or mouse CLDNl.
- FIGURE 8 Characterization of FL-J6/JFH-5 'Cl 9Rluc2AUbi.
- A Schematic of FL-J6/JFH-5'C19Rluc2AUbi.
- B Huh-7.5 cells were infected with FL- J6/JFH-5'C19Rluc2AUbi for the indicated times. At each timepoint, cells were harvested and luciferase activity determined. The dashed line indicates the background level of the assay from naive Huh-7.5 cells.
- Huh-7.5 cells were treated with bafilomycin Al either prior to infection (squares) or at 3 h postinfection (p.i.) (triangles) with FL-J6/JFH- 5'C19Rluc2AUbi. Samples were harvested for luciferase assays at 24 h p.i. The dashed line indicates the background level of the assay from na ⁇ ve Huh-7.5 cells.
- Huh-7.5 cells were treated with concanamycin A and infected as above. [0047] FIGURE 10. HCV infectivity is resistant to acidic pH.
- HCVcc or Sindbis was diluted in citric acid buffer (15 mM citric acid, 150 mM NaCl) at pH 7 (white bars) or pH 5 (black bars) for 10 min at 37oC. Samples were neutralized and used to infect Huh-7.5 cells. Samples were harvested at 24 h (SIN) or 48 h (HCV). [0048] FIGURE 11. Incubation at 37 0 C allows HCV to enter bafilomycin-treated cells. Huh-7.5 cells were treated with bafilomycin Al (+Baf) and infected with HCVcc at 4 0 C for 2 h.
- FIGURE 12 10049J FIGURE 12.
- FIGURE 13 Mapping of determinants for claudin specific HCV entry determinants.
- A ClustalW alignment of CLDNl (top) and CLDN7 (bottom) protein sequences, with matches shown as dots and differences as labeled. The predicted transmembrane domains are boxed and the extracellular loops (ELl and EL2) are indicated with dashed lines.
- B Diagram of proposed CLDNl (dark) and CLDN7 (light) chimeras. Full length, wild type clones (i) are represented by a single color. Chimeras with a single junction (ii) or precise swaps of one (iii) or both (iv) extracellular loop are diagramed with regions from each protein corresponding to the respective color.
- FIGURE 14 Extracellular loop-1 (ELl) of CLDNl contains specific HCV entry determinants. Reciprocal CLDN1/CLDN7 chimeras were constructed to map the sequence differences responsible for their different infectivity phenotypes. (A) The extracellular loops of either the HCV permissive CLDNl (dark lines in claudin diagram below graph) or nonpermissive CLDN7 (light lines in claudin diagram below graph) were swapped into the reciprocal gene. These chimeras were constructed as GFP-claudin fusions in the TRIP lentiviral vector.
- FIGURE 15 Deletions within the first two-thirds of CLDNl ELl disrupt HCV entry.
- CLDNl encoding deletions of ELl were tested for abilities to mediate HCVpp entry.
- Total cellular protein lysates were prepared from transduced 293T cells, resolved by SDS-PAGE gel electrophoresis, transferred to nitrocellulose membrane, and immunoblotted with an anti-GFP (top) or ⁇ -actin (bottom) mouse monoclonal antibody.
- C Illustration of FLAG insertions within CLDNl ELl tested above.
- FIGURE 17 CLDNl with epitopes insertions within ELl can be blocked by epitope specific antibodies.
- epitope inserted CLDNl constructs that remain functional for HCV entry were tested for HCVpp infectivity in the presence of anti-FLAG antibodies.
- 293T cells expressing GFP- CLDNl proteins with FLAG insertions within ELl that maintain the ability to promote HCV entry were preincubated (1 h, 37C) with media containing the indicated concentration of anti-FLAG M2 monoclonal antibody (Sigma), followed by infection with luciferase encoding pseudoparticles.
- FIGURE 18 Co-immunoprecipation of E2 with FLAG-CLDNl.
- Na ⁇ ve or stably transduced FLAG-CLDNl 293T cells were either mock transfected or transfected with an E1-E2 expression plasmid.
- Lysates prepared 48 h post transfection were immunoprecipitated with FLAG M2 monoclonal antibody conjugated agarose (Sigma).
- the insoluble (pellet) and soluble fractions of the lysate (representing 1% of IP input), and the IP, eluted from antibody conjugated agarose by boiling in SDS, were resolved by SDS-PAGE, transferred to membranes and immunoblotted for E2 (above panel) or CLDNl (lower panel).
- Approximate molecular masses are labeled to the left and the positions of relevant species are labelled to the right. (Note: some heavy chain from the anti-FLAG antibody has eluted with the IP, visible in all IP lanes.)
- FIGURE 19 Both mouse and human CLDNl mediate HCVcc entry with comparable efficiency.
- Human 293T cells were transduced to express human or mouse CLDNl and then infected with HCVcc at different dilutions. After 72h immunohistochemical staining for NS 5 A was performed and NS 5 A positive foci were enumerated. While none were found in mock transduced 293T cells (open circles). Comparable numbers of foci were seen in cells expressing human (filled circles) and mouse (filled triangles) CLDNl . These data argue against CLDNl being the factor that determines the host range restriction of HCV to higher primates. It is likely that one or more factor(s) that restricts HCV entry into non-human cells still remain(s) to be identified.
- FIGURE 20 Silencing of CLDNl expression in Hep3B cells specifically inhibits HCVpp.
- Human Hep3B hepatoma cells were transfected twice with siRNA against irrelevant, CD81 or one of three different siRNA's targeting CLDNl. After the second transfection HCVpp infection was performed and expression levels for CD81 and CLDNl were determined by whole-cell FACS staining (solid line - si-irr treated cells; dashed line - cells treated with specific siRNA; grey shaded area - isotype control stain). With all CLDNl targeting siRNAs a clear reduction both in CLDNl protein levels and in HCVpp susceptibility but not VSV-Gpp susceptibility was observed.
- FIGURE 21 CLDNl expression is associated with susceptibility to HCVpp in human cell lines.
- the indicated human cell lines were either mock transduced or transduced to express CLDNl ('pTRIP-CLDNl'), CD81 ('pTRIP-CD81'), or both and then challenged with HCVpp (black bars) and VSV-Gpp (white bars) encoding a GFP reporter. Background readings, that is, percentage of GFP-positive cells seen with Env2pp, were subtracted for each population (mean of n53; error bars, s.d.).
- CLDNl expression was assessed by immunoblotting. Approximate molecular weight (kDa) marker positions are indicated to the left of each blot.
- Cell surface CD81 expression ('endog. CD81 ') was determined before transduction, by flow cytometry using anti-CD81 1.3.3.22 as a primary antibody.
- FIGURE 22 HCVpp susceptibility depends on residues in the first extracellular loop of CLDNl.
- A GFP-reporter HCVpp (black) and VSV-Gpp (white) infection of 293T cells expressing CLDNl, 3 or 7. Background readings, that is, percentage of GFP-positive cells seen with Env2pp, were subtracted for each population (mean of n53, error bars, s.d.).
- B Immunoblot for CLDNl, 3 and 7. Approximate molecular weight (kDa) marker positions are indicated to the left of each blot.
- C CLDNl topology.
- the x axis labels refer to the GFP-CLDNl or GFP-CLDN7 fusion backbone encoding ('w/') the indicated region of the other claudin protein.
- N and C indicate either the amino or carboxy terminal, respectively, piece of CLDNl ELl, swapped into CLDN7.
- Readings are normalized to 293T cells expressing wild-type CLDNl (mean of n54; error bars, s.d.).
- F Immunoblot for GFP.
- G Alignment of the N-terminal half of ELl in CLDNl and CLDN7. Identical sequences are represented by a full- stop, numbering represents amino acid position in full-length CLDNl .
- H 293T cells expressing the indicated point mutants were tested for susceptibility as described above (mean of n54; error bars, s.d.).
- FIGURE 23 Mutational analysis of CLDNl protein interaction and membrane localization motif requirements for HCV entry.
- A Diagram f CLDNl specific features that were disrupted by mutagenesis. The location where a stop codon was inserted to create a CLDNl mutant lacking the carboxylterminal intracellular tail is indicated (Del C-term). The deleted region contains putative phosphorylation sites, the modification of which may influence TJ assembly, and a PDZ-binding domain that mediates interactions with intracellular TJ components.
- Claudin 14 has been shown to be palmitoylated on cysteine residues proximal to the second and fourth transmembrane domains28. Such modifications may regulate protein interactions or direct the molecule to specific regions of the plasma membrane.
- FIGURE 24 Mutational analysis of CLDNl domains required for HCV entry functions.
- A Illustration of CLDNl ELl deletion and insertion mutants. Lines above the CLDNl ELl sequence represent regions that were deleted. Arrowheads below the sequence indicate the position of individual FLAG epitope insertions (B, C).
- 293T cells transduced with lentiviruses to express the indicated GFP-fusion deletion (B) and insertion (C) mutants were challenged with either Luc- HCVpp (black bars) or VSV-Gpp (white bars).
- FIGURE 25 Analysis of CLDNl function in the HCV entry process.
- FIGURE 26 Identification of CLDNl ELl epitope insertion mutants that allow antibody blocking of HCV entry.
- 293T cells transduced to express the indicated CLDNl FLAG epitope insertion mutants were preincubated with increasing concentrations of anti-FLAG M2 monoclonal antibody for 30 min at 37°C before being challenged with luc-HCVpp in the continued presence of antibody.
- Media was changed 18 h post infection and luciferase activities were determined after 72 h. Values are reported as means +/- s.d. of data (n 4) from two independent experiments and normalized to infection in the absence of antibody.
- FIGURE 27 Low pH treatment affect syncytia size, but not quantity of GFP positive loci in the cell-cell fusion assay.
- acceptor CLDNl expressing 293T cells encoding GFP under control of the HIV-I promoter
- 293T donor cells expressing Tat and either the VSV- G or HCV E1E2 viral glycoproteins.
