WO1990000204A1

WO1990000204A1 - Novel protein and coding sequence for detection and differentiation of siv and hiv-2 group of viruses

Info

Publication number: WO1990000204A1
Application number: PCT/US1989/002536
Authority: WO
Inventors: Louis E. Henderson; Raoul E. Benveniste; Raymond C. Ii Sowder; Terry D. Copeland; Stephen Oroszlan
Original assignee: The United States Of America, Represented By The Secretary, United States Department Of Commerce
Priority date: 1988-06-13
Filing date: 1989-06-12
Publication date: 1990-01-11
Also published as: EP0452319A1; JPH0782017B2; AU3864489A; JPH03504132A; AU617201B2; EP0452319A4; IL90553A0

Abstract

A novel protein and coding sequence for detection and differentiation of SIV and HIV-2 group of viruses from other similar viruses are disclosed.

Description

NOVEL PROTEIN AND CODING SEQUENCE FOR DETECTION AND

DIFFERENTIATION OF SIV AND HIV-2 GROUP OF VIRUSES

The present invention is related generally to the field of isolation and characterization of proteins and their coding sequence. More particularly, the present invention is related to the identification, characterization and diagnostic use of a protein unique to the SIV and HIV-2 group of viruses and the gene encoding said protein.

BACKGROUND OF THE INVENTION

Simian immunodeficiency viruses (SIVs) cause a fatal disease in susceptible primate species with symptoms similar to human AIDS caused by human immunodeficiency viruses type 1 (HIV-1) and type 2 (HIV-2). Strains of SIV were originally isolated from rhesus monkeys with immunodeficiency or lymphoma (SIV/mac) and subsequently from asymptomatic mangabey monkeys (SIV/SMM, SIV/SMLV, SIV/Delta) and from Macaca nemestrina with lymphoma (SIV/Mne). A strain of SIV originally thought to be obtained from African green monkeys ( STLV-III/agm) has since been shown to be SIV/mac. SIV strains are closely related to each other (greater than 90% identity) and also partially related to HIV-1 (40% nucleotide sequence identity), but are more closely related to HIV-2 (75% overall nucleotide sequence identity).

The genomic organizations of HIV-1, HIV-2 and SIV are very similar, each containing open reading frames (ORFs) designated gag, pol, env, Q, R, trs, tat, and F. However, HIV-2 and SIV each contain an ORF designated X that is not found in HIV-1 [ Chakrabarti, et al, Nature 328, 543 (1987); Franchini, et al, Nature 328, 539 (1987); and Guyader, et al, Nature 326, 662 (1987)]. The X-ORF is located in the central region of the genome between the pol-ORF and the env-ORF. However, the nature and properties of X-ORF, its translational product and their significance have not heretofore been known or described. SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide an isolated, substantially pure protein designated herein as the "p14" or "sid-protein."

It is a further object of the present invention to identify a coding sequence which directs the synthesis of the sid-protein in a suitable expression vector.

It is another object of the present invention to provide a diagnostic kit for differential detection of HIV-2 and SIV group of viruses from other similar viruses, such as the group of HIV-1 strains.

A further object of the present invention is to provide a method for differentiating between HIV-2, SIV and HIV-1 infection.

Other objects and advantages of the present invention will become apparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein:

Figure 1 represents SDS gel electrophoresis showing protein products of the X-ORFs in SIV/Mne, SIV/mac and HIV-2 and the purified protein (p14) obtained from SIV/Mne. Figure 1(a) shows a coomassie stained gel of SIV/Mne (lane 1) and purified p14 (lane 2). Viral gag proteins (p.28, p16, p8, and p6) are identified in lane 1. Figure 1(b) shows a coomassie stained gel of HIV-1 (lane 1), HIV-2 (lane 2), SIV/Mne (lane 3) and SIV/mac (lane 4). Figure 1(c) shows the results of immunoblot analysis of separated viral proteins (SDS gel identical to panel-b) using rabbit antiserum to purified SIV/Mne p14. After SDS gel electrophoresis, proteins were transferred to nitro-cellulose and probed with antiserum at 1 to 500 dilution. Antigen-antibody complexes were detected by radioautography after reaction with ¹²⁵Ilabeled staphylococcal protein A as described by Henderson, et al, Virol 61, 1116 (1987). Approximately 50 μ q of total protein was applied to each viral lane (panel b and c);

