Partial restoration of replication of simian immunodeficiency virus by point mutations in either the dimerization initiation site (DIS) or Gag region after deletion mutagenesis within the DIS.

We used the simian immunodeficiency virus (SIV) molecular clone SIVmac239 to generate a deletion construct, termed SD2, in which we eliminated 22 nucleotides at positions +398 to +418 within the putative dimerization initiation site (DIS) stem. This SD2 deletion severely impaired viral replication, due to adverse effects on the packaging of viral genomic RNA, the processing of Gag proteins, and viral protein patterns. However, long-term culture of SD2 in either C8166 or CEMx174 cells resulted in restoration of replication capacity, due to two different sets of three compensatory point mutations, located within both the DIS and Gag regions. In the case of C8166 cells, both a K197R and a E49K mutation were identified within the capsid (CA) protein and the p6 protein of Gag, respectively, while the other point mutation (A423G) was found within the putative DIS loop. In the case of CEMx174 cells, two compensatory mutations were present within the viral nucleocapsid (NC) protein, E18G and Q31K, in addition to the same A423G substitution as observed with C8166 cells. A set of all three mutations was required in each case for restoration of replication capacity, and either set of mutations could be substituted for the other in both the C8166 and CEMx174 cell lines.

Hydrophobic amino acids in the human immunodeficiency virus type 1 p2 and nucleocapsid proteins can contribute to the rescue of deleted viral RNA packaging signals.

An RNA fragment of 75 nucleotides, which is located between the primer binding site and the 5' major splice donor site in human immunodeficiency virus type 1, has been shown to participate in specific encapsidation of viral RNA. Compensation studies have identified two second-site mutations, namely, MP2 (a T12I substitution in p2) and MNC (a T24I substitution in the nucleocapsid [NC] protein) that were involved in the rescue of various deletions in the aforementioned RNA region (i.e., BH-D1, BH-D2, and BH-LD3). To study whether the MP2 and MNC point mutations exert their compensatory effects in a cis manner, production of Gag proteins was blocked by insertion of stop codons into LD3, LD3-MP2-MNC, and wild-type BH10 such that the constructs generated, i.e., LD3-DG, LD3-MP2-MNC-DG, and BH-DG, only provided RNA transcripts for packaging. The results of cotransfection experiments showed that the LD3-MP2-MNC-DG viral RNA was packaged as inefficiently as LD3-DG; in contrast, BH-DG was efficiently packaged. Therefore, nucleotide substitutions in MP2 and MNC did not act in a cis manner to correct the packaging deficits in LD3. Next, we deliberately changed the T12 in p2 or the T24 in the NC to each of 19 other amino acids. We found that amino acids with long hydrophobic side chains, i.e., V, L, I, and M, were favored at either position 12 in p2 or at position 24 in NC to compensate for the above-mentioned deletions. Further studies showed that only a few amino acids could not be used at these two sites by the wild-type virus due to decreased RNA levels in the virion or abnormal Gag protein processing. In this case, W, D, and E could not substitute for T12 in p2, and S, D, and N could not substitute for T24 in NC, without affecting viral infectivity. Therefore, the long hydrophobic side chains of V, L, I, and M are necessary for these amino acids to rescue the BH-D1, BH-D2, and BH-LD3 mutated viruses.

Role of distal zinc finger of nucleocapsid protein in genomic RNA dimerization of human immunodeficiency virus type 1; no role for the palindrome crowning the R-U5 hairpin.

Genomic RNA isolated from HIV-1 variously mutated in nucleocapsid protein (NC) was characterized by nondenaturing gel electrophoresis. Mutations in the C-terminal, the N-terminal, and the linker regions had no effect on genomic RNA dimerization [they are R7R10K11S, P31L, R32G, S3(32-34), and K59L], while a C36S/C39S mutation in the distal zinc knuckle (Cys-His box or zinc finger) inhibited genome dimerization as much as disrupting the kissing-loop domain. The four mutations which inhibited tRNA(Lys3) genomic placement (i.e., the in vivo placement of tRNA(Lys3) on the primer binding site) had no effect on genome dimerization. Among five mutations which inhibited genome packaging, four had no effect on genome dimerization. Thus the N-terminal and linker regions of NC control genome packaging/tRNA(Lys3) placement (two processes which do not require mature NC) but have little influence on genome dimerization and 2-base extension of tRNA(Lys3) (two processes which are likely to require mature NC). It has been suggested, based on electron microscopy, that the AAGCUU82 palindrome crowning the R-U5 hairpin stimulates genomic RNA dimerization. To test this hypothesis, we deleted AGCU81 from wild-type viruses and from viruses bearing a disrupted kissing-loop hairpin or kissing-loop domain; in another mutant, we duplicated AGCU81. The loss of AGCU81 reduced dimerization by 2.5 +/- 4%; its duplication increased it by 3 +/- 6%. Dissociation temperature was left unchanged. We reach two conclusions. First, the palindrome crowning the R-U5 hairpin has no impact on HIV-1 genome dimerization. Second, genomic RNA dimerization is differentially influenced by NC sequence: it is Zn finger dependent but independent of the basic nature of the N-terminal and linker subdomains. We propose that the NC regions implicated in 2-base extension of tRNA(Lys3) are required for a second (maturation) step of tRNA placement. Genome dimerization and mature tRNA placement would then become two RNA-RNA interactions sharing similar NC sequence requirements.