- Cocultures were briefly treated with either pH 7 or 5 buffers 24 h post seeding. Shown are representative examples of typical GFP+ loci observed 48 h post pH treatment.
- FIGURE 28 Expression of human SR-BI, but not human CLDNl or human CD81, enhances HCVcc binding to CHO cells.
- FIGURE 29 Expression of human SR-BI, but not human CLDNl or human CD81, enhances HCVcc binding to CHO cells.
- 1 protein is any protein that comprises the amino acid residues of Claudin-1 extracellular loop 1 that provide for HCV entry and additional sequences that provide for extracellular presentation of the amino acid residues of Claudin-1 extracellular loop 1.
- HCV refers to any major HCV genotype, subtype, isolate, and/or quasispecie. HCV genotypes include, but are not limited to, genotypes
- 1, 2, 3, 4, 5 and 6 and HCV subtypes include, but are not limited to, subtypes Ia, Ib,
- an "endogenous Claudin-1 protein” comprises the protein of
- the phrase "conservative amino acid substitutions” refers to one or more changes in amino acids in a sequence is (are) that are replaced with another amino acid(s), the charge and polarity of which is similar to that of the native amino acid.
- Conservative substitutes for an amino acid within a protein, a peptide,or peptidomimetic compound are made with members of the group to which the originally occurring amino acid belongs.
- Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids.
- amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conservative amino acid changes can be made by substituting one amino acid within one of these groups with another amino acid within the same group.
- an "antibody” is any of a polyclonal antibody, a monoclonal antibody, a single chain antibody, or a synthetic antibody
- a "monoclonal antibody” is any antibody derived from any source that recognizes a single epitope.
- single chain antibody is any light chain antibody, any heavy chain antibody, or any fragment thereof comprising an antigen recognition site.
- Single chain antibodies can be derived from any source.
- a 'synthetic antibody is any antibody that is produced by recombinant DNA technology. Synthetic antibodies thus include, but are not limited to, humanized antibodies, mutagenized antibodies, and antibodies derived from human, bacterial, yeast or bacteriophage expression libraries.
- peptidomimetic compound refers a peptide analog containing one or more non-naturally occurring amino acids (e.g., non-natural side chains, non-natural chiralities, N-substituted amino acids, or beta amino acids), non-natural topologies (e.g., cyclic or branched) and/or peptide analogues with partially or totally substituted amide (peptide) bonds with ester, thioester or other linkages.
- non-naturally occurring amino acids e.g., non-natural side chains, non-natural chiralities, N-substituted amino acids, or beta amino acids
- non-natural topologies e.g., cyclic or branched
- peptide analogues with partially or totally substituted amide (peptide) bonds with ester, thioester or other linkages.
- the term "corresponding”, when used in the context of comparing, aligning, or identifying equivalent amino acids in one polypeptide sequence with another polypeptide, peptide,or peptidomimetic compound sequence, refers to the comparison or alignment that will yield the highest percent identity when aligned with the other polypeptide sequence. For example, in an alignment of SEQ
- membrane bound protein refers to any protein that is bound to a cell membrane under physiological pH and salt concentrations.
- Binding of the membrane bound protein can be either by direct binding to the phospholipid bilayer of by binding to a protein, glycoprotein, or other intermediary that is bound to the membrane.
- extracellular refers to the external, non- cytoplasmic region of a cell.
- interaction of a region of a Hepatitis C Virus with a region of a Claudin-1 protein encompasses any step in the process by which an HCV virion or component derived therefrom is recognized, bound and/or internalized by a cell.
- interactions include any direct or indirect function function of Claudin-1 required for HCV entry.
- a variety of methods for inhibiting HCV infection by inhibiting endogenous Claudin-1 function are contemplated by this invention. Such methods can comprise either blockage of regions of endogenous Claudin-1 that provide for cellular interactions required for HCV entry or blockage of the regions of HCV that provide for interactions with endogenous Claudin-1 or a Claudin-1 containing protein complex.
- Such methods can comprise either blockage of regions of endogenous Claudin-1 that provide for cellular interactions required for HCV entry or blockage of the regions of HCV that provide for interactions with endogenous Claudin-1 or a Claudin-1 containing protein complex.
- Provided herein is the identification of the Claudin-1 extracellular loop 1 region and specific amino acid residues within extracellular loop 1 which are key elements required for HCV entry.
- the identification of extracellular loop 1 as a key cellular factor involved in HCV entry into a cell provides for both blockage of endogenous Claudin-1 regions and blockage of HCV regions to inhibit HCV infection.
- endogenous Claudin-1 extracellular loop 1 (ELl) and extracellular loop 2 (EL2) are likely in close proximity to and perhaps interacting with each other in the endogenous Claudin-1 protein, it is also contemplated that the binding of inhibitors to extracellular loop 2 may also interfere with HCV infection, either through direct or indirect effects.
- agents or compounds that interact with regions of Claudin-1 other than either extracellular loop 1 or extracellular loop 2 can also disrupt Claudin- 1 function and provide for prevention, inhibition, or mitigation of HCV infection.
- Blockage of Claudin-1 extracellular loop 1 interactions with HCV can be effected by binding of an agent to any region within an endogenous Claudin-1 extracellular loop 1 or by binding of an agent that binds elsewhere but alters the structure of Claudin-1 extracellular loop 1 such that it no longer functions in HCV entry.
- endogenous Claudin-1 is understood herein to comprise the protein of SEQ ID NO:1 (NCBI Accession Number NP_066924) and any naturally occurring variants commonly found in HCV permissive subject populations.
- Extracellular loop- 1 of endogenous Claudin 1 comprises amino acid residues 28 to 81 of a Claudin-1 protein as shown in SEQ ID NO:1 and any naturally occurring variants commonly found in HCV permissive subject populations when found in a subject.
- binding of an antibody to a sequence that is immediately adjacent to Claudin-1 extracellular loop 1 is shown herein to be sufficient to block the ability of a Claudin-1 protein to function in entry of HCVpp.
- binding of the agent to Claudin-1 extracellular loop 1 is believed to sterically inhibit productive interactions of Claudin- 1 that permit cellular entry of HCV and HCV infection.
- any region of the Claudin-1 extracellular loop 1 can be bound by the agent.
- smaller agents may need to bind specific regions of the Claudin-1 extracellular loop 1 region to inhibit HCV infection.
- binding to residues 28 to 49 of an endogenous Claudin-1 protein as shown in SEQ ID NO.l can inhibit HCV infection.
- interaction of the agent with amino acid residues 32 is (isoleucine) and 48 (glutamic acid) of an endogenous Claudin-1 protein as shown in SEQ ID NO:1 can block HCV infection.
- an aptamers that bind to Claudin-1 extracellular loop 1 can be used to inhibit HCV infection.
- an aptamers can comprise any DNA, RNA, oligonucleotide, or chemically modified oligonucleotide that binds to a target. Isolation and identification of aptamers has been disclosed (U.S. Patent No. 5,582,981, U.S. Patent No. 6,867,289, U.S. Patent No. 7,179,894).
- recombinant binding proteins that bind an endogenous Claudin-1 extracellular loop 1 can be used to inhibit HCV infection.
- "recombinant binding proteins” are any non-naturally occurring proteins obtained by recombinant DNA or polymerase chain reaction-mediated reactions that bind to a target.
- Recombinant binding proteins can comprise polypeptide binding regions of immunoglobulin heavy chains variable domains, immunoglobulin light chain variable domains, V.alpha./V.beta. domains of T cell receptor proteins, or combinations thereof. Isolation and identification of recombinant binding proteins has been disclosed (U.S. Patent No. 6,010,884 and U.S. Patent No. 6,297,053).
- Blockage of HCV regions to inhibit HCV infection can also be effected by agents that mimic the region of Claudin-1 extracellular loop 1 that interacts with HCV.
- agents that mimic the region of Claudin-1 extracellular loop 1 that interacts with HCV.
- contacting HCV with agents that mimic the critical region of Claudin-1 extracellular loop 1 is expected to inhibit productive interactions of HCV with endogenous Claudin-1 that permit cellular entry of HCV and HCV infection.
- These agents are expected to competitively inhibit interactions with HCV and endogenous Claudin-1 EL-I .
- Claudin-1 extracellular loop 1 mimicking agents are that they can be optimized such that their interactions with the critical regions of HCV are potentiated while their interactions with any other endogenous cellular ligands that recognize endogenous Claudin-1 extracellular loop-1 (ELl) are minimized. Optimization of Claudin-1 mimicking agents is expected to provide for inhibition, prevention or mitigation of HCV infection while minimizing undesirable side effects.
- Claudin-1 mimicking agents can comprise a peptide or peptidomimetic compound that comprise or are derived from amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1.
- a key feature of such a peptide or peptidomimetic compound is the conservation of amino acids corresponding to residue 32 as isoleucine and to residue 48 as glutamic acid. It is anticipated that other residues in regions corresponding to amino acid residues 28 to 49 of a Claudin-1 protein can be substituted by conservative amino acids in the mimicking agents. Such conservative amino acid substitutions in mimicking agents can be tested for result effective inhibition of HCV interactions with Claudin 1 with any of the in vitro or cell-based assay methods provided herein.
- Mimicking agents include, but are not limited to, peptides or peptidomimetics where the valine at corresponding residue 41 can be conservatively substituted with other hydrophobic amino acids including, but not limited to, isoleucine. It is also contemplated that the arginine at residue 31 can be substituted by a glutamine and that the tyrosine at residue 33 can be substituted by a serine.
- peptidomimetic compounds or chemical derivatives that comprise amino acid residues 28 to 49 of a Claudin-1 protein to antagonize or otherwise inhibit interactions of HCV with endogenous Claudin-1 in a subject is also contemplated. It is anticipated that such peptidomimetics or chemical derivatives can confer pharmaceutically useful properties such as increased stability and/or specificity on the peptide, peptidomimetic compound or chemical derivative.