Figure 2 shows amino acid sequences of tryptic peptides from p14 and alignment with the protein predicted by the HIV-2 X-ORF. Figure 2(a): Purification of Tryptic Peptides: Purified p14 (Fig. 1, lane 2) (200 μg) was dissolved in 0.5 ml of 0.1 M Tris-HCl (pH 7.4) and 20 μg of trypsin (Cooper Biomedical) added. The digestion was continued for 8 hr. and stopped by adding trifluoroacetic acid (TFA) to pH 2.0. Peptides were separated by RP-HPLC at 1.0 ml/min on u-Bondapak C18 (3.9 x 300 mm column) at pH 2.0 (0.05% TFA) with a linear gradient of acetonitrile (- - - ) and detected by u.v. absorption

( ) at 206nm. Peaks containing peptides taken for further analysis are indicated by the letters "a" through "1".

Figure 2(b): Tryptic peptides of SIV/Mne p14 aligned with the amino acid sequence predicted by the XORF of HIV-2. Tryptic peptides derived from p14 (as in Fig. 2(a) were analyzed for amino acid content (Pico Tag system, Waters Inc. Milford, MA.) and sequence (gas phase sequencer, model 470A equipped with model 120A analyzer, Applied Biosystems, Foster City, CA. ) and the results compared to the amino acid sequence predicted by the XORF in the nucleotide sequence of HIV-2 [Guyader, et al, Nature 326, 661 (1987)]. Peptides are indicated by brackets and dashed lines [— ]. Arrowheads to the right (- - -

>) indicate the last residue identified by amino acid sequence analysis. Arrows to the left (<- --) indicate residues determined by digestion of p14 with carboxypeptidase-p. Residues in parentheses and separated by commas represent residues confirmed by amino acid content of purified peptides. Lower case letters (a, b, c, etc.) refer to peptides purified in Fig. 2(a). Asterisks (*) show where the amino acid sequences differ. The unknown blocking group on the N-terminal end of p14 is indicated by x; and

Figure 3 demonstrates that purified p14 binds to polyethenoadenylic acid. Purified SIV/Mne proteins including p28, p2, p8, p6 and p14 were tested for single stranded nucleic acid binding activity by the fluorescence enhancement assay using 2.19 μ M polyethenoadenine as described by Karpel, et al, [J . Biol. Chem. 262, 4961 (1987)]. Of the viral proteins tested only p14 and p8 produced significant enhancement of fluorescence when added to solutions of polyethenoadenine.

DETAILED DESCRIPTION OF THE INVENTION

The above and various other objects and advantages of the present invention are achieved by an isolated, substantially pure protein which, in whole or in part, possesses specific binding affinity only for antibody to sid-protein without cross-reacting with antibodies to HIV-1 strain of viruses.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art.

The term "substantially pure" as used herein means as pure as can be obtained by standard purification techniques well known to one of ordinary skill in the art.

Isolation and Characterization of the Protein

In order to grow the SIV/Mne virus, a single cell clone of Hut-78 cells infected with SIV/-Mne (clone E11S) was cultured for propagation of the virus and the virus then purified by standard sucrose density gradient centrifugation. Viral proteins were then fractionated, isolated and purified by standard procedures well known in the art, including reversed phase high pressure liquid chromatography (RP-HPLC) until a homogeneous preparation was obtained. The protein was then characterized by NH₂ and COOH amino acid sequence analysis as described, for example, by Henderson, et al, J. Virol 52:492 (1984).

