Role of Naturally Occurring Basic Amino Acid Substitutions in the Human Immunodeficiency Virus Type 1 Subtype E Envelope V3 Loop on Viral Coreceptor Usage and Cell Tropism

To assess the role of naturally occurring basic amino acid substitutions in the V3 loop of human immunodeficiency virus type 1 (HIV-1) subtype E on viral coreceptor usage and cell tropism, we have constructed a panel of chimeric viruses with mutant V3 loops of HIV-1 subtype E in the genetic background of HIV-1LAI. The arginine substitutions naturally occurring at positions 8, 11, and 18 of the V3 loop in an HIV-1 subtype E X4 strain were systematically introduced into that of an R5 strain to generate a series of V3 loop mutant chimera. These chimeric viruses were employed in virus infectivity assays using HOS-CD4 cells expressing either CCR5 or CXCR4, peripheral blood mononuclear cells, T-cell lines, or macrophages. The arginine substitution at position 11 of the V3 loop uniformly caused the loss of infectivity in HOS-CD4-CCR5 cells, indicating that position 11 is critical for utilization of CCR5. CXCR4 usage was conferred by a minimum of two arginine substitutions, regardless of combination, whereas arginine substitutions at position 8 and 11 were required for T-cell line tropism. Nonetheless, macrophage tropism was not conferred by the V3 loop of subtype E R5 strain per se. We found that the specific combinations of amino acid changes in HIV-1 subtype E env V3 loop are critical for determining viral coreceptor usage and cell tropism. However, the ability to infect HOS-CD4 cells through either CXCR4 or CCR5 is not necessarily correlated with T-cell or macrophage tropism, suggesting that cellular tropism is not dictated solely by viral coreceptor utilization.     

Langerhans cell tropism of human immunodeficiency virus type 1 subtype A through F isolates derived from different transmission groups.

To test the hypothesis that some subtypes of human immunodeficiency virus type 1 (HIV-1), especially subtype E, are more likely to infect mature Langerhans cells (mLC), we titrated a panel of 26 primary HIV-1 isolates of subtypes A through F on peripheral blood mononuclear cells (PBMC) and mLC. The majority of HIV-1 isolates from heterosexually infected patients did not show a preferred tropism for mLC compared to homosexually transmitted HIV-1 isolates. Only 6 of 26 isolates, 2 from patients infected by homosexual contact and 4 from patients infected by heterosexual contact, showed a higher infectivity for mLC than for PBMC. Both syncytium-inducing and non-syncytium-inducing isolates were able to infect mLC which express mRNA for the chemokine receptors CCR3, CCR5, and CXCR4.     

Significantly longer envelope V2 loops are characteristic of heterosexually transmitted subtype B HIV-1 in Trinidad

Background

In Trinidad and the wider Caribbean, subtype B Human Immunodeficiency Virus-type 1 (HIV-1B) overwhelmingly accounts for HIV infection among heterosexuals; this contrasts with the association of HIV-1B with homosexual transmission and injecting drug use globally. The HIV envelope contains genetic determinants of cell tropism and evasion from immune attack. In this study we investigate the genetic properties of the env V1-C4 of HIV-1B soon after transmission to Trinidadian heterosexuals. This will reveal distinctive genetic features of the strains that cause the HIV-1B epidemic in Trinidad and generate insights to better understand their properties.

Methodology/Principal Findings

Quasispecies sampling was performed on the env V1-C4 of HIV-1B strains soon after transmission to heterosexual Trinidadians in a cohort of seroconverters. Phylogenetic relationships were determined for these quasispecies and the length and number of asparagine (N) linked glycosylation sites (NLGS) in their variable loops compared to that for HIV-1B globally. Signature amino acids within the constant domains of the env V1-C4 were identified for heterosexually transmitted HIV-1B from Trinidad relative to HIV-1B globally. HIV-1B obtained from Trinidadian heterosexuals soon after seroconversion had significantly longer V2 loops with one more glycosylation site, shorter V3 loops and no significant difference in V1 or V4 when compared to HIV-1B obtained soon after seroconversion from infected individuals in the rest of the world. HIV-1B soon after seroconversion and during chronic infection of Trinidadians was not significantly different, suggesting that distinctly long V2 loops are characteristic of HIV-1B in Trinidad. A threonine deletion at position 319 (T319-) along with the substitutions R315K and S440R were found to be distinctly associated with HIV-1B from Trinidad compared to HIV-1B globally.

