Mosaic Structure of the Human Immunodeficiency Virus Type 1 Genome Infecting Lymphoid Cells and the Brain: Evidence for Frequent In Vivo Recombination Events in the Evolution of Regional Populations

In addition to immunodeficiency, human immunodeficiency virus type 1 (HIV-1) can cause cognitive impairment and dementia through direct infection of the brain. To investigate the adaptive process and timing of HIV-1 entry into the central nervous system, we carried out an extensive genetic characterization of variants amplified from different regions of the brain and determined their relatedness to those in lymphoid tissue. HIV-1 genomes infecting different regions of the brain of one study subject with HIV encephalitis (HIVE) had a mosaic structure, being assembled from different combinations of evolutionarily distinct lineages in p17(gag), pol, individual hypervariable regions of gp120 (V1/V2, V3, V4, and V5), and gp41/nef. Similar discordant phylogenetic relationships were observed between p17(gag) and V3 sequences of brain and lymphoid tissue from three other individuals with HIVE. The observation that different parts of the genome of HIV infecting a particular tissue can have different evolutionary histories necessarily limits the conclusions that can be drawn from previous studies of the compartmentalization of distinct HIV populations in different tissues, as these have been generally restricted to sequence comparisons of single subgenomic regions. The complexity of viral populations in the brain produced by recombination could provide a powerful adaptive mechanism for the spread of virus with new phenotypes, such as antiviral resistance or escape from cytotoxic T-cell recognition into existing tissue-adapted virus populations.     

Identification of Shared Populations of Human Immunodeficiency Virus Type 1 Infecting Microglia and Tissue Macrophages outside the Central Nervous System

Infection of microglia and other cells of the macrophage/monocyte lineage in the central nervous system (CNS) by human immunodeficiency virus type I (HIV-1) underlies the development of giant cell encephalitis (GCE). It is currently unknown whether GCE depends on the emergence of virus populations specifically adapted to replicate in cells of the monocyte/macrophage lineage and whether this also leads to the specific targeting of macrophages in other nonlymphoid tissues. Autopsy samples from lymph node, brain (frontal region), lung, and full-thickness colon sections were obtained from nine study subjects with GCE and from nine without. The two groups showed no significant differences in CD4 counts, disease progression, or treatment history before death. Genetic relatedness between variants recovered from lymph node and nonlymphoid tissues was assessed by sequence comparison of V3 and p17(gag) regions using a newly developed method that scores the sample composition at successive nodes in a neighbor-joining tree. The association index enabled objective, numerical comparisons on the degree of tissue compartmentalization to be made. High proviral loads and p24 antigen expression in the brain were confined to the nine individuals with GCE. GCE was also associated with significantly higher proviral loads in colon samples (median of the GCE(+) group: 1,010 copies/10(6) cells; median of GCE(-) group, 10/10(6) cells; P = 0.006). In contrast, there were no significant differences in proviral load between the GCE(+) and GCE(-) groups in lymph node or lung samples, where HIV infection was manifested predominantly by infiltrates of lymphoid cells. V3 sequences from brain samples of individuals with GCE showed the greatest compartmentalization from those of lymph node, although samples from other tissues, particularly the colon, frequently contained variants phylogenetically related to those found in brain. The existence of shared, distinct populations of HIV specifically distributed in cells of the monocyte/macrophage lineage was further indicated by immunocytochemical detection of CD68(+), multinucleated giant cells expressing p24 antigen in samples of lung and colon in two individuals with GCE. This study provides the basis for future investigation of possible phenotypic similarities that underline the shared distributions of HIV variants infecting microglia and tissue macrophages outside the CNS.     

In vivo distribution and cytopathology of variants of human immunodeficiency virus type 1 showing restricted sequence variability in the V3 loop.