The homogenous preparation of the protein, as shown by SDS-PAGE analysis (Fig. 1(a), lane 2) was inert to Edman degradation (gas phase sequencer) suggesting that it had a derivatized NH₂- terminal residue (blocked NH₂-terminus). To obtain amino acid sequence, the protein was digested with trypsin and the purified peptide fragments thus obtained (Fig. 2(a), "a" through "1") were subjected to analysis for determining amino acid composition and sequence [Fig. 2(b)], The determined amino acid sequences and compositions were compared to the translated proviral DNA sequence of SIV/mac and HIV-2 and found to be highly homologous to predicted sequences located in the X-ORF of each virus. The SIV/Mne p14 peptides [Fig. 2(b)] align with residues predicted by the X-ORF of HIV-2 starting at position 2 and continue through position 112 except that peptides corresponding to predicted residues 69 through 70 and 85 through 88 were not isolated. Of the 105 amino acid residues of SIV/Mne p14 that were determined by analysis of purified peptides, 90 were identical to predicted residues in the HIV-2 X-ORF (86% identity) and 103 were found to be identical to the predicted residues in SIVmac X-ORF (98% identity). The amino acid sequence of SIV/Mne p14 differs from the sequence predicted by the SIV/mac X-ORF in that the SIV/Mne protein does not contain aspartic acid at position 3 and has- valine substituted for threonine at position 67. In addition, the mature p14 protein has undergone modifications resulting in removal of the initiator methionine and addition of a blocking group to the newly formed NH₂-terminal group. The nature of the modifying group remains to be determined. The COOH-terminal residues of SIV/Mne p14 were determined by the rate of release of residues by digestion with carboxypeptidase-P [2 min., Ala (0.30), Leu (0.22); 20 min., Ala (0.40), Leu (p.47)]. These results are consistent with a COOH-terminal amino acid sequence of -Leu-Ala-OH and are in agreement with the sequence deduced from the last two codons of the X-ORFs of HIV-2 and SIV/mac.

The degree of amino acid sequence identity between the protein predicted by the X-ORF of SIV/mac and SIV/Mne p14 (98% identities) is greater than the degree of identity found for proteins derived from the gag gene (92% identity) and also greater than the degree of identity found between the two viruses for a 1.6 Kb proviral DNA fragment including the 3'-LTR (93% identities). These results indicate that the X-ORF protein is highly conserved among SIV strains.

To estimate the molar amount of p14 present in the viral preparation and to relate this amount to the molar amounts of other known viral proteins, the p18, p16, p14, and p8 bands were cut from the gel shown in Fig. 1(a) (lane 1) and their protein content determined by amino acid analysis as described by Henderson, et al,

J. Virol 52, 492 (1984). The results were in agreement with the known amino acid content of each protein and showed that the gel contained the proteins in the following molar ratios: 1.0 : 1.2 : 1.1 : 0.8 (p28 : p16 : p8

: p14). Several preparations of SIV/Mne, including viruses obtained from an infectious molecular clone, were examined by visual inspection of bands after SDS PAGE and found to contain similar ratios of p14 to p16 as shown in Fig. 1(a) (lane 1).

A polyclonal rabbit antiserum to purified SIV/Mne p14 was used to detect p14 in SIV/Mne and probe for cross-reactive proteins in HIV-1, HIV-2 and SIV/mac by immunoblot anlaysis [Fig. 1(c)]. The serum detected a 14kD band in SIV/Mne (lane 3) and SIV/mac (lane 4) and 16kD band in HIV-2 (lane 2), but did not appear to react significantly with proteins in HIV-1 (lane 1). The detected antigen in SIV/mac has the same mobility in SDSPAGE as SIV/Mne p14. This evidence leads to the conclusion that p14 is the translational product of the SIV/mac X-ORF gene. The detected antigen in HIV-2 (p16) has a slower mobility in SDS-PAGE than the p14s of SIV/Mne and SIV/mac (Fig. 1C). However, SIV isolates from other primate species appear to have cross-reactive proteins with mobilities more similar to the protein detected in HIV-2 (data not shown). Without being bound to any theory, it is believed that the observed difference in the SDS-PAGE mobilities is probably due to differences in amino acid compositions of the proteins rather than differences in actual molecular weights. In any event, the data support the conclusion that the X-ORF translational products of HIV-2 and SIV/mac are found in the purified viruses and that HIV-1 does not contain a similar crossreactive protein.