Novel, live attenuated simian immunodeficiency virus constructs containing major deletions in leader RNA sequences.

We have constructed a series of simian immunodeficiency virus (SIV) mutants containing deletions within a 97-nucleotide (nt) region of the leader sequence. Deletions in this region markedly decreased the replication capacity in tissue culture, i.e., in both the C8166 and CEMx174 cell lines, as well as in rhesus macaque peripheral blood mononuclear cells. In addition, these deletions adversely affected the packaging of viral genomic RNA into virions, the processing of Gag precursor proteins, and patterns of viral proteins in virions, as assessed by biochemical labeling and polyacrylamide gel electrophoresis. Different levels of attenuation were achieved by varying the size and position of deletions within this 97-nt region, and among a series of constructs that were generated, it was possible to rank in vitro virulence relative to that of wild-type virus. In all of these cases, the most severe impact on viral replication was observed when the deletions that were made were located at the 3' rather than 5' end of the leader region. The potential of viral reversion over protracted periods was investigated by repeated viral passage in CEMx174 cells. The results showed that several of these constructs showed no signs of reversion after more than 6 months in tissue culture. Thus, a series of novel, attenuated SIV constructs have been developed that are significantly impaired in replication capacity yet retain all viral genes. One of these viruses, termed SD4, may be appropriate for study with rhesus macaques, in order to determine whether reversions will occur in vivo and to further study this virus as a candidate for attenuated vaccination.

HIV drug resistance and implications for the introduction of antiretroviral therapy in resource-poor countries.

The development and transmission of HIV drug-resistant viruses is of serious concern and has been shown to significantly diminish the effectiveness of antiretroviral therapy. In addition, cross-resistance between drugs of the same class can seriously limit therapeutic options and may potentially be most problematic in resource-poor settings where new drugs are not widely available. Strategies based on avoidance of virological failure are therefore essential for the long-term success of therapy. In this regard, regionally adapted programs to facilitate proper adherence with therapy need to be urgently implemented, concomitant with expanded access to new antiretroviral drugs. The value of genotypic resistance testing as a prognostic tool to help guide therapeutic decisions has been established. However, the relatively high cost of this novel technology does not warrant its routine utilization at this time in resource-poor countries. Lastly, the genetic barrier of the antiretroviral agents that are prescribed is also an important consideration that needs to be integrated with knowledge of HIV-1 subtypes, drug pharmacology, and medical management of concurrent illnesses. The selection of appropriate first-line antiretroviral combination regimens may be an even more important consideration in developing than developed countries, given that options in the aftermath of treatment failure may be more limited in such settings.

Roles of Pr55(gag) and NCp7 in tRNA(3)(Lys) genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1.

To study in vivo tRNA(3)(Lys) genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1, total viral RNA isolated from either wild-type or protease-negative (PR(-)) virus was used as the source of primer tRNA(3)(Lys)/genomic RNA templates in an in vitro reverse transcription assay. At low dCTP concentrations, both the rate and extent of the first nucleotide incorporated into tRNA(3)(Lys), dCTP, were lower with PR(-) than with wild-type total viral RNA. Transient in vitro exposure of either type of primer/template RNA to NCp7 increased PR(-) dCTP incorporation to wild-type levels but did not change the level of wild-type dCTP incorporation. Exposure of either primer/template to Pr55(gag) had no effect on initiation. These results indicate that while Pr55(gag) is sufficient for tRNA(3)(Lys) placement onto the genome, exposure of this complex to mature NCp7 is required for optimum tRNA(3)(Lys) placement and initiation of reverse transcription.