The distribution, cell tropism, and cytopathology in vivo of human immunodeficiency virus (HIV) was investigated in postmortem tissue samples from a series of HIV-infected individuals who died either of complications associated with AIDS or for unrelated reasons while they were asymptomatic. Proviral sequences were detected at a high copy number in lymphoid tissue of both presymptomatic patients and patients with AIDS, whereas significant infection of nonlymphoid tissue such as that from brains, spinal cords, and lungs were confined to those with AIDS. V3 loop sequences from both groups showed highly restricted sequence variability and a low overall positive charge of the encoded amino acid sequence compared with those of standard laboratory isolates of HIV type 1 (HIV-1). The low charge and the restriction in sequence variability were comparable to those observed with isolates showing a non-syncytium-inducing (NSI) and macrophage-tropic phenotype in vitro. All patients were either exclusively infected (six of seven cases) or predominantly infected (one case) with variants with a predicted NSI/macrophage-tropic phenotype, irrespective of the degree of disease progression. p24 antigen was detected by immunocytochemical staining of paraffin-fixed sections in the germinal centers within lymphoid tissue, although little or no antigen was found in areas of lymph node or spleen containing T lymphocytes from either presymptomatic patients or patients with AIDS. The predominant p24 antigen-expressing cells in the lungs and brains of the patients with AIDS were macrophages and microglia (in brains), frequently forming multinucleated giant cells (syncytia) even though the V3 loop sequences of these variants resembled those of NSI isolates in vitro. These studies indicate that lack of syncytium-forming ability in established T-cell lines does not necessarily predict syncytium-forming ability in primary target cells in vivo. Furthermore, variants of HIV with V3 sequences characteristic of NSI/macrophage-tropic isolates form the predominant population in a range of lymphoid and nonlymphoid tissues in vivo, even in patients with AIDS.     

Biological characterization of human immunodeficiency virus type 1 clones derived from different organs of an AIDS patient by long-range PCR.

In order to characterize the biological properties of human immunodeficiency virus type 1 (HIV-1) variants from different tissues (peripheral blood mononuclear cells [PBMC], lymph node, spleen, brain, and lung) of one patient, we have chosen long-range PCR to amplify virtually full-length HIV proviruses and to construct replication-competent viruses by adding a patient-specific 5' long terminal repeat. To avoid selection during propagation in CD4+ target cells, we transfected 293 cells and used the supernatants from these cells as challenge viruses for tropism studies after titration on human PBMC. Despite differences in the V3 loop of the major variants found in brain and lung compared to lymphoid tissues all recombinant HIV clones obtained showed identical cell tropism and replicative kinetics. After infection of human PBMC these viruses replicated with similar kinetics, with a slow/low-titer, non-syncytium-inducing phenotype. In contrast to the prediction of macrophage tropism, drawn from the V3 loop sequence, none of these viruses infected monocyte-derived macrophages. The challenge of blood dendritic cells by these recombinant viruses in the presence of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-4 resulted in a productive infection only after adding stimulated CD4+ T lymphocytes. Therefore, the biological properties of the HIV-1 variants derived from nonlymphoid tissue of this patient did not differ from those of HIV-1 variants from lymphoid tissue with respect to tropism for primary cells such as PBMC, macrophages, and blood dendritic cells.     

Neuronal Death Induced by Brain-Derived Human Immunodeficiency Virus Type 1 Envelope Genes Differs between Demented and Nondemented AIDS Patients

Human immunodeficiency virus type 1 (HIV-1) infection of the brain results in viral replication primarily in macrophages and microglia. Despite frequent detection of viral genome and proteins in the brains of AIDS patients with and without HIV dementia, only 20% of AIDS patients become demented. To investigate the role of viral envelope gene variation in the occurrence of dementia, we examined regions of variability in the viral envelope gene isolated from brains of AIDS patients. Brain-derived HIV-1 V1-V2 envelope sequences from seven demented and six nondemented AIDS patients displayed significant sequence differences between clinical groups, and by phylogenetic analysis, sequences from the demented group showed clustering. Infectious recombinant viruses containing brain-derived V3 sequences from both clinical groups were macrophagetropic, and viruses containing brain-derived V1, V2, and V3 sequences from both clinical groups spread efficiently in macrophages. In an indirect in vitro neurotoxicity assay using supernatant fluid from HIV-1-infected macrophages, recombinant viruses from demented patients induced greater neuronal death than viruses from nondemented patients. Thus, the HIV-1 envelope diversity observed in these patient groups appeared to influence the release of neurotoxic molecules from macrophages and might account in part for the variability in occurrence of dementia in AIDS patients.     