To visualize and estimate the amounts of viral proteins in each viral preparation a SDS-PAGE gel, identical to that used for immunoblotting, was stained with coomassie brilliant blue [Fig. 1(b)]. Coomassie stained bands at 14kD and 16kD (p14 and p16-gag) are readily apparent in SIV/Mne (lane 3) and SIV/mac (lane 4); however in HIV-2 (lane 2) the 14kD band is absent and there is a prominent 16kD band. The 16kD band in the HIV-2 lane contains both p16 gag (data not shown) and the X-ORF protein. The staining intensities of the major gag proteins (HIV-1 p24 and p17, HIV2 p26 and SIV p28s and p16s) may be taken as relative indicators of the amount of virus applied to each lane. A comparison of the intensities of the bands in the immunoblot analysis [Fig. 1(c)] to the intensifies of the coomassie stained bands [Fig. 1(b)] suggests that SIV/mac and HIV-2 may contain as much of the translational products from their X-ORFs as is observed for p14 in SIV/Mne.

The established structure and genetic origin of p14 as demonstrated here shows that the X-ORF functions as a gene in SIV/Mne. Since the gene and gene product (p14) were first recognized in SIV/Mne and appear unique to simian immunodeficiency (sid) and closely related virsues, this gene is herein designated as the sid gene.

The amino acid sequences of SIV and HIV-2 sid proteins contain conserved cysteine residues in positions 73, 87 and 89 and a histidine residue in position 82 [Fig. 2(b)]. Similar cysteine-histidine motifs are believed to play a role in the nucleic acid binding properties of retroviral [Henderson, et al, J. Biol. Chem. 256, 8400 (1981)] and other proteins [J. Berg, Science 232, 485 (1986)]. Purified p14 was tested for nucleic acid binding activity by the method of fluorescence enhancement using polyethenoadenine as a single stranded polynucleotide template [Karpel, et al, J. Biol. Chem. 262, 4961 (1987)]. Figure 3 shows the titration curve obtained by adding p14 to a solution of polyethenoadenine. Also included in Fig. 3 are the titration results obtained for SIV/Mne p8 gag nucleocapsid protein and p28 gag. The data show that p14 and p8 bind to the template and indicate that these proteins are capable of binding to a single stranded RNA. The shape of the titration curve is dependent upon the degree and nature of cooperative binding which is different for each protein. The capacity of p14 to bind to single stranded nucleic acids may in part account for its apparent high concentration in the purified virus. However, the fact that p14 is capable of binding to single stranded nucleic acids suggests that it may function in vivo as a specific RNA binding protein.

Because the sid protein is unique to the HIV-2 and SIV group of viruses, diagnostic procedures utilizing this protein are developed to unequivocally distinguish between HIV-1, HIV-2 and strains of SIV. A diagnostic kit comprises containers separately containing sid protein and anti-p14 antibodies, the latter reacting only with those viruses which have antigenic components homologous, in whole or in part, to the sid protein. Since the HIV-1 strains do not have antigenic components which cross-react with sid antibodies, a positive antigen-antibody reaction between a biological sample and the components of the diagnostic kit (antibody or antigen) indicates the presence of a virus which has "sid" homology.

