Acid-labile formylation of amino terminal proline of human immunodeficiency virus type 1 p24(gag) was found by proteomics using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

HIV-1(LAV-1) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified human immunodeficiency virus type 1 (HIV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained, and the stained spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-five proteins were identified as the proteins inside the virion and the acid-labile formyl group of an amino terminal proline residue of HIV-1(LAV-1) p24(gag) was determined by MALDI-TOF MS before and after weak-acid treatments (0.6 N hydrochloric acid) and confirmed by post-source decay (PSD) of the N-formylated N-terminal tryptic peptide (N-formylated Pro(1)-Arg(18)). The role of formylation has been unclear so far, but it is surmised that the acid-labile formylation of HIV-1(LAV-1) p24(gag) may play a critical role in the formation of the HIV-1 core for conferring HIV-1 infectivity.     

Persistent infection of chimpanzees with human immunodeficiency virus: serological responses and properties of reisolated viruses.

Persistent infection by human immunodeficiency virus (HIV-1) in the chimpanzee may be valuable for immunopathologic and potential vaccine evaluation. Two HIV strains, the tissue culture-derived human T-cell lymphotropic virus type IIIB (HTLV-IIIB) and in vivo serially passaged lymphadenopathy-associated virus type 1 (LAV-1), were injected intravenously into chimpanzees. Two animals received HTLV-IIIB as either virus-infected H9 cells or cell-free virus. A third animal received chimpanzee-passaged LAV-1. Evaluation of their sera for virus-specific serologic changes, including neutralizations, was done during a 2-year period. During this period all animals had persistently high titers of antibodies to viral core and envelope antigens. All three animals developed a progressively increasing type-specific neutralizing LAV-1 versus HTLV-IIIB antibody titer during the 2-year observation period which broadened in specificity to include HTLV-HIRF, HTLV-IIIMN, and HTLV-IIICC after 6 to 12 months. The antibody titers against both viruses were still increasing by 2 years after experimental virus inoculation. Sera from all animals were capable of neutralizing both homologously and heterologously reisolated virus from chimpanzees. A slightly more rapid type-specific neutralizing response was noted for the animal receiving HTLV-IIIB-infected cells compared with that for cell-free HTLV-IIIB. Sera from all persistently infected chimpanzees were capable of mediating group-specific antibody-mediated complement-dependent cytolysis of HIV-infected cells derived from all isolates tested. Viruses reisolated from all three animals at 20 months after inoculation revealed very similar peptide maps of their respective envelope gp120s, as determined by two-dimensional chymotrypsin oligopeptide analysis. One peptide, however, from the original HTLV-IIIB-inoculated virus was deleted in viruses from all three animals, and in addition, we noted the appearance of a new or modified peptide which was common to LAV-1 as well as to HTLV-IIIB reisolated from infected chimpanzees. It thus appears that a group-specific neutralizing antibody response as well as a group-specific cytotoxic response can develop in chimpanzees after an inoculation of a single HIV variant. This finding suggests that a common, less immunodominant determinant(s) is present on a single HIV strain which could induce group-specific antibodies during viral infection and replication. The identification of this group-specific epitope and the induction of analogous immunity may be relevant to vaccine development against human acquired immunodeficiency syndrome.     

