Extensive Diversification of Human Immunodeficiency Virus Type 1 Subtype B Strains during Dual Infection of a Chimpanzee That Progressed to AIDS

A chimpanzee (C-499) infected for more than 9 years with two subtype B isolates of human immunodeficiency virus type 1 (HIV-1), one (HIV-1_SF2) that replicates poorly and one (HIV-1_LAV-1b) that replicates efficiently in chimpanzees, died of AIDS 11 years after initial infection (F. J. Novembre et al., J. Virol. 71:4086–4091, 1997). Nucleotide sequence and phylogenetic analyses of the C2 to V5 region of env (C2-V5^env) in proviral DNA from peripheral blood lymphocytes obtained 22 months before death revealed two distinct virus populations. One of these populations appeared to be a recombinant in env, having the V3 loop from HIV-1_SF2 and the V4-V5 region from HIV-1_LAV-1b; the other population had evolved from HIV-1_LAV-1b. In addition to C2-V5^env, the entire p17^gag and nef genes were sequenced; however, based on nucleotide sequences and phlyogeny, whether the progenitor of the p17^gag and nef genes was SF2 or LAV-1b could not be determined. Compared to the two original viruses, the divergence of all clones of C2-V5^env ranged from 9.37 to 20.2%, that of p17^gag ranged from 3.11 to 9.29%, and that of nef ranged from 4.02 to 7.9%. In contrast, compared to the maximum variation of 20.2% in C2-V5^env for C-499, the maximum diversities in C2-V5^env in proviruses from two chimpanzees infected with HIV-1_LAV-1b for 9 and 10 years were 9.65 and 2.48%, respectively. These results demonstrate that (i) two distinct HIV-1 populations can coexist and undergo extensive diversification in chimpanzees with progressive HIV-1-induced disease and (ii) recombination between two subtype B strains occurred even though the second strain was inoculated 15 months after the first one. Furthermore, evaluation of env genes from three chimpanzees infected with the same strain suggests that the magnitude of HIV-1 diversification could be related to higher viral burdens, manifestations of disease, and/or dual infection.     

Restricted Genetic Diversity of HIV-1 Subtype C Envelope Glycoprotein from Perinatally Infected Zambian Infants

Background

Mother-to-child transmission of HIV-1 remains a significant problem in the resource-constrained settings where anti-retroviral therapy is still not widely available. Understanding the earliest events during HIV-1 transmission and characterizing the newly transmitted or founder virus is central to intervention efforts. In this study, we analyzed the viral env quasispecies of six mother-infant transmission pairs (MIPs) and characterized the genetic features of envelope glycoprotein that could influence HIV-1 subtype C perinatal transmission.

Methodology and Findings

The V1-V5 region of env was amplified from 6 MIPs baseline samples and 334 DNA sequences in total were analyzed. A comparison of the viral population derived from the mother and infant revealed a severe genetic bottleneck occurring during perinatal transmission, which was characterized by low sequence diversity in the infant. Phylogenetic analysis indicates that most likely in all our infant subjects a single founder virus was responsible for establishing infection. Furthermore, the newly transmitted viruses from the infant had significantly fewer potential N-linked glycosylation sites in Env V1-V5 region and showed a propensity to encode shorter variable loops compared to the nontransmitted viruses. In addition, a similar intensity of selection was seen between mothers and infants with a higher rate of synonymous (dS) compared to nonsynonymous (dN) substitutions evident (dN/dS<1).

Conclusions

Our results indicate that a strong genetic bottleneck occurs during perinatal transmission of HIV-1 subtype C. This is evident through population diversity and phylogenetic patterns where a single viral variant appears to be responsible for infection in the infants. As a result the newly transmitted viruses are less diverse and harbored significantly less glycosylated envelope. This suggests that viruses with the restricted glycosylation in envelope glycoprotein appeared to be preferentially transmitted during HIV-1 subtype C perinatal transmission. In addition, our findings also indicated that purifying selection appears to predominate in shaping the early intrahost evolution of HIV-1 subtype C envelope sequences.     

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.     

Evolution of CCR5 use before and during coreceptor switching

The envelope gene (env) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1- to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants.     

Sexually-Transmitted/Founder HIV-1 Cannot Be Directly Predicted from Plasma or PBMC-Derived Viral Quasispecies in the Transmitting Partner

Objective

Characterization of HIV-1 sequences in newly infected individuals is important for elucidating the mechanisms of viral sexual transmission. We report the identification of transmitted/founder viruses in eight pairs of HIV-1 sexually-infected patients enrolled at the time of primary infection (“recipients”) and their transmitting partners (“donors”).

Methods

Using a single genome-amplification approach, we compared quasispecies in donors and recipients on the basis of 316 and 376 C2V5 env sequences amplified from plasma viral RNA and PBMC-associated DNA, respectively.