UTITLITY OF THE PRESENT INVENTION

A. DIAGNOSIS OF SIV/HIV-2 INFECTION

1. Detection of antibodies with protein from virus.

a. As illustrated herein above, the sid protein can be purified from any strain of the SIV/HIV-2 group by chromatography such as RP-HPLC or any other suitable protein purification method offering sufficient resolving power. The purified protein is then used to detect specific antibodies in patients' blood (sera) employing ELISA or similar techniques. The sensitivity of this method is below the microgram range and nanogram quantities will be sufficient for a single test. Current available diagnostic tests do not readily differentiate between HIV-1 and HIV-2 infection. In contrast, the sid protein provides an unequivocal differential diagnosis of HIV2. False positives are virtually eliminated when purified protein or its fragments are used. False negatives may appear oanly at very early stages of infection due to the absence of antibodies. But once immunreactive amounts of antibodies are produced, false negatives are not probable.

b. The present invention now allows sensitive diagnostic kits. A kit having sid protein or its fragments as one of the components can identify any virus which has a domain reactive with the sid protein.

2. Recombinant sid protein

The sid gene of the SIV/HIV-2 group of virus can be cloned by routine laboratory techniques. The complete gene or its fragments can be expressed in either prokaryotic or eukaryotic expression vectors and the products used for diagnostic tests as described herein supra. 3. Synthetic Protein

Sid protein or fragments of sid protein can be synthesized by conventional techniques of peptide synthesis and the resultant products used in diagnostic tests to detect antibody in patient sera as described herein supra.

B. TESTS FOR THE DIFFERENTIATION OF NEW VIRUS ISOLATES

1. Monoclonal or polyclonal antibodies specific for sid protein can be prepared with any of the available technology. The antibody can be used for identification and typing of new isolates from any animal species including humans, employing western blot analysis or other suitable detection techniques for the presence of sid protein in the virus isolate. Localization of the site of infection can be achieved by immunohistological, immunoradiological and other methods. Such techniques are well known to one of ordinary skill in the art. The results readily determine whether the new virus is a member of the SIV/HIV-2 group and whether the infection is due to HIV-2 or SIV. Viruses which belong to HIV-1 group will not show positive reaction.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

HIV-2_ROD sid GENE SEQUENCE

atgacagaccccagagagacagtaccaccaggaaacagcggcgaagagactatcgga gaggccttcgcctggctaaacaggacagtagaagccataaacagagaagcagtgaat cacctaccccgagaacttattttccaggtgtggcagaggtcctggagatactggcat gatgaacaagggatgtcagaaagttacacaaagtatagatatttgtgcataatacag aaagcagtgtacatgcatgttaggaaagggtgtacttgcctggggaggggacatggg ccaggagggtggagaccagggcctcctcctcctccccctccaggtctggtctaa

SIV_Mac sid GENE SEQUENCE

ATGtcaga tcccagggag agaatcccac ctggaaacag tggagaagag acaataggag aagccttcga gtggctaaac agaacagtag aggagataaa cagagaggcg gtaaacca cc taccgaggga gctaattttc caggtttggc aaaggtcttg ggaatactgg caTGA tgaac aagggatgtc acaaagctat acaaaataca gatacttgtg tttaatacaa aa ggctttat ttatgcattg caagaaagga tgtagatgta taggggaagg acacggggca gggggatgga gaccaggacc tcctcctcct ccccctccag gactagca TA.

Claims

WHAT IS CLAIMED IS:

1. An isolated, substantially pure protein which, in whole or in part, possesses specific binding affinity for antibody to sid protein, said protein being without cross-reactivity with antibodies to the HIV-1 group of viruses.

2. The protein of claim 1 having the following amino acid sequence before posttranslational modification to remove the first residue (Met) and attachment of a blocking group:

10

Het-Ser-Asp-Pro-Arg-GlU-Arg-Ile-Pro-Pro-Gly-Asn-Ser-Gly-Glu-Glu-Thr-

20 30

Ile-Gly-Glu-Ala-Phe-Glu-Trp-Leu-Asn-Arg-Thr--Val-Glu-Glu-Ile-Asn-Arg-

40 50

Glυ-Ala-Val-Asn-His-Leu-Pro-Arg-Glu-Leυ-Il e-Phe-Gln-Val-Trp-Gln-Arg-

60

Ser-Trp-Glu-Tyr-Trp-His-Asp-GTu-Gln-Gly-Het-Ser-Gln-Ser-Tyr-Thr-Lys-

70 80

Tyr-Arg-Tyr-Leυ-Cys-Leu-Ile-Gln-LyS-Ala-Leu-Phe-Het-His-Cys-LyS-Lys-

90 100

Gly-Cys-Arg-Cys-Leu-Gly-Glu-Gly-His-Gly-Ala-Gly-Gly-Trp-Arg-Pro-Gly-

110

Pro-Pro-Pro-Pro-Pro-Pro-Pro-Gly-Leu-Ala-OH

SUBSTITUTE S f_τ££-

3. Isolated, substantially pure antibody having specific binding affinity for sid protein and being without cross-reactivity with antigens of the HIV-1 group of viruses.

4. A diagnostic kit for differential identification of viral infection, comprising containers separately containing isolated, substantially pure sid protein and anti-sid antibodies.

5. A method for differentiating between HIV-2, SIV and HIV-1 group of viruses, comprising reacting a biological sample with an imunoreactive amount of substantially pure sid-antigen, a positive immunological reaction being indicative of the presence of only HIV-2 or SIV viral infection.

6. The method of claim 5 wherein said immunological reaction is either histological or in vitro.

7. A coding sequence for translational expression of sid protein having, in whole or in part, the following nucleotide sequence: ATGtcaga tcccagggag agaatcccac ctggaaacag tggagaagag acaataggag aagccttcga gtggctaaac agaacagtag aggagataaa cagagaggcg gtaaacca cc taccgaggga gctaattttc caggtttggc aaaggtcttg ggaatactgg caTGA tgaac aagggatgtc acaaagctat acaaaataca gatacttgtg tttaatacaa aa ggctttat ttatgcattg caagaaaggc tgtagatgtc taggggaagg acacggggca gggggatgga gaccaggacc tcctcctcct ccccctccag gactagca TA.

8. The protein of claim 1 having the following amino acid sequence before posttranslational modification to remove the first residue (Met) and attachment of a blocking group:

10

Het-Thr-Asp-Pro-Arg-Glu-Thr-Val-Pro-Pro-Gly-Asn-Ser-Gly-Glu-Glu-Thr-

20 30

Ile-Gly-Glu-Ala-Phe-Ala-Trp-Leu-Asn-Arg-Thr-Val-Glu-Ala-Ile-Asn-Arg-

40 50

Glu-Ala-Val-Asn-His-Leυ-Pro-Arg-Glu-Leu-Ile-Phe-Gln-Val-Trp-Gln-Arg-

60

Ser-Trp-Arg-Tyr-Trp-His-Asp-Glu-Gln-Gly-Het-Ser-Glu-Ser-Tyr-Thr-Lys-

70 80

Tyr-Arg-Tyr-Leu-Cys-Ile-Ile-Gln-Lys-Ala-Val-Tyr-Het-His-Val-Arg-Lys-

90 100

Gly-Cys-Thr-Cys-Leu-Gly-Arg-Gly-His-Gly-Pro-Gly-Gly-Trp-Arg-Pro-Gly-

110

Pro-Pro-Pro-Pro-Pro-Pro-Pro-Gly-Leu-Val-OH

9. A coding sequence for translational expression of sid protein having, in whole or in part, the following nucleotide sequence: atgacagaccccagagagacagtaccaccaggaaacagcggcgaagagactatcgga gaggccttcgcctggctaaacaggacagtagaagccataaacagagaagcagtgaat cacctaccccgagaacttattttccaggtgtggcagaggtcctggagatactggcat gatgaacaagggatgtcagaaagttacacaaagtatagatatttgtgcataatacag aaagcagtgtacatgcatgttaggaaagggtgtacttgcctggggaggggacatggg ccaggagggtggagaccagggcctcctcctcctccccctccaggtctggtctaa