Development of HIV/AIDS vaccine using chimeric gag-env virus-like particles

We attempted to develop a candidate HIV/AIDS vaccine, by using unprocessed HIV-2 gag pr45 precursor protein. We found that a 45 kDa unprocessed HIV-2 gag precursor protein (pr45), with a deletion of a portion of the viral protease, assembles as virus-like particles (VLP). We mapped the functional domain of HIV-2 gag VLP formation in order to find the minimum length of gag protein to form VLP. A series of deletion mutants was constructed by sequentially removing the C-terminal region of HIV-2 gag precursor protein and expressed truncated genes in Spodoptera frugiperda (SF) cells by infecting recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not affect VLP formation. There is a proline-rich region at the amino acid positions 373 to 377 of HIV-2 gag, and replacement of these proline residues by site-directed mutagenesis completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein, and zinc finger domains, are dispensable for gag VLP assembly, but the presence of at least one of the three prolines at amino acid positions 373, 375 or 377 of HIV-2NIH-Z is required for VLP formation. Animals immunized with these gag particles produced high titer antibodies and Western blot analyses showed that anti-gag pr45 rabbit sera react with p17, p24 and p55 gag proteins of HIV-1. We then constructed chimeric gag genes, which carry the hypervariable V3 region of HIV-1 gp120, because the V3 loop is known to interact with chemokine receptor as a coreceptor, and known to induce the major neutralizing antibodies and stimulate the cytoxic T lymphocyte responses in humans and mice. We expressed chimeric fusion protein of HIV-2 gag with 3 tandem copies of consensus V3 domain that were derived from 245 different isolates of HIV-1. In addition, we also constructed and expressed chimeric fusion protein that contains HIV-2 gag with V3 domains of HIV-1IIIB, HIV-1MN, HIV-1SF2 and HIV-1RF. The chimeric gag-env particles had a spherical morphology, and the size was slightly larger than that of a gag particle. Immunoprecipitation and Western blot analyses show that these chimeric proteins were recognized by HIV-1 positive human sera and antisera raised against V3 peptides, as well as by rabbit anti-gp120 serum. We obtained virus neutralizing antibodies in rabbits by immunizing these gag-env VLPs. In addition, we found that gag-env chimeric VLPs induce a strong CTL activity against V3 peptide-treated target cells. Our results indicate that V3 peptides from all major clades of HIV-1 carried by HIV-2 gag can be used as a potential HIV/AIDS vaccine.     

A p6Pol-protease fusion protein is present in mature particles of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) protease (PR) and p6(Pol) are translated as part of the Gag-Pol polyprotein after a ribosomal frameshift. PR is essential to virus replication and is responsible for cleaving Gag and Gag-Pol precursors, but the role of p6(Pol) in HIV-1 infection is poorly understood. Here, we report that (i) PR is present in mature HIV-1 virions primarily as a p6(Pol)-PR fusion protein; (ii) HIV-1 PR cleaves viral precursor proteins expressed in bacterial cells at the Phe-Leu bond (positions 1639 to 1642) located at the junction of the NC and p6(Pol) proteins, releasing the p6(Pol)-PR fusion protein; and (iii) purified p6(Pol)-PR fusion protein undergoes autocleavage in vitro at at least three sites.     

Reversal of P-glycoprotein-mediated multidrug resistance by 5,6,7,3',4'-pentamethoxyflavone (Sinensetin)

Vif, one of the six accessory genes expressed by HIV-1, is essential for the productive infection of natural target cells. Previously we suggested that Vif acts as a regulator of the viral protease (PR): It prevents the autoprocessing of Gag and Gag-Pol precursors until virus assembly, and it may control the PR activity in the preintegration complex at the early stage of infection. It was demonstrated before that Vif, and specifically the 98 amino acid stretch residing at the N'-terminal part of Vif (N'-Vif), inhibits both the autoprocessing of truncated Gag-Pol polyproteins in bacterial cells and the hydrolysis of synthetic peptides by PR in cell-free systems. Linear synthetic peptides derived from N'-Vif specifically inhibit and bind HIV-1 PR in vitro, and arrest virus production in tissue culture. Peptide mapping of N'-Vif revealed that Vif88-98 is the most potent PR inhibitor. Here we report that this peptide inhibits both HIV-1 and HIV-2, but not ASLV proteases in vitro. Vif88-98 retains its inhibitory effect against drug-resistant HIV-1 PR variants, isolated from patients undergoing long-term treatment with anti-PR drugs. Variants of HIV protease bearing the mutation G48V are resistant to inhibition by this Vif-derived peptide, as shown by in vitro assays. In agreement with the in vitro experiments, Vif88-98 has no effect on the production of infectious particles in cells infected with a G48V mutated virus.     

Down-regulation of N-myristoyl transferase expression in human T-cell line CEM by human immunodeficiency virus type-1 infection

The present study focuses on the expression level of N-myristoyl transferase (NMT) in the course of human immunodeficiency virus type-1 (HIV-1) infection. HIV-1 structural proteins were gradually expressed during the process of infection of the human T-cell line CEM, whereas the expression levels of NMT subsequently decreased under the same conditions. In addition, the chronically HIV-1-infected T-cell line CEM/LAV-1 exhibited low expression levels of NMT. We hypothesize that the decrease in the expression level of NMT due to HIV-1 infection may be related to the virus' strategy that leads to its persistent replication.     