Conclusions

This finding of distinctive genetic features that are characteristic of HIV-1B strains from Trinidad is consistent with the Trinidad epidemic being established by a founder strain or closely related founder strains of HIV-1B.     

Genetic heterogeneity of HIV-1 in Greece

The aim of this study was to detect and determine the genetic variation of HIV-1 in Greece and to analyze the phylogenetic relationships and transmission dynamics of identified variants. Eighty-six blood samples from HIV-1 seroconverted patients of different risk groups were collected from the AIDS clinic, AHEPA Hospital, Thessaloniki, Greece. Retroviral DNA was extracted from uncultured peripheral blood mononuclear cells. HIV-1 DNA sequences encoding a 500-bp fragment of the gp120 C2-C3 region were amplified from each study subject, and they were genetically subtyped by heteroduplex mobility assay and DNA sequencing. Genetic distances and phylogenetic relationships of DNA sequences were estimated using PHYLIP software. Our results revealed that 82 out of 86 (95.3%) subjects carried subtype B sequences, while four (4.7%) carried subtype A sequences. Subtype A in Greek individuals not having traveled abroad was documented. An average of intrasubtype B genetic divergence of 15% was noted. Our findings demonstrate the presence of at least two genetic subtypes of HIV-1 in northern Greece--subtype B and subtype A. The predominant subtype is subtype B, which was transmitted into Greece by multiple sources. Our observations lend support to the argument that the distribution of HIV-1 subtypes is determined by founder effects or other processes rather than any tropism for particular cell types or mode of transmission.     

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.     

广西壮族自治区HIV-1流行毒株的基因序列测定和亚型分析

使用ＰＣＲ技术对１４份广西ＨＩＶ－１阳性感染者外周血单核细胞（ＰＢＭＣｓ）样品进行扩增,获得ＨＩＶ－１膜蛋白（ｅｎｖ）基因的核酸片段,并对其Ｃ２－Ｖ３及邻区３５０－４５０个核苷酸序列进行了测定和分析。结果表明,１４份样品中９份为泰国Ｂ（Ｂ′）亚型,５份为Ｅ亚型毒株。其中Ｂ′亚型毒株的基因离散率为４．２％,与Ａ－Ｅ参考亚型及部分Ｂ亚型代表株序列相比较,与包括泰国、缅甸及云南德宏在内的Ｂ亚型毒株序列十分接近,相互之间基因离散率在３．０％－４．４％的范围内;而Ｅ亚型毒株的基因离散率为２．１％,与国际Ｅ亚型毒株的基因离散率最近,为５．６％,与其它国际参考亚型基因离散率很远,在２１．１％－２７．３％。根据以上数据及其它资料提示,广西存在Ｂ′和Ｅ两种亚型的ＨＩＶ－１的流行,且其Ｂ′亚型毒株的传入,与流行在云南德宏州的相同亚型ＨＩＶ－１毒株密切相关,而Ｅ亚型毒株则可能是由泰国经越南传入广西的     

Inflammatory Genital Infections Mitigate a Severe Genetic Bottleneck in Heterosexual Transmission of Subtype A and C HIV-1

The HIV-1 epidemic in sub-Saharan Africa is driven largely by heterosexual transmission of non-subtype B viruses, of which subtypes C and A are predominant. Previous studies of subtype B and subtype C transmission pairs have suggested that a single variant from the chronically infected partner can establish infection in their newly infected partner. However, in subtype A infected individuals from a sex worker cohort and subtype B individuals from STD clinics, infection was frequently established by multiple variants. This study examined over 1750 single-genome amplified viral sequences derived from epidemiologically linked subtype C and subtype A transmission pairs very early after infection. In 90% (18/20) of the pairs, HIV-1 infection is initiated by a single viral variant that is derived from the quasispecies of the transmitting partner. In addition, the virus initiating infection in individuals who were infected by someone other than their spouse was characterized to determine if genital infections mitigated the severe genetic bottleneck observed in a majority of epidemiologically linked heterosexual HIV-1 transmission events. In nearly 50% (3/7) of individuals infected by someone other than their spouse, multiple genetic variants from a single individual established infection. A statistically significant association was observed between infection by multiple genetic variants and an inflammatory genital infection in the newly infected individual. Thus, in the vast majority of HIV-1 transmission events in cohabiting heterosexual couples, a single genetic variant establishes infection. Nevertheless, this severe genetic bottleneck can be mitigated by the presence of inflammatory genital infections in the at risk partner, suggesting that this restriction on genetic diversity is imposed in large part by the mucosal barrier.     