- peptidomimetic compounds derived from Claudin-1 amino acid residues 28 to 49 containing one or more non-naturally occurring amino acids e.g., non-natural side chains, non-natural chiralities, N-substituted amino acids, or beta amino acids
- non-natural topologies e.g., cyclic or branched
- peptide analogues with partially or totally substituted amide (peptide) bonds with ester, thioester or other linkages is contemplated.
- peptidomimetic compounds comprising any of residues 29-49, 30-49, 31-49, and 32-49 or residues 29-48, 30-48, 31-48, or 32- 48 of amino acid residues 28 to 49 of a Claudin-1 protein can be used to antagonize or otherwise inhibit interactions of HCV with endogenous Claudin-1.
- Chemical modifications to amino acid residues 28 to 49 of a Claudin-1 protein can comprise any covalent modification that does not inhibit the capacity of that peptide or derived peptidomimetic to interact with HCV.
- Covalent modifications contemplated include, but are not limited to, acetylation, amidation, sulfation, succinylation, methylation, chelator linkage or terminal blockage.
- the peptidomimetic library would be focused on the relevant amino acid residues of a Claudin-1 protein as described immediatedly above (i.e., peptidomimetics comprising any of residues 29- 49, 30-49, 31-49, and 32-49 or residues 29-48, 30-48, 31-48, or 32-48 of amino acid residues 28 to 49 of a Claudin-1 protein).
- peptidomimetics comprising any of residues 29- 49, 30-49, 31-49, and 32-49 or residues 29-48, 30-48, 31-48, or 32-48 of amino acid residues 28 to 49 of a Claudin-1 protein.
- Other methods that can be used to obtain peptidomimetics are rational design methods such as those described in U.S. Patent No. 5,947,847 or U.S. Patent No. 5,331,573.
- compositions comprising these active agents can be administered by a variety of techniques.
- Such pharmaceutical compositions may be formulated in various ways known in the art for administration purposes.
- an effective amount of the particular compound, in base or acid salt form, as the active ingredient is combined with one or more pharmaceutically acceptable carriers and delivery vehicles.
- pharmaceutically acceptable carriers and delivery vehicles include aluminum stearate, lecithin, serum proteins, such as human serum albumin; buffer substances such as the various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids; water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts; colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene, polyoxypropylene-block polymers, polyethylene glycol and wool fat, and the like.
- buffer substances such as the various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids
- water, salts or electrolytes such as protamine sulfate, disodium hydrogen
- compositions described herein may further be prepared in unitary dosage form suitable for administration orally, percutaneously, by parenteral injection (including subcutaneous, intramuscular, intravenous and intradermal), topically, intranasally, by inhalation, or for application to a medical device, such as an implant, catheter, or other device.
- parenteral injection including subcutaneous, intramuscular, intravenous and intradermal
- a medical device such as an implant, catheter, or other device.
- any of the pharmaceutically acceptable carriers known in the art may be used, such as water, glycols, oils, alcohols and the like in the case of carriers that permit oral delivery of liquid preparations such as suspensions, syrups, elixirs and solutions.
- solid pharmaceutically acceptable carriers When solid pharmaceutically acceptable carriers are desired that permit oral or rectal administration, starches, sugars, kaolin, lubricants, binders, cellulose and its derivatives, and disintegrating agents and the like may be used to prepare, for example, powders, pills, capsules and tablets.
- the pharmaceutically acceptable carriers For pharmaceutically acceptable carriers that permit parenteral administration, the pharmaceutically acceptable carriers often comprise sterile water, which may be supplemented with various solutes to, for example, increase solubility.
- injectable solutions may be prepared in which the pharmaceutically acceptable carrier comprises saline solution, glucose solution, or a mixture thereof, which may include certain well-known anti-oxidants, buffers, bacteriostats, and other solutes that render the formulation isotonic with the blood of the intended patient.
- HCV genotypes include, but are not limited to, genotypes 1, 2, 3, 4, 5 and 6 and HCV subtypes include, but are not limited to, subtypes Ia, Ib, 2a, 2b, 2c, 3a, 4a-4f, 5a and 6a.
- In vitro assays comprise any assay wherein binding, interaction or association of a recombinant Claudin-1 protein with HCV or components of HCV is determined. It is understood that the binding of recombinant Claudin-1 with HCV or components of HCV can be either direct or indirect. Indirect binding would entail binding of Claudin-1 to HCV through an intermediary.
- a recombinant Claudin 1 protein refers to any protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO: 1 or conservative amino acid substitutions thereof.
- the recombinant Claudin-1 protein used can comprise amino acids 28 to 81 or amino acids 1 to 102 of a Claudin-1 protein as shown in SEQ ID NO:1, amino acids 28 to 102 of a Claudin-1 protein as shown in SEQ ID NO:1, amino acids 1-102 or amino acids 28 to 102 and any number of additional Claudin-1 amino acid residues between 102 and 211, a Claudin-1 protein of SEQ ID NO:1, derivatives of any of the foregoing proteins wherein conservative amino acid substitutions have been made, or derivatives of any of the foregoing proteins wherein insertions or deletions that do not affect the capacity of the protein to interact with HCV have been made.
- Recombinant Claudin-1 protein can be used in the in vitro binding or interaction assays in either a soluble form or in insoluble forms such as liposomes. Still other recombinant Claudin-1 proteins can comprise or be derived from the murine Claudin-1 (SEQ ID NO:2), Claudin- 1 /Claudin-7 chimeras, or Claudin-7 proteins wherein residues corresponding to residues 32 and 48 of the Claudin-7 protein are substituted with the corresponding amino acid residues found in Claudin-1 (i.e., wherein residue 32 is isoleucine and residue 48 is glutamic acid). Claudin-7 is provided herein as SEQ ID NO:3.
- insoluble forms that can be used in binding assays include forms where the recombinant Claudin-1 protein is coupled to a solid support.
- Solid supports include beads, microtiter plates, column matrices, or any other materials suitable for immobilizing proteins for binding assays.
- Soluble forms of the recombinant Claudin- *1 can further comprise sequences that facilitate solubility, detection and/or retension. Sequences that can facilitate solubility include, but are not limited to, sequences from glutathione-S-transferases or E.coli maltose binding proteins. Sequences that facilitate detection include any reporter protein, any epitope or any protein-binding domain.
- any of the foregoing sequences that promote solubility or detection can also facilitate retension.
- Retension is typically used in binding assays to associate the protein or protein ligand to a solid support.
- the recombinant protein can also further comprise additional sequences for retension such as FLAGTM epitopes (Stratagene, La Jolla, CA, USA), myc epitopes, histidine tags and the like,
- Binding or interaction of the recombinant Claudin -1 protein to any HCV or HCV derived material such as HCV, HCVcc, HCVpp, semi-purified HCV components, purified HCV proteins or recombinant HCV protein(s) can be determined in the methods contemplated herein. Binding or interaction of recombinant Claudin-1 to HCV El and E2 proteins is specifically contemplated. Such binding may be either direct or indirect. Either the recombinant Claudin-1 or the HCV or HCV derived materials can be detectably labeled to facilitate the binding assay.
- the recombinant Claudin-1 and the HCV or HCV derived materials are labeled with distinct detectable labels permitting simultaneous detection of each.
- the recombinant Claudin-1 protein is typically contacted by the HCV or HCV derived material, subjected to some form of buffer exchange, and binding determined. Binding may be determined by any suitable technique or combination of techniques, including but not limited to, detection of a bound label, surface plasmon resonance, or scintillation proximity assays.
- a recombinant Claudin-1 protein is a membrane bound protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO: 1 or conservative amino acid substitutions thereof such that the Claudin-1 amino acid residues 28 to 49 are extracellularly located.
- the recombinant, membrane bound protein that provides for extracellular presentation of at least amino acid residues 28 to 49 of a Claudin-1 protein used can comprise amino acids 28 to 81 or amino acids 1 to 102 of a Claudin-1 protein as shown in SEQ ID NO:1, amino acids 28 to 102 of a Claudin- 1 protein as shown in SEQ ID NO:1, amino acids 1-102 or amino acids 28 to 102 and any number of additional Claudin-1 amino acid residues between 102 and 211, a Claudin-1 protein of SEQ ID NOrI 5 derivatives of any of the foregoing proteins wherein conservative amino acid substitutions have been made, or derivatives of any of the foregoing proteins wherein insertions or deletions that do not affect the capacity of the protein to interact with HCV have been made.
- membrane bound Claudin-1 proteins can comprise or be derived from the murine Claudin-1 (SEQ ID NO:2), CIaudin-l/Claudin-7 chimeras, or Claudin-7 proteins wherein residues corresponding to residues 32 and 48 of the Claudin-7 protein are substituted with the corresponding amino acid residues found in Claudin-1 (i.e., wherein residue 32 is isoleucine and residue 48 is glutamic acid).
- Claudin-7 is provided herein as SEQ ID NO:3.
- Membrane bound forms of any of the foregoing proteins can be obtained by any method.
- the Claudin-1 residues and transmembrane domains located between amino acids 1-27 and residues 82-102 can provide for membrane bound, for extracellular presentation of any of the foregoing recombinant Claudin-1 protein.
- a signal peptide encoding sequence can be operably linked to at least amino acid residues 28 to 49 of a Claudin-1 protein to provide for extracellular localization of that sequence. Operable linkage of the signal peptide sequence would typically be to the N-terminus of the Claudin-1 amino acid sequences.
- membrane binding functionalities can be provided by a variety of sequences.
- the membrane binding functionality can be provided by any combination of transmembrane domains (located C-terminal to the Claudin-1 amino acid sequences), domains that bind to extracellularly located protein or glycosylated domains of other transmembrane proteins, or domains that provide for post- translational modifications that result in membrane binding.