Three isoforms of cyclophilin A associated with human immunodeficiency virus type 1 were found by proteomics by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry

Human immunodeficiency virus type 1 (HIV-1) strain LAV-1 (HIV-1(LAV-1)) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified HIV-1(LAV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained. The 2D gel electrophoresis image suggested that 24 proteins can be identified inside the virion. Furthermore, the stained protein spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Peptide mass fingerprinting data suggested that two isoforms of cyclophilin A (CyPA), one with an isoelectric point (pI) of 6.40 and one with a pI of 6.53, are inside the viral membrane; that another isoform, with a pI of 6.88, is outside the viral membrane; and that the CyPA isoform with a pI of 6.53 is N acetylated. The mechanisms that permit the redistribution of CyPA on the viral surface have not yet been clarified, but it is surmised that the CyPA isoform with a pI of 6.88 may play a critical role in the attachment of virions to the surface of target cells and that both CyPA isoforms with pIs of 6.40 and 6.53 may regulate the conformation of the HIV-1 capsid protein.     

Specific reactions between purified HIV-1 particles and CD4+ cell membrane fragments in a cell-free system of virus fusion or entry

The initial step of human immunodeficiency virus type 1 (HIV-1) infection has been studied by Env-mediated fusion or entry assays with appropriate cells expressing CD4 or CXCR4/CCR5 receptors in cultures, where many factors underlying cellular activities likely regulate the fusion/entry efficiency. Here we attempted to develop a more simplified in vitro cell-free fusion/entry reaction that mimics HIV-1 infection in cultures. Membrane fragments of target cells and intact infectious HIV-1 particles were purified, mixed and incubated. The core p24 protein was released from the purified virions and detected by ELISA without detergents in the supernatant of the reaction mixtures. This release reaction proceeded temperature-dependently and in a dose-dependent manner between the virion and membrane fractions, and was specific for HIV-1 Env and CD4. Env-deleted or VSV-G-pseudotyped HIV-1 released little p24, if any. Pretreatment of the membrane fragments with anti-CD4 antibodies inhibited the p24 induction from both X4-tropic and R5-tropic HIV-1. Furthermore, X4 but not R5 HIV-1 reacted with the membrane prepared from intrinsically CXCR4-positive HeLa-CD4 cells, whereas both viruses reacted with that prepared from CCR5-transduced HeLa-CD4 cells, indicating that this cell-free reaction mimics coreceptor usage of HIV-1 infection. Therefore, a potent entry inhibitor of X4 HIV-1, SDF-1alpha, blocked the release from X4 but not R5 HIV-1. Inversely, a weak entry inhibitor of R5 HIV-1, MIP-1beta, partially affected only the release from R5 HIV-1. These results suggest that this cell-free reaction system provides a useful tool to study biochemical fusion/entry mechanisms of HIV-1 and its inhibitors.     

Identification of a key target sequence to block human immunodeficiency virus type 1 replication within the gag-pol transframe domain

Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed. Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3' end of the HIV-1 gag-pol transframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6(Gag) protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNA(PR2). A disrupted translation of gag-pol mRNA induced at the PNA(PR2)-annealing site resulted in a decreased synthesis of Pr160(Gag-Pol) polyprotein, hence the viral protease, a predominant expression of Pr55(Gag) devoid of a fully functional p6(Gag) protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly. Treatment with PNA(PR2) abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype. This particular segment of the gag-pol transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target.     

Anti-HIV-1 activity of an ionically modified porous polypropylene membrane determined by filtration of a viral suspension