Characterization of human immunodeficiency virus type 1 (HIV-1) envelope variation and neutralizing antibody responses during transmission of HIV-1 subtype B

We analyzed neutralization sensitivity and genetic variation of transmitted subtype B human immunodeficiency virus type 1 (HIV-1) in eight recently infected men who have sex with men and the virus from the six subjects who infected them. In contrast to reports of heterosexual transmission of subtype C HIV-1, in which the transmitted virus appears to be more neutralization sensitive, we demonstrate that in our study population, relatively few phenotypic changes in neutralization sensitivity or genotypic changes in envelope occurred during transmission of subtype B HIV-1. We suggest that limited genetic variation within the infecting host reduces the likelihood of selective transmission of neutralization-sensitive HIV.     

Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from approximately 20% in early infection to approximately 50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial). None of 33 subtype A or 10 A/D-recombinant viruses used the CXCR4 coreceptor. In contrast, nine (36%) of 25 subtype D viruses used both CXCR4 and CCR5 coreceptors. Clonal analyses of the nine subtype D samples with dual or mixed tropism revealed heterogeneous viral populations comprised of X4-, R5-, and dual-tropic HIV-1 variants. In five of the six samples with dual-tropic strains, V3 loop sequences of dual-tropic clones were identical to those of cocirculating R5-tropic clones, indicating the presence of CXCR4 tropism determinants outside of the V3 loop. These dual-tropic variants with R5-tropic-like V3 loops, which we designated "dual-R," use CCR5 much more efficiently than CXCR4, in contrast to dual-tropic clones with X4-tropic-like V3 loops ("dual-X"). These observations have implications for pathogenesis and treatment of subtype D-infected individuals, for the association between V3 sequence and coreceptor tropism phenotype, and for understanding potential mechanisms of evolution from exclusive CCR5 use to efficient CXCR4 use by subtype D HIV-1.     

Cross-subtype neutralizing antibodies induced in baboons by a subtype E gp120 immunogen based on an R5 primary human immunodeficiency virus type 1 envelope

Global human immunodeficiency virus type 1 (HIV-1) diversity may require engineering vaccines to express antigens representing strains prevalent in the target population of vaccine testing. The majority (90%) of incident infections in Thailand are genetic subtype E, with a small percentage of subtype B infections in the intravenous drug user populations. We have evaluated and compared the binding and HIV-1 neutralizing properties of serum antibodies induced in baboons by CHO cell-expressed monomeric gp120 derived from a CCR5-using (R5) subtype E primary HIV-1CM235 or a CXCR4-using (X4) subtype B T-cell line-adapted (TCLA) HIV-1SF2 isolate. In contrast to the subtype-specific HIV-1 neutralizing antibodies induced with recombinant HIV-1SF2 gp120 (rgp120SF2), rgp120CM235 immunization induced antibodies capable of neutralizing both subtype E and subtype B TCLA HIV-1 isolates. However, neither immunogen induced antibodies capable of neutralizing primary HIV-1 isolates. Antibody induced by rgp120CM235 preferentially bound natively folded gp120 and retained strong cross-reactivity against multiple gp120 strains within subtype E as well as subtype B. In contrast, antibody responses to rgp120SF2 were directed predominantly to linear epitopes poorly exposed on native gp120 and had more limited cross-recognition of divergent gp120. Fine epitope mapping revealed differences in antibody specificities. While both rgp120CM235 and rgp120SF2 induced antibodies to regions within C1, V1/V2, V3, and C5, unique responses were induced by rgp120CM235 to multiple epitopes within C2 and by rgp120SF2 to multiple epitopes within C3, V4, and C4. These data demonstrate that strain and/or phenotypic differences of HIV-1 subunit gp120 immunogens can substantially alter antibody binding specificities and subsequent HIV-1 neutralizing capacity.     