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.     

Phenotypic and genotypic characteristics of human immunodeficiency virus type 1 from patients with AIDS in northern Thailand.

Primary human immunodeficiency virus type 1 (HIV-1) isolates were obtained from 22 patients with AIDS from northern Thailand, where HIV-1 is transmitted primarily through the heterosexual route. Viral sequences were determined for the 22 patients with AIDS, and all were subtype E HIV-1 on the basis of sequence analysis of a region from the envelope protein gp120. Syncytium-inducing (SI) viruses were detected for 16 of 22 patients with AIDS by using MT-2 cells. Characteristics of amino acid sequences in V3 which have not been reported previously for subtype B SI HIV-1 were associated with the subtype E HIV-1 SI phenotype. The SI viruses from our study population contain predominantly a GPGR or GPGH motif at the tip of the V3 loop, in contrast to the previously described subtype E HIV-1 from Thailand which contained predominantly GPGQ. All the SI viruses lost a potential N-linked glycosylation site in V3 which is highly conserved among previously described subtype E HIV-1 isolates from asymptomatic patients from Thailand. HIV-1 envelope sequences including V3 from some patients with AIDS were significantly more divergent than viruses from asymptomatic patients in Thailand characterized 2 years ago or earlier. These results suggest that emergence of subtype E SI HIV-1 variants is associated with the development of AIDS, as it is for subtype B HIV-1. The divergence of subtype E HIV-1 in patients with AIDS as the disease progresses, and the divergence of subtype E HIV-1 in the infected population as the epidemic continues in Thailand, may have important implications for vaccine development.     

Host-specific driving force in human immunodeficiency virus type 1 evolution in vivo.

To investigate the process of human immunodeficiency virus type 1 (HIV-1) evolution in vivo, a total of 179 HIV-1 V3 sequences derived from cell-free plasma were determined from serial samples in three epidemiologically linked individuals (one infected blood donor and two transfusion recipients) over a maximum period of 8 years. A systematic analysis of pairwise comparisons of intrapatient sequences, both within and between each sample time point, revealed a preponderance and accumulation of nonsynonymous rather than synonymous substitutions in the V3 loop and flanking regions as they diverged over time. This strongly argues for the dominant role that positive selection for amino acid change plays in governing the pattern and process of HIV-1 env V3 evolution in vivo and nullifies hypotheses of purely neutral or mutation-driven evolution or completely chance events. In addition, different rates of evolution of HIV-1 were observed in these three different individuals infected with the same viral strain, suggesting that the degree of positive pressure for HIV-1 amino acid change is host dependent. Finally, the observed similar rate of accumulation in divergence within and between infected individuals suggests that the process of genetic divergence in the HIV epidemic proceeds regardless of host-to-host transmission events, i.e., that transmission does not reset the evolutionary clock.     

Virus population homogenization following acute human immunodeficiency virus type 1 infection

Understanding the properties of human immunodeficiency virus type 1 (HIV-1) variants capable of establishing infection is critical to the development of a vaccine against AIDS. Previous studies of men have shown that the HIV-1 env gene is homogeneous early in infection, leading to the suggestion that infection is established by a single transmitted variant. However, we report here that all of eight homosexual men evaluated beginning 3.7 to 9 weeks following onset of symptoms of acute infection harbored diverse virus populations in their blood, with median genetic distances averaging 1.08% in the env C2V5 region and 0.81% in the gag p17 gene. Within another 4.7 to 11 weeks, the variant lineage in env became more homogeneous, while gag sequences continued to diversify. Thus, the homogenization that has been reported to characterize acute infection is actually preceded by the replication of multiple virus variants. This early selective process focuses on viral properties within Env but not Gag p17. Hence, the viral homogeneity observed early in HIV-1 infection results from a selective process that occurs during the establishment of infection.     