- the Claudin-1 amino acid sequences can be imbedded in an extracellular domain of another membrane bound protein to provide for presentation of the Claudin-1 sequences.
- the cell expressing the recombinant, membrane bound extracellular Claudin-1 amino acid residues is contacted by any of an HCV envelope protein, a cell expressing HCV envelope proteins El and E2, an HCV pseudotyped retroviral particle, an HCVcc particle, an ex vivo HCVcc particle or HCV.
- an HCV envelope protein(s) When the cell expressing the recombinant Claudin-1 is contacted by an HCV envelope protein(s), one can determine if binding of the envelope protein(s) is inhibited by a compound or agent by monitoring the presence or absence of the envelope protein following a buffer exchange. Such binding analyses are facilitated by providing detectably labelled envelope proteins.
- Binding of cells expressing envelope proteins, HCVpp, HCVcc, ex vivo HCVcc, or HCV to the cell expressing the recombinant Claudin-1 can also be monitored in the manner described for binding of envelope proteins. Infection by HCVpp or HCVcc can also be monitored by the use of reporter genes encoded by these species, which can be assayed for in cells following exposure to the agents or compounds.
- Interactions of HCV with Claudin-1 that result in entry of HCV into the cell can also be assayed to identify compounds or agents that interfere with any aspect of that process.
- Productive interaction or fusion of cells expressing envelope proteins, HCVpp, HCVcc, ex vivo HCVcc, or HCV to the cell expressing the recombinant Claudin-1 can also be assayed by techniques that monitor transfer of proteins or nucleic acids from the cells expressing envelope proteins, HCVpp, HCVcc, ex vivo HCVcc, or HCV to the cell expressing the recombinant Claudin-1. In certain embodiments, transfer of a reporter gene is monitored.
- the reporter When the reporter is transferred from HCVpp or any other suitable viral vector, the reporter will be expressed only upon entry into the cell expressing the recombinant Claudin-1 protein.
- the reporter When the reporter is present in a distinct cell that fuses to the cell expressing the recombinant Claudin-1 protein, the reporter gene will only be expressed when the cells fuse and the reporter is exposed to a factor in the cell expressing the recombinant Claudin-1 protein.
- Methods that provide for expression of a reporter upon cell fusion include, but are not limited to, operable linkage of the reporter to a promoter that is regulated by a trans-acting transcription factor present in the host cell.
- Entry of HCV, HCVcc, or ex vivo HCVcc can also be determined by any hybridization or polymerase-chain reaction based method for measuring the associated HCV RNA. Entry of replication-competent HCV RNA into cells that permit HCV RNA replication results in a substantial signal amplification due to HCV RNA replication.
- the HCV-derived PCR product can be detected by use of any labelled polynucleotide probes, by use of an intercalating dye such as ethidium bromide or SYBR green, use of a hybridization probe containing a fluorophore and a quencher such that emission from the fluorophore is only detected when the fluorophore is released by the 5' nuclease activity of the polymerase used in the PCR reaction (i.e., a TaqMan TM reaction; Applied Biosystems, Foster City, CA) or use of methods where the fluorophore and quencher are displaced by polymerase mediated synthesis of the complementary strand (i.e., Scorpion TM or Molecular Beacon TM probes).
- an intercalating dye such as ethidium bromide or SYBR green
- a hybridization probe containing a fluorophore and a quencher such that emission from the fluorophore is only detected when the fluoro
- RNA quantitation techniques such as Quantitative Nucleic Acid Sequence Based Amplification (Q-NASBA TM) can be used to quantitate TIC807 protein-encoding mRNA and identify expressing plants.
- kits for quantitating HCV RNA include the COB ASTM TaqMan HCV Test (TaqMan HCV; Roche Molecular Systems Inc., Branchburg, N.J.).
- HCVcc that comprise reporter genes are also available, thus allowing the quantification of infection following challenge similarly to the methods described for HCVpp.
- Bacteriophage libraries comprising phage vectors that display antibody antigen recognition regions are one potential source of Claudin-1 recognition agents and have been described in U.S. Patent No. 6,265,150.
- Methods for generating libraries of antigen-combining proteins of high diversity are described in U.S. Patent 5780225 and U.S. Patent 6,303,313.
- Methods of using libraries to obtain antigen recognition regions are also described in U.S. Patent No. 5,395,750.
- Methods of obtaining a nucleic acid encoding a binding protein having a proteinaceous binding domain that binds a predetermined target material are described in U.S. Patent No.
- Yeast antibody display libraries can also be used to obtain antigen recognition regions and as described in U.S. Patent No. 6,300,065. Those skilled in the art will recognize that such antigen recognition sequences can subsequently be isolated from the recombinat page, bacterial or yeast vector and reengineered into a synthetic antibody appropriate for use in subjects. Reengineering of these recognition domains into humanized antibodies is particularly contemplated.
- Kits for Identifying Compounds Or Agent That Prevent Or Mitigate Interaction Of A Region Of A Hepatitis C Virus With A Region Of A Claudin-1 Protein [0098]
- the methods and kits detect interactions of a region of HCV with a Claudin-1 protein.
- a kit may contain any of : i) a recombinant protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, or ii) a recombinant vector that provides for expression of a membrane bound protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, wherein said amino acid residues of said Claudin-1 protein are located extracellularly to a cell expressing said membrane bound protein, or iii) a cell comprising said recombinant vector and instructions for using the kit.
- the kit may also contain reagent(s) for detecting an interaction between a sample comprising HCV, HCVcc, HCVpp, HCV envelope proteins and the aforementioned recombinant proteins, vectors or cells comprising a Claudin -1 protein.
- the provided reagent(s) can be radio-, spectrophotometrically-, fluorescently- or enzymatically-labeled.
- the provided reagents can also be detectbly labelled by other materials.
- the provided reagents may include a substrate that is converted to a product that can be detected by spectrophotometry, luminometry, or fluorescence.
- the kit can contain a known radiolabeled or hapten-labeled agent capable of binding or interacting with an antibody of the present invention.
- the reagent(s) of the kit may be provided as a liquid solution, attached or otherwise depositied in or on a solid support or as a dried powder.
- the liquid solution is an aqueous solution.
- the solid support can be chromatography media, a test plate having a plurality of wells, or a microscope slide.
- the reagent(s) provided are a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.
- the container will generally include a vial into which the recombinant protein, recombinant vector, cells or detection reagent may be placed, and preferably suitably aliquotted.
- the kits of the present invention will also typically include a means for containing the recombinant protein, recombinant vector and/or cells in a container in close confinement for commercial sale.
- Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
- kits may be assembled not for commercial sale, but for internal use within a research group. Thus the usefulness of such kits is not restricted to commercial sales.
- the instructions for the kit may either be enclosed in the kit or provided by way of reference to a external or internal website or other internal or external document or reference.
- the reference may include, but is not limited to, this patent application or Evans et al., Nature. 2007;446(7137):801-5 and associated supplementary information and data provided by Evans et al.
- the methods of the invention provide for useful cell culture compositions.
- These cell culture composition comprise: i) a cell comprising a recombinant vector that provides for expression of a membrane bound protein comprising at least amino acid residues 28 to 49 of a Claudin-1 protein as shown in SEQ ID NO:1 or conservative amino acid substitutions thereof, wherein said amino acid residues of said Claudin-1 protein are located extracellularly to said cell, and either ii) a cell comprising a recombinant vector that encodes HCV envelope proteins El and E2 or iii) any one of a HCV pseudotyped retroviral particle, a HCV cell culture particle, an ex vivo HCV cell culture particle, or an HCV particle.
- Such cell culture compositions can be used to determine if an added compound or agent can inhibit, mitigate, or prevent HCV interactions with Claudin-1.
- Compounds or agents identified by the use of the cell culture compositions can be used to prevent, inhibit or mitigate HCV infection in a subject.
- compositions Comprising Non-Naturally Occurring Agents Or Compounds Bound To Extracellular Loop 1 Of Claudin-1
- compositions comprising a non-naturally occurring agent or compound bound to extracellular loop 1 of a Claudin-1 protein are also provided herein.
- Such compositions can occur in a subject when the non-naturally occurring agent or non- naturally occurring compound is administered to the subject and are useful as they prevent,inhibit or mitigate HCV infection of the subject.
- Such compositions can also occur in the cell culture compositions of the invention when a non-naturally occurring agent or non-naturally occurring compound is added to the cell culture composition and are useful as they serve to identify the non-naturally occurring agent or compound for preventing, inhibiting or mitigating HCV infection of the subject.
- Example 1 Identification of CLDN-I as an Entry Factor [00107] A powerful iterative screening method to identify the HCV entry factor CLDN-I was conducted. A complementation screen, where cDNA from an HCV permissive cell is transduced into a nonpermissive cell was conducted. To select for cells permissive for HCV entry HCVpp carrying a drug resistance reporter gene were used, allowing isolation of successfully infected cell clones. Initial attempts, where a retroviral human liver cDNA library was used to complement a variety of HCVpp nonpermissive cell lines (Fig. 2, boxed region), were unsuccessful due to an inherent background of nonspecific infection with pseudoparticles (unpublished data).
- Modifying the provirus to include sequences, such as LoxP sites and bacterial origins of replication, may also be accomplished, which allows rescue of the sequence from infected genomic DNA as plasmids capable of replication in bacteria (Harmon et al., 1999, Science 283:1129-30; Koh et al., 2002, Nucleic Acids Res 30:el42; Li et al., 1996, Cell 85:319-29; Ohet al., 2002, J Virol 76:1762-8). [00109] A simpler approach that takes advantage of the intrinsic replication capacities of the retrovirus used to deliver the cDNAs was undertaken.
- retroviral vectors used for common gene-delivery applications are self-inactivating vectors that contain deletions in the long terminal repeat (LTR) elements.