Unequal evolutionary rates in the human immunodeficiency virus type 1 (HIV-1) pandemic: the evolutionary rate of HIV-1 slows down when the epidemic rate increases

HIV-1 sequences in intravenous drug user (IDU) networks are highly homogenous even after several years, while this is not observed in most sexual epidemics. To address this disparity, we examined the human immunodeficiency virus type 1 (HIV-1) evolutionary rate on the population level for IDU and heterosexual transmissions. All available HIV-1 env V3 sequences from IDU outbreaks and heterosexual epidemics with known sampling dates were collected from the Los Alamos HIV sequence database. Evolutionary rates were calculated using phylogenetic trees with a t test root optimization of dated samples. The evolutionary rate of HIV-1 subtype A1 was found to be 8.4 times lower in fast spread among IDUs in the former Soviet Union (FSU) than in slow spread among heterosexual individuals in Africa. Mixed epidemics (IDU and heterosexual) showed intermediate evolutionary rates, indicating a combination of fast- and slow-spread patterns. Hence, if transmissions occur repeatedly during the initial stage of host infection, before selective pressures of the immune system have much impact, the rate of HIV-1 evolution on the population level will decrease. Conversely, in slow spread, where HIV-1 evolves under the pressure of the immune system before a donor infects a recipient, the virus evolution at the population level will increase. Epidemiological modeling confirmed that the evolutionary rate of HIV-1 depends on the rate of spread and predicted that the HIV-1 evolutionary rate in a fast-spreading epidemic, e.g., for IDUs in the FSU, will increase as the population becomes saturated with infections and the virus starts to spread to other risk groups.     

Human immunodeficiency virus type 1 (HIV-1) diversity at time of infection is not restricted to certain risk groups or specific HIV-1 subtypes

African women frequently acquire several genetically distinct human immunodeficiency virus type 1 (HIV-1) variants from a heterosexual partner, whereas the acquisition of multiple variants appears to be rare in men. To determine whether newly infected individuals in other risk groups acquire genetically diverse viruses, we examined the viral envelope sequences in plasma samples from 13 women and 4 men from the United States infected with subtype B viruses and 10 men from Kenya infected with non-subtype B viruses. HIV-1 envelope sequences differed by more than 2% in three U.S. women, one U.S. man, and one Kenyan man near the time of seroconversion. These findings suggest that early HIV-1 genetic diversity is not exclusive to women from Africa or to infection with any particular HIV-1 subtype.     

Human immunodeficiency virus type 1 intersubtype (B/E) recombination in a superinfected chimpanzee.

Genetic characterization of a large number of human immunodeficiency virus type 1 (HIV-1) isolates indicates that at least 10% of all strains have mosaic genomes generated by recombination between viruses of the same or different subtypes or clades. What is not known, however, is the time between infection with the first and second HIV-1 strains as well as the time between infection with the second strain and the recombinational event. After 32 months of infection with HIV-1(LAI(IIIB)), a chimpanzee was inoculated intravenously and became infected with a subtype E strain, HIV-1(90CR402). With PCR amplification, DNA heteroduplex analysis, and DNA sequencing, both parental strains and two distinct recombinant proviruses were found in genomic DNA from lymph node tissue obtained 24 weeks after exposure to HIV-1(90CR402). These results show (i) that antiviral immune responses established by long-term infection with an HIV-1 subtype B strain did not prevent infection by a subtype E strain and (ii) that both strains actively replicated and produced sufficient quantities of virus to coinfect the same cell(s), resulting in recombinant viruses.     

Human immunodeficiency virus type 2 (HIV-2) seroprevalence and characterization of a distinct HIV-2 genetic subtype from the natural range of simian immunodeficiency virus-infected sooty mangabeys.