Impact of human immunodeficiency virus type 1 RNA dimerization on viral infectivity and of stem-loop B on RNA dimerization and reverse transcription and dissociation of dimerization from packaging.

The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop or dimerization initiation site (DIS) hairpin (nucleotides [nt] 248 to 270 in the human immunodeficiency virus type 1 strains HIV-1(Lai) and HIV-1(Hxb2)), seated on top of a 12-nt stem-internal loop called stem-loop B (nt 243 to 247 and 271 to 277). Destroying stem-loop B reduced genome dimerization by approximately 50% and proviral DNA synthesis by approximately 85% and left unchanged the dissociation temperature of dimeric genomic RNA. The most affected step of reverse transcription was plus-strand DNA transfer, which was reduced by approximately 80%. Deleting nt 241 to 256 or 200 to 256 did not reduce genome dimerization significantly more than the destruction of stem-loop B or the DIS hairpin. We conclude that the KLD is nonmodular: mutations in stem-loop B and in the DIS hairpin have similar effects on genome dimerization, reverse transcription, and encapsidation and are also "nonadditive"; i.e., a larger deletion spanning both of these structures has the same effects on genome dimerization and encapsidation as if stem-loop B strongly impacted DIS hairpin function and vice versa. A C258G transversion in the palindrome of the kissing-loop reduced genome dimerization by approximately 50% and viral infectivity by approximately 1.4 log. Two mutations, CGCG261-->UUAA261 (creating a weaker palindrome) and a Delta241-256 suppressor mutation, were each able to reduce genome dimerization but leave genome packaging unaffected.

The effect of mutations in the HIV-1 nucleocapsid protein on strand transfer in cell-free reverse transcription reactions.

Interactions between the nucleocapsid protein (NC) and reverse transcriptase of HIV-1 have been shown to promote the initiation of reverse transcription. We assayed the effect of NC on later events, using a strand transfer system with donor and acceptor HIV RNA templates and found that the presence of NC resulted in increased synthesis of full-length strand-transferred (FLST) DNA. This effect also occurred with mutated forms of NC that lacked both zinc fingers, or that contained a point mutation (histidine-->cysteine) at amino acid 23. In contrast, NC-derived proteins containing only the proximal or distal zinc fingers, or lacking the N- and C-termini, were all unable to catalyze the synthesis of FLST DNA. Band-shift assays using both the mutated and wild-type forms of these proteins revealed that all the NC proteins promoted strand association between (-) strong-stop DNA [(-)ssDNA] and acceptor RNA. The zinc finger motifs were dispensable for full-length processive reverse transcription, and the N- and C-termini were required; however, all NC domains were dispensable for association of (-)ssDNA and acceptor RNA. This suggests that annealing is a less stringent reaction than DNA polymerization.

The M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1 impairs rescue of chain-terminated DNA synthesis.

Nucleoside analog chain terminators such as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxy-3'-thiacytidine (3TC) represent an important class of drugs that are used in the clinic to inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1. Recent data have suggested that mutant enzymes associated with AZT resistance are capable of removing the chain-terminating residue with much greater efficiency than wild-type RT and this may, in turn, facilitate rescue of DNA synthesis; these experiments were performed using physiological concentrations of pyrophosphate or nucleoside triphosphates, respectively. The present study demonstrates that the M184V mutation, which confers high-level resistance to 3TC, can severely compromise the removal of chain-terminating nucleotides. Pyrophosphorolysis on 3TC-terminated primer strands was not detectable with M184V-containing, as opposed to wild-type, RT, and rescue of AZT-terminated DNA synthesis was significantly decreased with the former enzyme. Thus, mutated RTs associated with resistance to AZT and 3TC possess opposing, and therefore incompatible, phenotypes in this regard. These results are consistent with tissue culture and clinical data showing sustained antiviral effects of AZT in the context of viruses that contain the M184V mutation in the RT-encoding gene.

Mechanism of action and in vitro activity of 1',3'-dioxolanylpurine nucleoside analogues against sensitive and drug-resistant human immunodeficiency virus type 1 variants.