- LTR long terminal repeat
- No packaging competent retroviral RNA transcripts are generated from such integrated proviruses. Instead transgene expression is driven by an internal non-retroviral promoter.
- the retroviral promoter should be active in transduced cell and a full length viral RNA should be expressed.
- Expression of the packaging components, gag-pol and envelope, in these cells allows packaging of this RNA into pseudoparticles capable of transducing na ⁇ ve cells (Fig. 3).
- CPR cyclic packaging rescue
- CSPW puromycin
- CSZW zeocin
- HIV-I provirus In a single round HIV-I pseudoparticle production, the viral RNA can be expressed from an exogenous promoter, alleviating the need for these accessory proteins.
- CPR with an HIV-I provirus requires the use of the HIV-I promoter and expression of accessory proteins.
- an HIV-I provirus termed Vl, where the gag-pol and envelope genes were deleted, but most of the accessory protein transcripts remained intact (Cowan et al., 2002, Proc Natl Acad Sci USA 99:11914-9; Simon et al., 2005, PLoS Pathog 1 :e6).
- Vl-GFP a control plasmid, which encoded GFP in place of a cDNA.
- Vl-GFP HIV gag-pol and VSVG plasmids were cotransfected into 293T cells high titers of released pseudoparticles were obtained (7x105 to 3x107 transducing units (TU)/ml depending on the target cell line).
- TU 3x107 transducing units
- AU tested cells lines produced re-packaged pseudoparticles, which were then titered on a na ⁇ ve population of the same cell line. Although titers varied by several orders of magnitude between cell lines, all were >lxlO4 TUJmI, demonstrating the capacity of this lenti viral system to undergo CPR and its utility for the screen.
- cDNA library was constructed from the hepatocarcinoma Huh-7.5 cell line, a subclone of the Huh-7 cell line highly permissive for HCV subgenomic replicons (Blight et al., 2002, J. Virol. 76:13001-14).
- Huh-7.5 cells are susceptible to both HCVcc and HCVpp and thus must express all molecules required for HCV infection.
- the final Vl-Huh-7.5 cDNA library contained 2.5x106 clones, 95% of which had inserts averaging 970 bp in size.
- Vl vector does not encode a reporter gene
- pseudoviruses carrying this library were first titered alongside a stock of Vl-GFP virus on the TZM HeLa indicator cell line, where an integrated lacZ gene is transcriptionally regulated by the HIV-I LTR (Derdeyn et al., 2000, J Virol 74:8358- 67; Platt et al., 1998, J Virol 72:2855-64; Wei et al., 2002, Antimicrob Agents Chemother 46: 1896-905). Infection of a TZM cell by a Vl containing pseudotype results in Tat expression from the Vl provirus and lacZ activation.
- the ratio of infectivity of these two viruses (Vl -library and Vl-GFP) on TZM cells was then used to approximate the effective titer of the library virus on the cell line of interest, based on the measurable infectivity of Vl-GFP.
- the target cell line for the screen was chosen based on several criteria. As stated above, the primary requirement was that (1) the cell line must express as many of the putative HCV entry factors as possible, most importantly CD81 and SR-BI, but be non-susceptible to HCVpp infection. (2) To minimize nonspecific background, cell lines with lower uptake of HCVpp and "no envelope" pseudoparticles were preferred.
- Vl-Huh-7.5 cDNA library was co-transfected with HTV gag-pol and VSVG into 293T cells to generate lentiviral pseudoparticles carrying the cDNA library and bearing VSVG on their surface (Vl/library-VSVGpp).
- a GFP carrying control (Vl/GFP-VSVGpp) was generated in parallel.
- Both Vl/library-VSVGpp and Vl/GFP-VSVGpp were titered on TZM cells; Vl/GFP-VSVGpp was also titered on 293T cells.
- the calculated titer of Vl/library-VSVGpp on 293T cells was 4x106 TU/ml.
- CSPW-HCVpp was used for infection, and selected with Puro.
- the number of PuroR colonies was not strikingly different between library and mock transduced cells. PuroR colonies were pooled for each population (library and mock) and infected a fraction of each population with a GFP- carrying HCVpp (CSGW-HCVpp) as well as "no envelope" (CSGW-"noenv”pp) and VSVG bearing CSGW (CSGW-VSVGpp) pseudoparticles (Fig. 4, second row).
- Genomic DNA was prepared from a pool of the selected clones and used as template in PCR across the Vl provirus cDNA-cloning site. Two distinct species were amplified, which were subsequently cloned and sequenced. The major, larger species was identified as the full-length cDNA for CLDNl . The lesser, smaller species was identified as myosin light chain polypeptide 6 (myl6) transcript variant 2.
- myl6 myosin light chain polypeptide 6
- Vl-CLDNl transduction enhanced susceptibility to HCVpp (pNL4.3-HCV) by 1000-fold while V1-MYL6 and Vl-GFP (negative control) had no significant effect (Fig. 5).
- Transduction with CLDNl had no effect on VSVGpp susceptibility.
- HCVpp permissive cell lines Huh-7.5, Hep3B, HepG2/hCD81
- non-permissive cell lines that been considered for the screen (293T, HepH, SW13, HeLa) 5 pre- and post- CLDNl transduction, for HCVpp permissiveness.
- HCVpp permissive cell lines Huh-7.5, Hep3B, HepG2/hCD81
- non-permissive cell lines that been considered for the screen
- HepH cells displayed low level susceptibility to HCVpp upon expression of CLDNl even though transgene expression was very high.
- the HepH cell line is highly permissive to VSV Gpp suggesting that other HCV entry factors may be missing or limiting in this line.
- CLDNl is important for HCVpp entry in a range of human cells.
- CLDN7 which has the highest sequence identity to CLDNl (60% identity, 78% similarity; BLASTP 2.2.12, BLOSUM62 matrix), and CLDN3, which is the closest CLDNl relative that is preferentially expressed in the liver (49% identity, 66% similarity to CLDNl).
- Both coding sequences were cloned into the pTRIP provirus, packaged, and transduced into na ⁇ ve 293T cells. While cells transduced with pTRIP-CLDNl became permissive to HCVpp, neither the CLND3- nor CLDN7-transduced cells were infectable with HCVpp (Fig. 7B).
- HCVcc Using the recently developed HCVcc system (Lindenbach et al., 2005, Science 309:623-6), we have begun examining several aspects of the HCV entry pathway into Huh-7.5 cells.
- an HCV virus termed FL- J6/JFH-5'C19Rluc2Aubi that encodes the Renilla luciferase reporter gene (Fig. 8A). Luciferase activity from this virus is proportional to replication and hence provides a sensitive assay for detection of HCV infection (Fig. 8B).
- bafilomycin Al and concanamycin A inhibitors of vacuolar H+-type ATPases.
- HCV entry therefore appears to be pH-dependent requiring an acidified intracellular compartment. This result confirmed what was previously suggested from studies using HCVpp, which were also sensitive to inhibitors of endosomal acidification (Hsu et al., 2003, Proc. Natl. Acad. Sci. USA 100:7271-76; Zhang et al., 2004, J Virol 78:1448-55).
- acidic pH triggers an irreversible conformational change, which promotes virion-endosomal membrane fusion. Such viruses are often inactivated by low pH.
- CLDNl specific reagents will be developed, in particular, antibodies directed against the CLDNl extracellular loops, which will be raised in chicken using highly purified peptides representing epitopes of the predicted extracellular loops of CLDNl.
- functional tagged derivatives of CLDNl will be developed. Using the pTRIP lentivirus vector we have constructed FLAG epitope (FLAG- CLDNl) or GFP (GFP-CLDNl) fusions to the CLDNl N-terminus as well as a C- terminal GFP fusion (CLDNl-GFP) GFP fusion (Fig. 12A).
- a nested set of chimeras will be created encoding N-terminal segments of increasing length from one claudin, with the remaining C-terminal sequence from the other. Since the extracellular loops are of obvious interest, junctions will be chosen flanking the domains between TMl /ELl, EL1/TM2, TM3/EL2, and EL2/TM4 (Fig 13B, ii). It is possible that interactions between claudin segments, such as the TM domains, will not be sufficiently conserved to allow the collect folding, higher order structure or localization needed for HCV entry. For these reasons, precise swaps, alone and in combination, of the extracellular loops will also be made (Fig 14B, iii and iv).
- these chimeras will be constructed as N- terminal GFP fusions as described above (see section D Ia2) for CLDNl .
- Further subdomain chimeras may allow a finer resolution mapping of functional CLDNl determinants.
- site-directed mutagenesis of the CLDNl domain of interest can be performed. For example, since the CLDNl extracellular loops are only 53 and 24 amino acids, it would be straightforward to mutate these regions, initially in sets of five amino acids and eventually individually, to CLDNl or CLDN7 residues (or alternative amino acids such as alanine) and test for entry function. Deletion mutants, of either entire loops or smaller portions of loops, will also be constructed and tested for their capacity to allow HCV entry. [00127] A catalogue of insertional mutants throughout the CLDNl region of interest were assembled (Figs. 14, 15, 16, and 17).
- Such insertional mutants can be constructed in a defined manner, where a FLAG epitope sequence will be inserted every three to four amino acid residues. 293T cells will be transduced with each insertional mutant separately and then challenged with HCVpp. Mutants that disrupt HCV infection may reside in regions important for the interaction between CLDNl and the virus. Furthermore, mutants that retain the ability to mediate HCVpp infection can be retested in the presence of an anti-FLAG monoclonal antibody (Sigma).
- a site within CLDNl that is capable of tolerating a small insertion may thus be turned into the epitope for an antibody that, if bound, could block a putative virus-CLDNl interaction. Since we currently do not have antibodies directed against the extracellular loops of CLNDl, these conditional CLDNl mutants may prove important for additional studies proposed below (section D3).