Selection for human immunodeficiency virus type 1 envelope glycosylation variants with shorter V1-V2 loop sequences occurs during transmission of certain genetic subtypes and may impact viral RNA levels

Designing an effective human immunodeficiency virus type 1 (HIV-1) vaccine will rely on understanding which variants, from among the myriad of circulating HIV-1 strains, are most commonly transmitted and determining whether such variants have an Achilles heel. Here we show that heterosexually acquired subtype A HIV-1 envelopes have signature sequences that include shorter V1-V2 loop sequences and fewer predicted N-linked glycosylation sites relative to the overall population of circulating variants. In contrast, recently transmitted subtype B variants did not, and this was true for cases where the major risk factor was homosexual contact, as well as for cases where it was heterosexual contact. This suggests that selection during HIV-1 transmission may vary depending on the infecting subtype. There was evidence from 23 subtype A-infected women for whom there was longitudinal data that those who were infected with viruses with fewer potential N-linked glycosylation sites in V1-V2 had lower viral set point levels. Thus, our study also suggests that the extent of glycosylation in the infecting virus could impact disease progression.     

Molecular epidemiology of HIV-infection in drug addicts of Southern Ural

The analysis of HIV-1 variants circulating among drug addicts in 16 settlements of the Sverdlov, Chelyabinsk and Orenburg regions (72, 90 and 42 patients respectively) was carried out. As shown by the serological analysis, the spread of HIV-1 variant IDU-A among the drug addicts in this area continued in this area and could be detected in 99.5% of the samples (203 out of 204 samples). These data were confirmed by the results of the analysis of 35 samples by the Heteroduplex Mobility Assay for genes env and gag. The analysis of nucleotide sequences of gene env revealed that HIV-1 variants in the Southern Urals, where the greatest outbreak of HIV infection in Russia had been registered, were genetically related to viruses of subtype A, detected earlier in this group of risk in other regions of Russia, as well as in Ukraine, Belarus and other East European countries.     

Macrophage tropism of human immunodeficiency virus type 1 isolates from brain and lymphoid tissues predicts neurotropism independent of coreceptor specificity

The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.     

Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIVmac239

Background

The ability of emerging pathogens to infect new species is likely related to the diversity of pathogen variants present in existing reservoirs and their degree of genomic plasticity, which determines their ability to adapt to new environments. Certain simian immunodeficiency viruses (SIVcpz, SIVsm) have demonstrated tremendous success in infecting new species, including humans, resulting in the HIV-1 and HIV-2 epidemics. Although SIV diversification has been studied on a population level, the essential substrates for cross-species transmission, namely SIV sequence diversity and the types and extent of viral diversification present in individual reservoir animals have not been elucidated. To characterize this intra-host SIV diversity, we performed sequence analyses of clonal viral envelope (env) V1V2 and gag p27 variants present in individual SIVsm-infected sooty mangabeys over time.

Results

SIVsm demonstrated extensive intra-animal V1V2 length variation and amino acid diversity (le 38%), and continual variation in V1V2 N-linked glycosylation consensus sequence frequency and location. Positive selection was the predominant evolutionary force. Temporal sequence shifts suggested continual selection, likely due to evolving antibody responses. In contrast, gag p27 was predominantly under purifying selection. SIVsm V1V2 sequence diversification is at least as great as that in HIV-1 infected humans, indicating that extensive viral diversification in and of itself does not inevitably lead to AIDS.

Conclusion

Positive diversifying selection in this natural reservoir host is the engine that has driven the evolution of the uniquely adaptable SIV/HIV envelope protein. These studies emphasize the importance of retroviral diversification within individual host reservoir animals as a critical substrate in facilitating cross-species transmission.     