We describe here a unique anti-HIV-1 membrane, derived from a chemically modified porous polypropylene (PP) membrane, which lowers viral infectivity upon the filtration of HIV-1 suspension. A cationic polymer, polyethyleneimine (PEI) was graft-polymerized onto the PP filter membrane (PP-PEI), and infectious HIV-1(HTLV-IIIB) derived from MOLT-4/HIV-1(HTLV-IIIB) cells (HIV-1(HTLV-IIIB(MOLT-4)) was applied. When a viral suspension of high titer (10(3.93) TCID50 ml(-1) was filtered, efficient reduction (>99%) of gag p24 antigen levels and infectious titer resulted. In a viral suspension of medium titer (10(2.37) TCID50 ml(-1), a significant decrease in the p24 antigen did not occur, although the titer was markedly reduced (>95%). Electron microscopic observation suggested that PEI induced viral aggregations under high titer conditions, and under medium titer conditions, PEI deprived HIV-1(HTLV-IIIB(MOLT-4)) of its infectivity alone to avoid virus adsorption. In contrast, HIV-1 propagated in human peripheral blood mononuclear cells (PBMC) such as HIV-1(HTLV-III(PBMC)) was more efficiently trapped by PP-PEI at lower titers as compared with HIV-1(HTLV-IIIB(MOLT-4)) from MOLT-4/HIV-1(HTLV-IIIB) cells. These data suggest host cell modification in the interactions between PP-PEI and HIV-1 strains. Since HIV-1(HTLV-IIIB(MOLT-4)) and HIV-1(HTLV-IIIB(PBMC)) were almost electrically neutral and negative, respectively, we concluded that the divergent effect of PEI on each HIV-1(HTLV-IIIB) resulted from their different electrical characteristics.     

Fluoroquinolones protect the human lymphocyte CEM cell line from HIV-1-mediated cytotoxicity

Infection of the human lymphocyte CEM cell line with the HIV-1 (human immunodeficiency virus-1, LAV-1 strain) results in cell death. A fluoroquinolone antibiotic, ofloxacin, protected the infected cells from HIV-1-mediated cytolysis. Other fluoroquinolones, e.g. ciprofloxacin, norfloxacin, and enoxacin, also protected the infected cells from HIV-1-mediated cytolysis. The d-isomer of ofloxacin (DR-3354) was about 50-fold less effective than the l-isomer (DR-3355). Almost none of the rescued cells had detectable HIV-antigens and they could be maintained for long periods in vitro without drugs.     

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.     

Reduced glycosylation of human cell lines increases susceptibility to CD4-independent infection by human immunodeficiency virus type 2 (LAV-2/B).

The human immunodeficiency virus type 2 (HIV-2) strain LAV-2/B is able to infect a variety of human cell lines via a CD4-independent pathway. We have used the glycosylation inhibitors tunicamycin, swainsonine, and deoxymannojirimycin to further characterize this putative alternative receptor for HIV-2 (LAV-2/B). These antibiotics resulted in an increase (5- to 30-fold) in the susceptibility of a variety of CD4- human cell lines to infection by LAV-2/B (RD, HeLa, HT29, Rsb, Heb7a, Hos, and Daudi). Several nonprimate cell lines (mink Mv-1-lu, rabbit SIRC, hamster a23, mouse NIH 3T3, cat CCC, and rat HSN) remained resistant to infection by LAV-2/B after treatment with glycosylation inhibitors, suggesting that they do not express the HIV-2 CD4-independent receptor. Two of these nonprimate cell lines are readily infected by HIV-2 when they express CD4 (Mv-1-lu and CCC). Treatment of human cells with neuraminidase had no effect on subsequent infection by LAV-2/B, suggesting that the increase in susceptibility to infection of deglycosylated cells is not due to a change in the electrostatic charge of the cell surface. Treatment of RD CD4- cells and HeLa CD4+ cells with a variety of proteases resulted in a 75 to 90% decrease in infection by LAV-2/B when compared with untreated cells. Taken together, all these data suggest that HIV-2 can utilize a membrane glycoprotein other than CD4 to attach and fuse with a variety of human cells.     

Antimyristoylation of the gag proteins in the human immunodeficiency virus-infected cells with N-myristoyl glycinal diethylacetal resulted in inhibition of virus production

The gag proteins of human retroviruses such as human immunodeficiency virus (HIV-1) are specifically myristoylated in their amino termini (1, 2, 3). N-myristoyl glycinal diethylacetal (N-Myr-GOA) and other N-Myr-compounds (N-Myr-Gly-GOA, N-Myr-Gly-Gly-GOA and N-Myr-Gly-Gly-Gly-GOA) were newly synthesized and investigated for activity of antimyristoylation of these gag proteins and for influence on viral replication. Of the N-Myr-compounds tested, N-Myr-GOA most severely inhibited the protein myristoylation; N-Myr-Gly-GOA also inhibited it, but moderately. Furthermore, it was observed that N-Myr-GOA at 20 microM caused noticeable inhibition (about 80%) of the production of mature HIV in the HIV-1-infected MT-4 cells. In this system, N-Myr-GOA substantially inhibited more than 90% of the N-myristoylation of p17 gag protein produced in the HIV-1-infected MT-4 cells. These results suggest that the N-myristoylation of p17 gag protein of HIV-1 may be essential in its structural assembly or maturation.     

Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne).

A simian immunodeficiency virus (SIV) designated SIVMne was isolated from a pig-tailed macaque with lymphoma housed at the University of Washington Regional Primate Research Center, Seattle. To better establish the relationship of SIVMne to other immunodeficiency viruses, we purified and determined the partial amino acid sequences of six structural proteins (p1, p2, p6, p8, p16, and p28) from SIVMne and compared these amino acid sequences to the translated nucleotide sequences of SIVMac and human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). A total of 125 residues of SIVMne amino acid sequence were compared to the predicted amino acid sequences of the gag precursors of SIV and HIVs. In the compared regions 92% of the SIVMne amino acids were identical to predicted residues of SIVMac, 83% were identical to predicted residues of HIV-2, and 41% were identical to predicted residues of HIV-1. These data reveal that the six SIVMne proteins are proteolytic cleavage products of the gag precursor (Pr60gag) and that their order in the structure of Pr60gag is p16-p28-p2-p8-p1-p6. Rabbit antisera prepared against purified p28 and p16 were shown to cross-react with proteins of 60, 54, and 47 kilodaltons present in the viral preparation and believed to be SIVMne Pr60gag and intermediate cleavage products, respectively. SIVMne p16 was shown to contain covalently bound myristic acid, and p8 was identified as a nucleic acid-binding protein. The high degree of amino acid sequence homology between SIVs and HIV-2 around proven proteolytic cleavage sites in SIV Pr60gag suggests that proteolytic processing of the HIV-2 gag precursor is probably very similar to processing of the SIV gag precursor. Peptide bonds cleaved during proteolytic processing of the SIV gag precursor were similar to bonds cleaved during processing of HIV-1 gag precursors, suggesting that the SIV and HIV viral proteases have similar cleavage site specificities.     

Characterization of the gag/fusion protein encoded by the defective Duplan retrovirus inducing murine acquired immunodeficiency syndrome.

Murine acquired immunodeficiency syndrome is induced by a defective retrovirus. Sequencing of this defective viral genome revealed a long open reading frame which encodes a putative gag/fusion protein, N-MA-p12-CA-NC-COOH, (D. C. Aziz, Z. Hanna, and P. Jolicoeur, Nature (London) 338:505-508, 1989). We raised a specific antibody to the unique p12 domain of this gag fusion precursor, Pr60gag. We found that Pr60gag was indeed encoded by the defective viral genome both in cell-free translation reticulocyte extracts and in infected mouse fibroblasts. Pr60gag was found to be myristylated, phosphorylated, and attached to the cell membrane, like other helper murine leukemia virus (MuLV) gag precursors. Pr60gag was not substantially cleaved within the nonproducer cells and was not released from these cells. However, in the presence of helper MuLV proteins, it formed phenotypically mixed particles. In these particles, Pr60gag was only partially cleaved. In helper MuLV-producing cells harboring the defective virus, a gag-related p40 intermediate was generated both intracellularly and extracellularly. In these cells, Pr60gag appeared to behave as a dominant negative mutant, interfering with proper cleavage of helper Pr65gag. Our data indicate that Pr60gag is a major (and possibly the only) gene product of the defective murine acquired immunodeficiency syndrome virus and is likely to harbor some determinants of pathogenicity of this virus.     

Cepharanthine inhibited HIV-1 cell–cell transmission and cell-free infection via modification of cell membrane fluidity

The anti-HIV-1 activity of cepharanthine (CEP), a natural product derived from Stephania cepharantha Hayata, was evaluated. CEP stabilized plasma membrane fluidity and inhibited HIV-1 envelope-dependent cell-to-cell fusion of HIV-1-infected cells as well as cell-free infection. It is suggested that CEP inhibited the HIV-1 entry process by reducing plasma membrane fluidity, and the plasma membrane is therefore an identical target to prevent viral infection.