The extent of zoonotic infections in rural Sierra Leone, where both feral and pet sooty mangabeys harbor divergent members of the human immunodeficiency virus type 2 (HIV-2)-sooty mangabey simian immunodeficiency virus (SIVsm) family, was tested in blood samples collected from 9,309 human subjects in 1993. Using HIV-1- and HIV-2-specific enzyme immunoassays and confirmatory Western blot analysis to test for antibodies to SIVsm-related lentiviruses, we found only nine subjects (0.096%) who tested positive for HIV: seven tested positive for HIV-1 and two tested positive for HIV-2. Compared with other rural West African communities, Sierra Leone displayed the lowest seroprevalence (0.021%) of HIV-2 infection yet reported, much lower than the previously reported seroprevalence in SIVsm-infected feral and household pet sooty mangabeys. Heteroduplex analysis demonstrated that two of the newly found HIV-1 strains belonged to subtype A, the most common HIV-1 subtype in Africa, but this is the first report of subtype A in Sierra Leone. The two HIV-2-infected individuals harbored two distinct HIV-2 strains, designated 93SL1 and 93SL2. Phylogenetic analysis indicated that HIV-2 93SL1 is a member of HIV-2 subtype A, the first strain of this HIV-2 subtype found in Sierra Leone. In contrast, HIV-2 93SL2 belongs to none of the five previously characterized HIV-2 subtypes (A to E) but is a new subtype, herein designated F, having the most divergent transmembrane sequences yet reported for HIV-2. The fact that both of the two most divergent HIV-2 subtypes known, E and F, are rare and found as single occurrences in persons from Sierra Leone may be related to the fact that this small region of West Africa also contains free-living and household pet sooty mangabeys with highly divergent variants of SIVsm. This finding provides support for the hypotheses that new HIV-2 subtypes result from independent cross-species transmission of SIVsm to the human population and that these single-occurrence transmission events had not spread widely into the population by 1993.     

山东省归国劳务人员中HIV-1感染毒株的序列特征和亚型分析

为了解自1992年以来,山东省归国劳务人员HIV-1感染毒株的分子流行病学特征。我们采集了11份确认为HIV-1感染者的全血分离单核细胞(PBMC),提取前病毒DNA,用套式PCR扩增HIV-1膜蛋白(env)基因,并对其env C2-V3区进行序列测定和分析。结果表明10名归国劳工人员及1名归国劳工配偶的样本经序列测定和基因分析发现了HIV-1的4种基因亚型A、B、C、E亚型。其中C亚型7名,B亚型2名,A和E亚型各1名。由此可见;山东省归国劳务人员中HIV-1毒株的感染情况较复杂,通过计算发现这些毒株间的基因离散率相差较大。提示应加强归国劳务人员的教育、检测和控制以防其它国家毒株传入。     

The replicative fitness of primary human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2 isolates

The main (M) group of human immunodeficiency virus type 1 (HIV-1) is responsible for the global AIDS epidemic while HIV-1 group O (outlier) and HIV type 2 are endemic only in west and central Africa. The failure of HIV-2 and especially HIV-1 group O to spread following the initial zoonotic jumps is not well understood. This study was designed to examine the relative replicative capacities between these human lentiviruses. A pairwise competition experiment was performed with peripheral blood mononuclear cells with eight HIV-2 isolates, 6 group O viruses, and 15 group M viruses of subtype A (2 viruses), B (5 viruses), C (4 viruses), D (2 viruses) and CRF01_AE (2 viruses). HIV-1 group M isolates of any subtype were typically 100-fold-more fit than group O or HIV-2 strains when competed in peripheral blood mononuclear cells from various humans. This order in replicative fitness was also observed when virus pairs were added to human dendritic cells and then cocultured with primary, quiescent T cells, which is the model for HIV-1 transmission. These results suggest that reduced replicative and transmission fitness may be contributing to the low prevalence and limited geographical spread of HIV-2 and group O HIV-1 in the human population.     

HIV-1 genetic diversity in Western Brittany,France

We describe human immunodeficiency virus type 1 (HIV-1) diversity in Western Brittany, France, and trace the dissemination of HIV-1 non-B subtype infection. The strategy for HIV-1 subtyping used involved subtype specific enzyme immunoassays, heteroduplex mobility assays and phylogenetic analysis of the sequences of env encoding the V3 loop region. Samples were obtained from 567 patients: 465 (82%) were of subtype B and 66 (11.6%) were not (20 were subtype A, 11 subtype C, four subtype D, seven subtype F, five subtype G and 19 others with circulating recombinant forms: 4CRF01_AE, 11CRF02_AG, 1H, 3CRF11_cpx). These findings are consistent with other studies of French populations. There is an epidemiological correlation between subtype B and homosexual or heterosexual contamination in France and between non-B subtype and heterosexual contamination in Africa.     

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.