Genetic and functional analysis of full-length human immunodeficiency virus type 1 env genes derived from brain and blood of patients with AIDS

The genetic evolution of human immunodeficiency virus type 1 (HIV-1) in the brain is distinct from that in lymphoid tissues, indicating tissue-specific compartmentalization of the virus. Few primary HIV-1 envelope glycoproteins (Envs) from uncultured brain tissues have been biologically well characterized. In this study, we analyzed 37 full-length env genes from uncultured brain biopsy and blood samples from four patients with AIDS. Phylogenetic analysis of intrapatient sequence sets showed distinct clustering of brain relative to blood env sequences. However, no brain-specific signature sequence was identified. Furthermore, there was no significant difference in the number or positions of N-linked glycosylation sites between brain and blood env sequences. The patterns of coreceptor usage were heterogeneous, with no clear distinction between brain and blood env clones. Nine Envs used CCR5 as a coreceptor, one used CXCR4, and two used both CCR5 and CXCR4 in cell-to-cell fusion assays. Eight Envs could also use CCR3, CCR8, GPR15, STRL33, Apj, and/or GPR1, but these coreceptors did not play a major role in virus entry into microglia. Recognition of epitopes by the 2F5, T30, AG10H9, F105, 17b, and C11 monoclonal antibodies varied among env clones, reflecting genetic and conformational heterogeneity. Envs from two patients contained 28 to 32 N-glycosylation sites in gp120, compared to around 25 in lab strains and well-characterized primary isolates. These results suggest that HIV-1 Envs in brain cannot be distinguished from those in blood on the basis of coreceptor usage or the number or positions of N-glycosylation sites, indicating that other properties underlie neurotropism. The study also demonstrates characteristics of primary HIV-1 Envs from uncultured tissues and implies that Env variants that are glycosylated more extensively than lab strains and well-characterized primary isolates should be considered during development of vaccines and neutralizing antibodies.     

Complex positive selection pressures drive the evolution of HIV-1 with different co-receptor tropisms

HIV-1 co-receptor tropism is central for understanding the transmission and pathogenesis of HIV-1 infection. We performed a genome-wide comparison between the adaptive evolution of R5 and X4 variants from HIV-1 subtypes B and C. The results showed that R5 and X4 variants experienced differential evolutionary patterns and different HIV-1 genes encountered various positive selection pressures, suggesting that complex selection pressures are driving HIV-1 evolution. Compared with other hypervariable regions of Gp120, significantly more positively selected sites were detected in the V3 region of subtype B X4 variants, V2 region of subtype B R5 variants, and V1 and V4 regions of subtype C X4 variants, indicating an association of positive selection with co-receptor recognition/binding. Intriguingly, a significantly higher proportion (33.3% and 55.6%, P<0.05) of positively selected sites were identified in the C3 region than other conserved regions of Gp120 in all the analyzed HIV-1 variants, indicating that the C3 region might be more important to HIV-1 adaptation than previously thought. Approximately half of the positively selected sites identified in the env gene were identical between R5 and X4 variants. There were three common positively selected sites (96, 113 and 281) identified in Gp41 of all X4 and R5 variants from subtypes B and C. These sites might not only suggest a functional importance in viral survival and adaptation, but also imply a potential cross-immunogenicity between HIV-1 R5 and X4 variants, which has important implications for AIDS vaccine development.     

Intrapatient sequence variation of the gag gene of human immunodeficiency virus type 1 plasma virions.

Because certain regions of the gag gene, such as p24, are highly conserved among human immunodeficiency virus (HIV) isolates, many therapeutic strategies have been directed at gag gene targets. Although intrapatient variation of segments of gag have been determined, little is known about the variability of the full-length gag gene for HIV isolated from a single individual. To evaluate intrapatient full-length gag variability, we derived the nucleotide sequences of at least 10 cDNA gag clones of virion RNA isolated from plasma for each of four asymptomatic HIV type 1-infected patients with relatively high CD4+ T-cell counts (300 to 450 cells per mm3). Mean values of intrapatient gag nucleotide variation obtained by pairwise comparisons ranged from 0.55 to 2.86%. For three subjects, this value was equivalent to that reported for intrapatient full-length env variation. The greatest range of intrapatient mean nucleotide variation for individual protein-coding regions was observed for p7. We did not detect any G-to-A hypermutation, as A-to-G and G-to-A transitions occurred at similar frequencies, accounting for 29 and 25%, respectively, of the changes. Mean variation values and phylogenetic analysis suggested that the extent of nucleotide variation correlated with the length of viral infection. Furthermore, no distinct subpopulations of quasispecies were detectable within an individual. The predicted amino acid sequences indicated that there were no regions within a gag protein that were comprised of clustered changes.     