- a CLDNl random insertion library can be constructed through a transposon based approach.
- CLDNl and many other claudins have a C-terrninal PDZ domain-interacting motif, thought to interact with a variety of TJ components and intracellular signaling molecules. It will be interesting to test if disruption of specific CLDNl motifs or sites of possible protein modification affect HCV entry. In particular, it will be important to determine if the interactions between CLDNl and other TJ components are required for this function. If so, then one may speculate that the cellular mechanisms of TJ regulation and internalization may be utilized by HCV for cell entry. If so, CLDNl mutants lacking this cellular function that maintain the ability to interact with HCV may function as dominant negative inhibitors of HCV entry in cells expressing wild type CLDNl. [00129] CLDNl levels should ultimately become limiting for HCV entry.
- Lentiviral transduction of CLDNl should produce cell clones expressing a range of CLDNl levels, mainly through variation in the copy number of integrated viruses and chromosome locus-dependent transcriptional activity.
- the functional GFP-CLDNl fusion protein can be employed to facilitate monitoring of expression levels and protein localization.
- Such a population will be challenged with various moi's of a red fluorescent protein expressing HCVpp and subjected to dual-color FACS to determine if cells that express higher CLDNl levels are more likely to be infected.
- GFP- CLDNl can be sorted by expression level and used for HCVpp challenge as a population or after cloning to produce individual cell lines with different CLDNl expression levels.
- a pTRIP provirus will be used where the internal cassette is expressed from a tetracycline regulated promoter (TRex system, Clontech) that can be used to infect the commercially available 293T cell lines expressing the tetracycline repressor.
- TRex system Clontech
- This system will allow doxycycline-mediated regulation of CLDNl expression to investigate how this affects HCV permissiveness.
- the J6/JFH genotype 2A chimeric virus has been described (Lindenbach et al., 2005, Science 309:623-6) and we have established a technique for quantifying viral infectivity by limiting dilution assay and NS5 A immunostaining (Lindenbach et al., 2005, Science 309:623-6). Additionally, a Renilla luciferase reporter, engineered in the backbone of the J6/JFH chimeric virus, termed FL-J6/JFH-5'C19Rluc2AUbi, has been used in studies of HCV entry (Tscherne et al., 2006, J Virol 80:1734-1741).
- HCVcc H77/JFH HCVcc, which expresses the H77 envelope glycoproteins.
- HCVcc Ex vivo HCVcc: We have obtained acute phase plasma and serum samples of HCVcc that have been passaged in either chimpanzees (chHCVcc) or SCID-uPA beige xenograft mice (muHCVcc) (Lindenbach et al., 2006, Proc. Natl. Acad. Sci. USA 103 In press) (see Appendix G). These ex-vivo samples are infectious in Huh-7.5 cells and provide a unique opportunity to compare the entry properties of functional virus produced in vivo with HCVcc and HCVpp.
- Soluble E2 and soluble E1E2 C-terminally truncated soluble E2 protein (sE2) is routinely generated in our lab by transfection of a sE2 expression construct into 293T cells and harvest of cell supernatants (Flint et al., 2000, J Virol 74:702-9). Expression constructs for generating E1E2 and methods for purifying large quantities of cell-associated E1E2 complexes have been described (Brazzoli et al., 2005, Virology 332:438-53; Frey et al., 2005, Presented at the 12th International Symposium on Hepatitis C Virus and Related Viruses, Montreal, Canada, Oct 2-6). Purified material for use in blocking experiments will be provided by Michael Houghton (Chiron).
- Permissive cells Huh-7.5 cells are permissive for HCVpp, HCVcc, chHCVcc, and muHCVcc infection and therefore possess all of the necessary factors for HCV entry. We also have characterized a battery of cell types, including HepG2 and 293T cells which are non-permissive for HCV infection, but become permissive when transduced with a particular entry factor, like CD81 or CLDNl, respectively. [00133] To analyze the specific roles of CD81, SR-BFII, and CLDNl in HCV entry, we will make use of blocking reagents in the form of antibodies, peptides, and soluble forms of protein molecules, as described below.
- CD81 GST-CD81 large extracellular loop (LEL) and anti-CD81 antibodies (Santa Cruz 1.3.3.22 and BD Pharmingen JS81, respectively) are established blocking reagents and readily available (Cormier et al., 2004 et al., Proc Natl Acad Sci USA 101:7270-4; Hsu et al., 2003, Proc. Natl. Acad. Sci. USA 100:7271-76; Lindenbach et al., 2005, Science 309:623-6) (unpublished data).
- SR-BI/II We have obtained a stable CHO cell line that expresses a recombinant, soluble form of the SR-Bl extracellular domain (SR-Bl-EC) fused to a C-terminal FLAG-epitope tag (Heo et al., 2004, J Immunol 173:446-55). SR-Bl-EC can be purified from cell lysates using an M2 anti-Flag-agarose column (Sigma) and will be tested for inhibitory activity against HCVpp and HCVcc. [00136] CLDNl : Blocking antibodies directed against the CLDNl extracellular loops will be obtained.
- a straightforward approach to begin to address the sequence of virus-host cell interactions is to bind virus on ice and then shift to 37°C; the blocking reagents described above that target different entry factors will be added at defined time points (during binding on ice, at the time of the temperature shift, or at various time points thereafter) and the infection signal recorded as a percentage of the signal in the absence of blocking reagents.
- a time course of antibody or peptide addition after shifting to 37°C can be used to dissect the virus-receptor and receptor-receptor interactions occurring at each stage of the HCV entry process.
- an antibody recognizing CLDNl or a CLDNl derived peptide with blocking activity corresponding to CLDNl ELl could be added to the cells at the time of shifting to 37oC and at defined later time points, to determine to what extent they perturb entry.
- the same experiment could be performed with reagents targeting CD81 or SR-BI/II.
- a blocking reagent is likely to lose activity after its target molecule has interacted with the virus.
- a reagent targeting an early event in the entry process should become ineffective earlier than one targeting a later step.
- using the blocking agents in various combinations at distinct timepoints at/after shifting to 37°C will allow the development of a temporal model of virus-entry molecule interactions during HCV entry.
- Non-human, non-permissive cells such as CHO cells can be transduced with constructs expressing CD81, SR-BI/II, and CLDNl, alone or in combination and asssayed for HCV binding.
- 293T cells used in the screen that identified CLDNl as an HCV receptor described above, are non- permissive for HCV infection but become permissive when transfected with CLDNl cDNA.
- 293T cells and 293T-CLDN1 cells can therefore be used to look specifically at the contribution of CLDNl to HCV binding.
- a similar approach has been taken to determine that the contribution of CD81 to HCVpp binding to HepG2 cells is low, making it an unlikely initial attachment receptor (since this cell line is fully HCV permissive when transduced with CD81 yet HCVpp binding is not affected (Cormier et al., 2004 et al., Proc Natl Acad Sci USA 101:7270-4)).
- permissive cells such as Huh-7.5 cells, we will examine the effect of incubation prior to HCV binding with CD81, SR-BI/II, and CLDNl antibodies, described in D3a, individually or in concert.
- the binding assay will also be performed using virus that has been incubated with peptides targeting distinct regions of CD81, SR-BI/II, and CLDNl, or soluble forms of these receptor molecules where available.
- Binding of sE2 or soluble E1E2 heterodimers to any of the above-mentioned cell-types may be another tool to study which molecules can function as receptors in mediating virus attachment.
- HCV fusion assay based on rhodamine (Rl 8) fluorescence dequenching (Lavillette et al., 2005, J Biol Chem).
- Rl 8 rhodamine
- fusion of HCVpp with liposomal membranes was observed with fluorescent probes incorporated into either HCVpp or the liposome.
- HCV gp-mediated fusion was shown to be pH- and temperature dependent. Surprisingly fusion did not require the presence of any protein or receptor on the liposome surface. However, the extent and kinetics of fusion were decreased compared to influenza HApp mediated fusion in the same assay. The authors speculate that HCVpp fusion may be suboptimal in the absence of receptor on the target membrane.
- HCVgp based cell-cell fusion assay HCV glycoproteins are mainly retained in the ER, although some El and E2 escape ER retention and transit to the cell surface (Bartosch et al., 2003, J Exp Med 197:633-642; Drummer et al., 2003, FEBS Lett 546:385-90; Flint et al., 2004, Virol 78:6875-82).
- HCV gp based cell fusion assay to study sequential receptor interactions. To study the sequential interaction of HCV with its receptors, we can use a variation of the HCV gp based cell fusion assay described in (2).
- a recombinant soluble receptor can be preincubated with effector cells prior to incubation with target cells.
- the effector cells will then be incubated with target cells containing the additional receptors but lacking CD81 or CLDNl 5 respectively. If interaction with the soluble receptor is required prior to interaction with the additional receptors, fusion should theoretically be catalyzed by the presence of such a factor in the media.
- This assay has been used previously to demonstrate that soluble CD4 can activate HIV Env for coreceptor-dependent fusion (Salzwedel et al., 2000, J Virol 74:326-33).
- co-IP co-immunoprecipation assay
- M ⁇ CD methyl- ⁇ -cyclodextrin
- the CD81 -negative hepatoma HepG2 line previously reported to become susceptible on CD81 expression, also expressed endogenous CLDNl and HCVpp entry remained CD81- dependent even when CLDNl was overexpressed.
- the human HeLa and HepH cell lines both CD811 SR-BIl CLDN 12
- expression of human CLDNl and/or CD81 in non-human cell lines did not allow HCVpp entry.
- murine CLDNl (90% amino acid identity to human) efficiently supported HCV entry, indicating that CLDNl is not a determinant of species host range (Fig. 19).
- Example 3 HCVpp susceptibility depends on residues in the first extracellular loop of CLDNl.
- CLDN7 the closest relative of CLDNl (60% amino acid identity), nor CLDN3, the closest liver-expressed relative (49% identity), rendered 293 T cells permissive to HCVpp (Figure 22), despite high levels of protein expression (Fig. 22b).