(-)-Beta-D-1',3'-Dioxolane guanosine (DXG) and 2,6-diaminopurine (DAPD) dioxolanyl nucleoside analogues have been reported to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1). We have recently conducted experiments to more fully characterize their in vitro anti-HIV-1 profiles. Antiviral assays performed in cell culture systems determined that DXG had 50% effective concentrations of 0.046 and 0.085 microM when evaluated against HIV-1(IIIB) in cord blood mononuclear cells and MT-2 cells, respectively. These values indicate that DXG is approximately equipotent to 2', 3'-dideoxy-3'-thiacytidine (3TC) but 5- to 10-fold less potent than 3'-azido-2',3'-dideoxythymidine (AZT) in the two cell systems tested. At the same time, DAPD was approximately 5- to 20-fold less active than DXG in the anti-HIV-1 assays. When recombinant or clinical variants of HIV-1 were used to assess the efficacy of the purine nucleoside analogues against drug-resistant HIV-1, it was observed that AZT-resistant virus remained sensitive to DXG and DAPD. Virus harboring a mutation(s) which conferred decreased sensitivity to 3TC, 2',3'-dideoxyinosine, and 2',3'-dideoxycytidine, such as a 65R, 74V, or 184V mutation in the viral reverse transcriptase (RT), exhibited a two- to fivefold-decreased susceptibility to DXG or DAPD. When nonnucleoside RT inhibitor-resistant and protease inhibitor-resistant viruses were tested, no change in virus sensitivity to DXG or DAPD was observed. In vitro drug combination assays indicated that DXG had synergistic antiviral effects when used in combination with AZT, 3TC, or nevirapine. In cellular toxicity analyses, DXG and DAPD had 50% cytotoxic concentrations of greater than 500 microM when tested in peripheral blood mononuclear cells and a variety of human tumor and normal cell lines. The triphosphate form of DXG competed with the natural nucleotide substrates and acted as a chain terminator of the nascent DNA. These data suggest that DXG triphosphate may be the active intracellular metabolite, consistent with the mechanism by which other nucleoside analogues inhibit HIV-1 replication. Our results suggest that the use of DXG and DAPD as therapeutic agents for HIV-1 infection should be explored.

Anti-human immunodeficiency virus type 1 activity, intracellular metabolism, and pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine.

The racemic nucleoside analogue 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC) is in clinical development for the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection. dOTC is structurally related to lamivudine (3TC), but the oxygen and sulfur in the furanosyl ring are transposed. Intracellular metabolism studies showed that dOTC is phosphorylated within cells via the deoxycytidine kinase pathway and that approximately 2 to 5% of dOTC is converted into the racemic triphosphate derivatives, which had measurable half-lives (2 to 3 hours) within cells. Both 5'-triphosphate (TP) derivatives of dOTC were more potent than 3TC-TP at inhibiting HIV-1 reverse transcriptase (RT) in vitro. The K(i) values for dOTC-TP obtained against human DNA polymerases alpha, beta, and gamma were 5,000-, 78-, and 571-fold greater, respectively, than those for HIV RT (28 nM), indicating a good selectivity for the viral enzyme. In culture experiments, dOTC is a potent inhibitor of primary isolates of HIV-1, which were obtained from antiretroviral drug-naive patients as well as from nucleoside therapy-experienced (3TC- and/or zidovudine [AZT]-treated) patients. The mean 50% inhibitory concentration of dOTC for drug-naive isolates was 1.76 microM, rising to only 2.53 and 2.5 microM for viruses resistant to 3TC and viruses resistant to 3TC and AZT, respectively. This minimal change in activity is in contrast to the more dramatic changes observed when 3TC or AZT was evaluated against these same viral isolates. In tissue culture studies, the 50% toxicity levels for dOTC, which were determined by using [(3)H]thymidine uptake as a measure of logarithmic-phase cell proliferation, was greater than 100 microM for all cell lines tested. In addition, after 14 days of continuous culture, at concentrations up to 10 microM, no measurable toxic effect on HepG2 cells or mitochondrial DNA replication within these cells was observed. When administered orally to rats, dOTC was well absorbed, with a bioavailability of approximately 77%, with a high proportion (approximately 16.5% of the levels in serum) found in the cerebrospinal fluid.

Reverse transcriptase inhibitors can selectively block the synthesis of differently sized viral DNA transcripts in cells acutely infected with human immunodeficiency virus type 1.