Diversity of human immunodeficiency virus type 1 subtype A and CRF03_AB protease in Eastern Europe: selection of the V77I variant and its rapid spread in injecting drug user populations

To characterize polymorphisms of the subtype A protease in the former Soviet Union, proviral DNA samples were obtained, with informed consent, from 119 human immunodeficiency virus type 1 (HIV-1)-positive untreated injecting drug users (IDUs) from 16 regions. All individuals studied have never been treated with antiretroviral drugs. The isolates were defined as IDU-A (n = 115) and CRF03_AB (n = 4) by using gag/env HMA/sequencing. The pro region was analyzed by using sequencing and original HIV-ProteaseChip hybridization technology. The mean of pairwise nucleotide distance between 27 pro sequences (23 IDU-A and 4 CRF03_AB) was low (1.38 +/- 0.79; range, 0.00 to 3.23). All sequences contained no primary resistance mutations. However, 13 of 23 (56.5%) subtype A isolates bore the V77I substitution known as the secondary protease mutation. V77I was associated with two synonymous substitutions in triplets 31 and 78, suggesting that all V77I-bearing viruses evolved from a single source in 1997. Hybridization analysis showed that 55 of 115 (47.8%) HIV-1 isolates contained V77I, but this variant was not found in any of 31 DNA samples taken from regions, where the HIV-1 epidemic among IDUs started earlier 1997, as well as in any of four CRF03_AB isolates. The results of analysis of 12 additional samples derived from epidemiologically linked subjects showed that in all four epidemiological clusters the genotype of the donor and the recipients was the same irrespective of the route of transmission. This finding demonstrates the transmission of the V77I mutant variant, which is spreading rapidly within the circulating viral pool in Russia and Kazakhstan. The continued molecular epidemiological and virological monitoring of HIV-1 worldwide thus remains of great importance.     

Predictions of linear T-cell and B-cell epitopes in proteins encoded by HIV-1, HIV-2 and SIVMAC and the conservation of these sites between strains

An important consideration in the design of vaccines to prevent HIV-1 infection effective against different strains is the amino acid sequence conservation of antigenic determinants. Even one amino acid change can destroy the antigenicity of a site for the antibody or T-cell receptor. The comparisons of predicted T- and B-cell epitopes between human HIV-1, HIV-2 and monkey SIVMAC AIDS viruses are presented. The three major gene products (env, gag and pol) were examined. A number of epitopes were identical between strains of HIV-1. Our analysis highlights the problem of designing an effective HIV-1 and HIV-2 vaccine and also the problem of testing human vaccines in monkey models.     

Evolution and biological characterization of human immunodeficiency virus type 1 subtype E gp120 V3 sequences following horizontal and vertical virus transmission in a single family

It has been suggested that immune-pressure-mediated positive selection operates to maintain the antigenic polymorphism on the third variable (V3) loop of the gp120 of human immunodeficiency virus type 1 (HIV-1). Here we present evidence, on the basis of sequencing 147 independently cloned env C2/V3 segments from a single family (father, mother, and their child), that the intensity of positive selection is related to the V3 lineage. Phylogenetic analysis and amino acid comparison of env C2/V3 and gag p17/24 regions indicated that a single HIV-1 subtype E source had infected the family. The analyses of unique env C2/V3 clones revealed that two V3 lineage groups had evolved in the parents. Group 1 was maintained with low variation in all three family members regardless of the clinical state or the length of infection, whereas group 2 was only present in symptomatic individuals and was more positively charged and diverse than group 1. Only virus isolates carrying the group 2 V3 sequences infected and induced syncytia in MT2 cells, a transformed CD4(+)-T-cell line. A statistically significant excess of nonsynonymous substitutions versus synonymous substitutions was demonstrated only for the group 2 V3 region. The data suggest that HIV-1 variants, possessing the more homogeneous group 1 V3 element and exhibiting the non-syncytium-inducing phenotype, persist in infected individuals independent of clinical status and appear to be more resistant to positive selection pressure.