- Fig. 22d To map CLDNl -specific entry determinants, we analysed a series of CLDNl- CLDN7 chimaeras (Fig. 22d) fused to the carboxyterminus of GFP. The parental fusion proteins functioned identically to untagged proteins (Fig. 22e) and were expressed similarly (Fig. 22f).
- CLDNl is a 211 amino acid protein with four transmembrane helices, intracellular amino and carboxy termini and two extracellular loops (Fig. 22c).
- Fig. 22d When extracellular loops were exchanged between CLDNl and CLDN7 (Fig. 22d) it was found that only those containing the CLDNl ELl enhanced HCVgp-dependent infection (Fig. 22e).
- Fig. 22e Progressively smaller exchanges identified the N-terminal third of the CLDNl ELl (ELl Nl/3) to be sufficient in an otherwise CLDN7 background to confer full susceptibility to HCVpp entry in 293T cells (Fig. 22e).
- Example 4 Blocking antibodies, ligands or antagonists are useful for probing the function of cellular molecules involved in viral entry.
- CLDNl -specific antibodies recognize the intracellular C-terminal segment of the protein and are thus not useful for such studies.
- HCVpp infection of 293T cells expressing this mutant was blocked in a dose-dependent manner by anti-Flag M2 monoclonal antibody.
- 293T cells seeded in a 6-well plate, were transfected with either mock Vl vector, to provide Tat expression, a glycoprotein expression vector (either H77 HCV E1E2 or VSV-G), or Vl plus glycoprotein. Transfections were performed with Fugene-6. At 36 h post transfection, combinations of donor and acceptor cells were co-seeded (1.5x106 cells each/well) in 2-well chamber slides (BD Falcon). 12 h later, cells were washed with citric acid buffer at either pH 5 or pH 7 (15 mM citric acid, 150 mM NaCl), followed by media to neutralize the pH, and incubated for an additional 36-48 h.
- citric acid buffer at either pH 5 or pH 7 (15 mM citric acid, 150 mM NaCl
- Example 6 Description of Various Techniques and Materials Used in the Examples [00153] 293T, HeLa, Hep3B, Hepal.6, HepG2, HepH, Huh-7.5, SW13 and TZM cells were maintained in DMEM with 10% fetal bovine serum (FBS). CHO cells were maintained in DMEM/F-12 with 10% FBS. HepG2 were grown on collagen coated plastic.
- FBS fetal bovine serum
- Mouse monoclonal antibodies against CLDNl (clone 2Hl ODlO), 3 and 7 were purchased from Zymed Laboratories (San Francisco, CA).
- Mouse anti-CD81 1.3.3.22 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
- Purified mouse IgGl was from BD Pharmingen (Franklin Lakes, NJ).
- the mouse anti-GFP, mouse anti- ⁇ -actin and HRP-anti-mouse-IgG used for irnmunoblotting were from Novus Biologicals (Littleton, CO), Sigma (St. Louis, MO), and Pierce (Rockford, IL), respectively.
- HRP detection was performed with SuperSignal West Pico Substrate (Pierce), according to the manufacturer's instructions.
- the mouse anti-NS5A antibody 9E10 has previously been described30.
- AlexaFluor488 conjugated anti- mouse IgG secondary antibodies were obtained from Invitrogen (Carlsbad, CA).
- the M2 anti-FLAG antibody was obtained from Sigma (St. Louis, MO), JS81 anti-CD81 and negative control mouse IgG antibodies were from BD Pharmingen (Franklin Lakes, NJ).
- the pNL4.3.1uc.R-.E- HIV genome has been described elsewhere 31, 32.
- the HIV proviruses expressing from the Spleen focus forming virus promoter GFP (CSGW), puromycin resistance (CSPW) and zeocin resistance (CSZW) reporter genes were constructed within a self-inactivating proviral genome encoding a deletion in the 3' LTR U3 region, resulting in inactivation the viral promoter during reverse transcription and integration.
- the CSGW and CSPW plasmids have been previously described 33.
- CSZW was constructed by PCR amplification of the zeocin resistance gene open reading frame (ORF) from pCDNA3.1Zeo (Invitrogen, Carlsbad, CA) with the oligos 5'-GGGATCCGGG ATG GCC AAG TTG ACC AGT GCC GTT CCG (SEQ ID NO:4) (start codon in bold) and 5'-CCCCTCGAGTCTAGA TCA GTC CTG CTC CTC GGC CAC GAA GTG (SEQ ID NO:5) (stop codon in bold).
- ORF zeocin resistance gene open reading frame
- This product was then digested with the BamHI and Xbal restriction endonucleases (New England Biolabs, Ipswich, MA) and cloned into a BamHI and partially Xbal digested CSGW plasmid, resulting in the replacement of the GFP coding sequence with that for zeocin resistance.
- BamHI and Xbal restriction endonucleases New England Biolabs, Ipswich, MA
- the CLDNl ORF was amplified from the cDNA clones isolated in the screen with the oligos 5'-GGGG GGATCC GTC ATG GCC AAC GCG GGG CTG CAG CTG TTG GGC (SEQ ID NO:6) and 5'-GGGG CTCGAG TCA CAC GTA GTC TTT CCC GCT GGA AGG TGC(SEQ ID NO:7) .
- This product was cloned as a BamHI and Xhol digested fragment into a likewise digested pTRIP plasmid.
- expressed sequence tag (EST) clones (ATCC numbers 6869006, 9035916, and 7156714, respectively) constructed by the Integrated Molecular Analysis of Genome Expression (I.M.A.G.E.) Consortium (image.llnl.gov) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).
- pTRIP-CLDN3 was constructed by amplification of the CLDN3 ORF with oligos 5'-GGGG GGATCC GCC ATG TCC ATG GGC CTG GAG ATC ACG GGC ACC (SEQ ID NO:8) and 5'-GGGG GTCGAC TTA GAC GTA GTC CTT GCG GTC GTA GCC TGT (SEQ ID NO:9) to generate a product that was cloned as a BamHI/Sall fragment into the BamHI/Sall digested pTRIP plasmid.
- ⁇ TRIP-CLDN7 was constructed by amplification of the CLDN7 ORF with oligos 5'-GGGG GGATCC ATG GCC AAT TCG GGC CTG CAG TTG CTG GGC (SEQ ID NO: 10) and 5'-GGGG GTCGAC TCA CAC ATA CTC CTT GGA AGA GTT GGA CTT (SEQ ID NO: 11) to generate a product that was cloned as a BamHI/Sall fragment into the BamHI/Sall digested pTRIP plasmid.
- the mouse CLDNl ORF was amplified with oligos 5'-GGGG GGATCC GTC ATG GCC AAC GCG GGG CTG CAG CTG CTG GGT (SEQ ID NO: 12) and 5'-GGGG CTCGAG TCA CAC ATA GTC TTT CCC ACT AGA AGG TGT TGG (SEQ ID NO: 13). This fragment was cloned as a BamHI/XhoI fragment into a likewise digested pTRIP plasmid.
- the GFP-CLDNl and CLDN7 fusions were constructed in the TRIP lentiviral vector by cloning of the GFP coding sequence, amplified with the oligos 5'- GGGG GGA TCC GGA ATG GTG AGC AAG GGC GAG GAG CTG TTC (SEQ ID NO:14) and 5'- GGGG AGA TCT CTT GTA CAG CTC GTC CAT GCC GAG AGT GAT (SEQ ID NO: 15) and digested with BamHI/Bglll, into the pTRIP-CLDNl and pTRIP-CLDN7 at the BamHI sites of each plasmid to make pTRIP-GFP-CLDNl and pTRIP-GFP-CLDN7, respectively.
- forward and reverse oligos that anneal to CLDNl and CLDN7 coding sequences at desired junctions were used in combination with the forward GFP oligo, described above, or the oligo specific for the 3' end of the coding sequence of interest, described above, were used for first-round PCR with pTRIP-GFP-CLDNl and pTRIP-GFP-CLDN7 plasmids as template. Each fragment was purified then amplified in a second round of PCR with only the most 5' and 3' specific oligo, thus generating fusion of claudin coding sequences at the desired junction.
- Claudin point mutants and ELl deletions and insertions were generated by essentially the same procedure, where outside GFP and claudin 3' end specific oligos were used in conjunction with internal mutation specific oligos (sequences available upon request) in two rounds of PCR to generate coding sequence fragments with desired changes.
- pTRIP-CLDNl FlO was constructed by amplification of pTRIP-GFP-CLDNl with the forward GFP oligo, described above, and the reverse oligo 5'- CTT GTC GTC ATC GTC CTT ATA GTC GAC CAA TGT GCT GCT CAG ATT CAG (SEQ ID NO: 16), which creates a Sail site and a single FLAG epitope at the 3' end, and, in a separate reaction, the forward oligo 5'-GAC TAT AAG GAC GAT GAC GAC AAG CTT CAA GCA ACG CGT GCC TTG ATG GTG (SEQ ID NO: 17) and the reverse oligo for the CLDNl coding sequence, which creates a 5' single FLAG epitope and a HindIII site.
- the products were then amplified with the outside oligos to create a fragment with an internal FLAG epitope fusion within ELl that was then cloned into pTRIP as a BamHI/XhoI digested fragment into a like digested vector.
- the forward oligo 5'-GGGG GTC GAC TAC AAA GAC CAT GAC GGT GAT TAT AAA GAT CAT GAT ATC GAC TAT AAG GAC GAT GAC GAC AAG CTT (SEQ ID NO: 18), encoding a Sail site, the triple epitope sequence, and a HindIII site, and the reverse oligo for the CLDNl coding sequence were used in a PCR with the pTRIP- CLDNl FlO plasmid as template.