We have recently reported that the in vitro inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcription by inhibitors of reverse transcriptase (RT) occurred most efficiently when the expected DNA products of RT reactions were long (Quan et al. , Nucleic Acids Res. 26:5692-5698, 1998). Here, we have used a quantitative PCR to analyze HIV-1 reverse transcription within acutely infected cells treated with RT inhibitors. We found that levels of minus-strand strong-stop DNA [(-)ssDNA] formed in acutely infected MT2 cells were only slightly reduced if cells were infected with viruses that had been generated in the presence of either azidothymidine or nevirapine (5 microM) and maintained in the presence of this drug throughout the viral adsorption period and thereafter. Control experiments in which virus inoculation of cells was performed at 4 degrees C, followed directly by cell extraction, showed that less than 1% of total (-)ssDNA within acutely infected cells was attributable to its presence within adsorbed virions. In contrast, synthesis of intermediate-length reverse-transcribed DNA products decreased gradually as viral DNA strand elongation took place in the presence of either of these inhibitors. This establishes that nucleoside and nonnucleoside RT inhibitors can exert similar temporal impacts in regard to inhibition of viral DNA synthesis. Generation of full-length viral DNA, as expected, was almost completely blocked in the presence of these antiviral drugs. These results provide insight into the fact that high concentrations of drugs are often needed to yield inhibitory effects in cell-free RT assays performed with short templates, whereas relatively low drug concentrations are often strongly inhibitory in cellular systems.

Mutations within four distinct gag proteins are required to restore replication of human immunodeficiency virus type 1 after deletion mutagenesis within the dimerization initiation site.

Human immunodeficiency virus type 1 (HIV-1) genomic RNA segments at nucleotide (nt) positions +240 to +274 are thought to form a stem-loop secondary structure, termed SL1, that serves as a dimerization initiation site for viral genomic RNA. We have generated two distinct deletion mutations within this region, termed BH10-LD3 and BH10-LD4, involving nt positions +238 to +253 and +261 to +274, respectively, and have shown that each of these resulted in significant diminutions in levels of viral infectiousness. However, long-term culture of each of these viruses in MT-2 cells resulted in a restoration of infectiousness, due to a series of compensatory point mutations within four distinct proteins that are normally cleaved from the Gag precursor. In the case of BH10-LD3, these four mutations were MA1, CA1, MP2, and MNC, and they involved changes of amino acid Val-35 to Ile within the matrix protein (MA), Ile-91 to Thr within the capsid (CA), Thr-12 to Ile within p2, and Thr-24 to Ile within the nucleocapsid (NC). The order in which these mutations were acquired by the mutated BH10-LD3 was MNC > CA1 > MP2 > MA1. The results of site-directed mutagenesis studies confirmed that each of these four substitutions contributed to the increased viability of the mutated BH10-LD3 viruses and that the MNC substitution, which was acquired first, played the most important role in this regard. Three point mutations, MP2, MNC, and MA2, were also shown to be sequentially acquired by viruses that had emerged in culture from the BH10-LD4 deletion. The first two of these were identical to those described above, while the last involved a change of Val-35 to Leu. All three of these substitutions were necessary to restore the infectiousness of mutated BH10-LD4 viruses to wild-type levels, although the MP2 mutation alone, but neither of the other two substitutions, was able to confer some viability on BH10-LD4 viruses. Studies of viral RNA packaging showed that the BH10-LD4 deletion only marginally impaired encapsidation while the BH10-LD3 deletion caused a severe deficit in this regard.

Effect of insulin-like growth factor I on HIV type 1 long terminal repeat-driven chloramphenicol acetyltransferase expression.

In this study, we have investigated the ability of insulin-like growth factor I (IGF-I) to inhibit HIV long terminal repeat (LTR)-driven gene expression. Using COS 7 cells cotransfected with tat and an HIV LTR linked to a chloramphenicol acetyltransferase (CAT) reporter, we observed that physiological levels of IGF-I (10(-9) M) significantly inhibited CAT expression in a concentration- and time-dependent manner. IGF-I did not inhibit CAT expression in COS 7 cells transfected with pSVCAT, and did not affect CAT expression in the absence of cotransfection with tat. Transfection of HIV-1 proviral DNA into COS 7 cells +/- IGF-I resulted in a significant decrease (p < 0.05) in infectious virion production. Both IGF-I and Ro24-7429 inhibited LTR-driven CAT expression, while TNF-alpha-enhanced CAT expression was not affected by IGF-I. On the other hand, a plasmid encoding parathyroid hormone-related peptide exhibited dramatic additivity of inhibition of CAT expression in COS 7 cells. Finally, we show that in Jurkat or U937 cells cotransfected with HIVLTRCAT/tat, IGF-I significantly inhibited CAT expression. Further, interleukin 4 showed in U937 cells inhibition of CAT expression that was not additive to IGF-I induced inhibition. Our data demonstrate that IGF-I can specifically inhibit HIVLTRCAT expression. This inhibition may occur at the level of the tat/TAR interaction. Finally, this IGF-I effect is seen in target cell lines and similar paths of inhibition may be involved in the various cell types employed.