- the triple-FLAG epitope was then cloned in place of the single epitope in the ELl insertion mutant plasmids at the Sall/Hindlll sites.
- the Huh-7.5 cell cDNA library was assembled in a minimal HIV-I provirus, termed Vl, where most genes were deleted, but the Tat, Rev, and Vpu ORF 's, as well as all necessary cis acting sequences remained intact36, 37.
- Vl minimal HIV-I provirus
- the Nef gene was also deleted and replaced with a cloning site where the cDNA of interest could be cloned and expressed.
- Vl vector used in this study include Vl-GFP, where the coding sequence for GFP was cloned into the Nef position, and VldTat-GFP, where the Vl-GFP plasmid was digested with Mfel, which cuts once 23 amino acids into the Tat open reading frame, filled-in with Klenow DNA polymerase, then religated to introduce a frame shift and thus prevents translation of Tat.
- SMART cDNA Library Construction Kit (Clontech, Mountain View, CA) protocol was essentially followed, with the following modifications: mRNA prepared from Huh-7.5 cells with Trizol Reagent (Invitrogen) and polyA selected with the Oligotex mRNA Maxi Kit (Qiagen, Valencia, CA) was subjected to poly-T primed first strand cDNA synthesis using the Superscript III First- Strand Synthesis System for RT-PCR (Invitrogen). Second strand cDNA was then synthesized and amplified using the TaqPlus Long PCR System (Stratagene, La Jolla, CA). Oligo sequences are identical to those described in the SMART cDNA Library Construction Kit.
- the resulting cDNA was the Sf ⁇ l digested, fractionated by cDNA Size Fractionation Columns (Invitrogen), and cloned into an Sfil digested Vl vector. All DNA precipitation steps were replaced by purification using the QIAquick PCR Cleanup Kit (Qiagen).
- the final Vl-Huh-7.5 cDNA library contained 2.5x106 clones with inserts averaging 970 bp in size.
- the JFHl El E2 sequence was amplified using the oligos 5' - CAC CAT GGG TTT CCC CTT TTC TAT CTT (SEQ ID NO: 19) and 5' - CTA CTA TGC TTC GGC CTG GCC CAA CAA GAT GAG CAT CCA (SEQ ID NO:20) and cloned into pcDNA3.1D/V5HisTOPO (Invitrogen, Carlsbad, CA).
- Other E1E2 sequences Conl, OH8, HC- J6 used for HCVpp generation have been described previously 39.
- 8xlO 5 293T cells were seeded in a 35 mm well.
- plasmid combinations and ratios were used: To generate luciferase reporter HCVpp and controls, equal amounts of pNL43.luc.R-.E- (encoding a provirus containing luciferase and HIV gag-pol) and either HCV E1E2, VSV-G or empty vector were co-transfected giving rise to HCVpp, VSV-Gpp and envelope- deficient pseudoparticles (Env-pp), respectively - as previously reported32. To generate GFP, puromycin or zeocin reporter HCVpp and controls, plasmids encoding
- a provirus encoding the respective reporter gene (CSGW, CSPW or CSZW), (2) HIV gag-pol, and (3) either HCV strain H77 E1E2, VSV-G or empty vector were transfected at a 1 :1:4 ratio.
- plasmids encoding (1) either a Vl or pTRIP provirus containing the desired transgene
- Infection assays with luciferase reporter pseudoparticles were performed in a 96-well format using 104 target cells per well. Cells were infected with pseudovirus supernatants diluted in fresh media (1 :5 for HCVpp and Env-pp; 1 :5000 for VSV- Gpp) and polybrene was added to a final concentration of 4 ⁇ g/ml. After 6-18 h the media was changed. Luciferase assays were performed 72 h after infection as previously described 32.
- GFP expression was quantified using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Except where noted otherwise, results of infection experiments are represented as the mean of greater than three independent replicate wells, and, in the case of the chimera and mutant claudin analysis, of at least two independently transduced populations. Error bars represent the standard deviation.
- cDNA cloned into the lentiviral Vl vector was packaged into pseudovirus particles bearing VSV-G glycoprotein as described above. Since the Vl vector does not encode a reporter gene, pseudoviruses carrying this library were first titered alongside a stock of Vl-GFP virus on the HeLa-derived TZM indicator cell line.
- TZM cells have an integrated lacZ gene that is transcribed via the HIV-I LTR.40, thus infection of a TZM cell by a Vl containing pseudotype results in Tat expression from the Vl provirus and lacZ activation.
- Vl-library and Vl-GFP viruses were measured on TZM cells using the lacZ reporter in a limiting dilution type assay41.
- the determined titers were then used to approximate the effective titer of the library virus on the cell line of interest, based on the measurable infectivity of Vl-GFP on the cell line of interest.
- VSV-Gpp carrying the Vl -library were then used to transduce 293T cells at a multiplicity of infection of about 1. Approximately 5x106 cells were transduced for the first round of screening; lower numbers were used in subsequent rounds. 293T cells expressing the library were challenged with HCVpp carrying a puromycin (CSPW) or zeocin (CSZW) reporter.
- CSPW puromycin
- CSZW zeocin
- HCVcc were generated as previously reported30. Briefly, a plasmid encoding the chimeric J6/JFH genome was linearized with Xbal and transcribed using MEGAscript T7 (Ambion, Austin, TX). RNA was electroporated into Huh-7.5 cells using a ECM 830 (BTX Genetronics). 72 h after electroporation the supernatant was transferred to na ⁇ ve Huh-7.5 cells. These cells were incubated for 72-96 h before the supernatant was again transferred to na ⁇ ve cells. This procedure was performed 3-4 times to generate high-titer HCVcc stocks.
- filtered supernatants were titered on Huh-7.5 using a limiting dilution assay as described30.
- 293T cells were seeded in 35 mm dishes. The next day HCVcc containing supernatant was applied.
- infection was detected by immunohistochemical staining for NS5A with the 9E10 anti-NS5A as primary and an HRP-conjugated anti-mouse-IgG (Immpress, Vector, Burlingame, CA) as secondary antibody.
- DAB chromogen DakoCytomation, Carpinteria, CA served as substrate.
- siRNA's Small interfering RNA oligonucleotides
- All siRNA's targeting CLDNl corresponded to 19 nucleotide regions in the CLDNl reference sequence (accession NM_021101): 5'- AGUGGAGGAUUUACUCCUA (subsequently referred to as "306") (SEQ ID NO:21); 5'-UGAAGUGUAUGAAGUGCUU (525) (SEQ ID NO:22); 5'- UGGUAUGGCAAUAGAAUCG (635) (SEQ ID NO:23); 5'- CACCAAGGCCCUAUCCAAA (804) (SEQ ID NO:24); 5'- UAACAUUAGGACCUUAGAA (921) (SEQ ID NO:25); 5'-
- UUCCAUAUUGAUGAAGAUG (1259) (SEQ ID NO:26).
- a sequence without close homology to any expressed human sequence (5'-UAGCAGCUAAACACAUCAA) (SEQ ID NO:27) was used as an irrelevant control.
- the siRNA targeting CD81 (5'- UGAUGUUCGUUGGCUUCCU) (SEQ ID NO:28) has previously been described 43.
- cells were transfected twice on days one and five. On the day before transfection 1.8x105 cells were seeded in 35 mm wells. For transfection 6 ⁇ l Lipofectamine2000 (Invitrogen, Carlsbad, CA) and 150 pmol siRNA were used per well. Between transfections, cells were passaged as needed to keep them subconfluent. Protein expression and infection assays were performed on day 8.
- CHO cells mock transduced, or transduced with human CD81, CLDNl, or SR-BI, seeded in triplicate wells, were incubated with J6/JFH HCVcc (MOI -0.5) for 2 h at 37°C. After binding, the cells were washed extensively with DPBS and total RNA was harvested using the RNeasy Mini kit (Qiagen). HCV RNA was amplified from 100 ng total RNA using the LightCycler RNA Amplification kit (Roche) and detected using a LightCycler 480 (Roche).
- the forward and reverse primer sequences were 5' CTT CAC GCA GAA AGC GTC TA 3' and 5' CAA GCA CCC TAT CAG GCA GT 3' (SEQ ID NO:30), respectively (Applied Biosystems).
- the probe sequence was 5' FAM-TAT GAG TGT CGT GCA GCC TC-MGBNFQ 3' (SEQ ID NO:31) (Applied Biosystems).
Abstract
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AU2007248493A AU2007248493B2 (en) | 2006-05-04 | 2007-05-04 | HCV coreceptor and methods of use thereof |
EP07809043.8A EP2016100B1 (en) | 2006-05-04 | 2007-05-04 | Hcv coreceptor and methods of use thereof |
US12/299,439 US8021835B2 (en) | 2006-05-04 | 2007-05-04 | HCV coreceptor and methods of use thereof |
CA002649962A CA2649962A1 (en) | 2006-05-04 | 2007-05-04 | Hcv coreceptor and methods of use thereof |
IL194777A IL194777A0 (en) | 2006-05-04 | 2008-10-22 | Hcv coreceptor and methods of use thereof |
US13/215,820 US8703149B2 (en) | 2006-05-04 | 2011-08-23 | HCV coreceptor and methods of use thereof |
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Also Published As
Publication number | Publication date |
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AU2007248493B2 (en) | 2010-09-30 |
EP2016100B1 (en) | 2014-07-09 |
IL194777A0 (en) | 2011-08-01 |
WO2007130646A3 (en) | 2008-11-20 |
CA2649962A1 (en) | 2007-11-15 |
US20120054879A1 (en) | 2012-03-01 |
AU2007248493A1 (en) | 2007-11-15 |
EP2016100A4 (en) | 2009-11-11 |
US8021835B2 (en) | 2011-09-20 |
EP2016100A2 (en) | 2009-01-21 |
US20090098123A1 (en) | 2009-04-16 |
US8703149B2 (en) | 2014-04-22 |
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