Deletion mutagenesis within the dimerization initiation site of human immunodeficiency virus type 1 results in delayed processing of the p2 peptide from precursor proteins.

Previous work has shown that deletions of genomic segments at nucleotide (nt) positions +238 to +253, i.e., construct BH10-LD3, or nt positions +261 to +274, i.e., construct BH10-LD4, within the human immunodeficiency virus type 1 (HIV-1) dimerization initiation site (DIS) destroyed DIS secondary structure and dramatically reduced viral replication capacity. Surprisingly, two point mutations located within the viral peptide 2 (p2) and nucleocapsid (NC) protein termed MP2 and MNC, respectively, were able to compensate for this defect. Since the MP2 mutation involves an amino acid substitution near the cleavage site between p2 and NC, we investigated the effects of the above-mentioned deletions on the processing of Gag proteins. Immunoprecipitation assays performed with monoclonal antibodies against viral capsid (CA) (p24) protein showed that p2 was cleaved from CA with less efficiency in viruses that contained the LD3 and LD4 deletions than in wild-type viruses. The presence of the two compensatory mutations, MP2 and MNC, increased the efficiency of the cleavage of p2 from CA, but neither mutation alone had this effect or was sufficient to compensate for the observed impairment in infectiousness. A virus that contained both of the above-mentioned deletions within the DIS was also impaired in regard to processing and infectiousness, and it could likewise be compensated by the MP2 and MNC point mutations. These results suggest that the DIS region of HIV-1 RNA plays an important role in the processing of Gag proteins.

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.

Heat shock protein-based therapeutic strategies against human immunodeficiency virus type 1 infection.

Heat shock proteins (hsps) and cyclophilins (CypA) are intracellular chaperone molecules that facilitate protein folding and assembly. These proteins are selectively expressed in cells following exposure to a range of stress stimuli, including viral infection. Hsp species are highly immunogenic, eliciting humoral, cytotoxic T lymphocyte (CTL), and natural killer (NK) cell responses against viruses, tumours, and infectious diseases. This review discusses the roles of stress proteins in immunity and viral life cycles, vis-à-vis the development of Hsp-based therapeutic strategies against human immunodeficiency virus type-1 (HIV-1) infection. Cumulative findings are cited implicating the requirement of CypA in HIV-1 replication and formation of infectious virions. Studies by our group show the upregulated expression of hsp27 and hsp70 during single-cycle HIV infections. These species redistribute to the cell surface following HIV-infection and heat stress, serving as targets for NK and antibody-dependent cellular cytotoxicity. Co-immunoprecipitation and Western blot studies show that hsp27, hsp70, and hsp78 complex with HIV-1 viral proteins intracellularly. Hsp70, hsp56, and CypA are assembled into HIV-1 virions. The ability of hsps to interact with HIV-1 viral proteins, combined with their inherent adjuvant and immunogenic properties, indicates that hsps may serve as vehicles for antigen delivery and the design of vaccines against acquired immunodeficiency syndrome.

Temporal coordination between initiation of HIV (+)-strand DNA synthesis and primer removal.

In this study, we have analyzed the interdependence between the polymerase and RNase H active sites of human immunodeficiency virus-1 reverse transcriptase (RT) using an in vitro system that closely mimics the initiation of (+)-strand DNA synthesis. Time course experiments show that RT pauses after addition of the 12th DNA residue, and at this stage the RNase H activity starts to cleave the RNA primer from newly synthesized DNA. Comparison of cleavage profiles obtained with 3'- and 5'-end-labeled primer strands indicates that RT now translocates in the opposite direction, i.e. in the 5' direction of the RNA strand. DNA synthesis resumes again in the 3' direction, after the RNA-DNA junction was efficiently cleaved. Moreover, we further characterized complexes generated before, during, and after position +12, by treating these with Fe2+ to localize the RNase H active site on the DNA template. Initially, when RT binds the RNA/DNA substrate, oxidative strand breaks were seen at a distance of 18 base pairs upstream from the primer terminus, whereas 17 base pairs were observed at later stages when the enzyme binds more and more DNA/DNA. These data show that the initiation of (+)-strand synthesis is accompanied by a conformational change of the polymerase-competent complex.