Results

Both DNA and RNA sequences indicated very homogeneous viral populations in all recipients, suggesting transmission of a single variant, even in cases of recent sexually transmitted infections (STIs) in donors (n = 2) or recipients (n = 3). In all pairs, the transmitted/founder virus was derived from an infrequent variant population within the blood of the donor. The donor variant sequences most closely related to the recipient sequences were found in plasma samples in 3/8 cases and/or in PBMC samples in 6/8 cases. Although donors were exclusively (n = 4) or predominantly (n = 4) infected by CCR5-tropic (R5) strains, two recipients were infected with highly homogeneous CXCR4/dual-mixed-tropic (X4/DM) viral populations, identified in both DNA and RNA. The proportion of X4/DM quasispecies in donors was higher in cases of X4/DM than R5 HIV transmission (16.7–22.0% versus 0–2.6%), suggesting that X4/DM transmission may be associated with a threshold population of X4/DM circulating quasispecies in donors.

Conclusions

These suggest that a severe genetic bottleneck occurs during subtype B HIV-1 heterosexual and homosexual transmission. Sexually-transmitted/founder virus cannot be directly predicted by analysis of the donor’s quasispecies in plasma and/or PBMC. Additional studies are required to fully understand the traits that confer the capacity to transmit and establish infection, and determine the role of concomitant STIs in mitigating the genetic bottleneck in mucosal HIV transmission.     

The human immunodeficiency virus type 1 envelope confers higher rates of replicative fitness to perinatally transmitted viruses than to nontransmitted viruses

Selection of a minor viral genotype during perinatal transmission of human Immunodeficiency virus type 1 (HIV-1) has been observed, but there is a lack of information on the correlation of the restrictive transmission with biological properties of the virus, such as replicative fitness. Recombinant viruses expressing the enhanced green fluorescent protein or the Discosoma sp. red fluorescent (DsRed2) protein carrying the V1 to V5 regions of env from seven mother-infant pairs (MIPs) infected by subtype C HIV-1 were constructed, and competition assays were carried out to compare the fitness between the transmitted and nontransmitted viruses. Flow cytometry was used to quantify the frequency of infected cells, and the replicative fitness was determined based on a calculation that takes into account replication of competing viruses in a single infection versus dual infections. Transmitted viruses from five MIPs with the mothers chronically infected showed a restrictive env genotype, and all the recombinant viruses carrying the infants' Env had higher replicative fitness than those carrying the Env from the mothers. This growth fitness is lineage specific and can be observed only within the same MIP. In contrast, in two MIPs where the mothers had undergone recent acute infection, the viral Env sequences were similar between the mothers and infants and showed no further restriction in quasispecies during perinatal transmission. The recombinant viruses carrying the Env from the infants' viruses also showed replication fitness similar to those carrying the mothers' Env proteins. Our results suggest that newly transmitted viruses from chronically infected mothers have been selected to have higher replicative fitness to favor transmission, and this advantage is conferred by the V1 to V5 region of Env of the transmitted viruses. This finding has important implications for vaccine design or development of strategies to prevent HIV-1 transmission.     

Identification of a Cluster of HIV-1 Controllers Infected with Low Replicating Viruses

Long term non-progressor patients (LTNPs) are characterized by the natural control of HIV-1 infection. This control is related to host genetic, immunological and virological factors. In this work, phylogenetic analysis of the proviral nucleotide sequences in env gene from a Spanish HIV-1 LTNPs cohort identified a cluster of 6 HIV-1 controllers infected with closely-related viruses. The patients of the cluster showed common clinical and epidemiological features: drug user practices, infection in the same city (Madrid, Spain) and at the same time (late 70’s-early 80’s). All cluster patients displayed distinct host alleles associated with HIV control. Analysis of the virus envelope nucleotide sequences showed ancestral characteristic, lack of evolution and presence of rare amino-acids. Biological characterization of recombinant viruses with the envelope proteins from the cluster viruses showed very low replicative capacity in TZMbl and U87-CD4/CCR5 cells. The lack of clinical progression in the viral cluster patients with distinct combinations of protective host genotypes, but infected by low replicating viruses, indicate the important role of the virus in the non-progressor phenotype in these patients.     

Phylogenetic Relatedness of Circulating HIV-1C Variants in Mochudi,Botswana

Background

Determining patterns of HIV transmission is increasingly important for the most efficient use of modern prevention interventions. HIV phylogeny can provide a better understanding of the mechanisms underlying HIV transmission networks in communities.

Methods

To reconstruct the structure and dynamics of a local HIV/AIDS epidemic, the phylogenetic relatedness of HIV-1 subtype C env sequences obtained from 785 HIV-infected community residents in the northeastern sector of Mochudi, Botswana, during 2010–2013 was estimated. The genotyping coverage was estimated at 44%. Clusters were defined based on relatedness of HIV-1C env sequences and bootstrap support of splits.

Results

The overall proportion of clustered HIV-1C env sequences was 19.1% (95% CI 17.5% to 20.8%). The proportion of clustered sequences from Mochudi was significantly higher than the proportion of non-Mochudi sequences that clustered, 27.0% vs. 14.7% (p = 5.8E-12; Fisher exact test). The majority of clustered Mochudi sequences (90.1%; 95% CI 85.1% to 93.6%) were found in the Mochudi-unique clusters. None of the sequences from Mochudi clustered with any of the 1,244 non-Botswana HIV-1C sequences. At least 83 distinct HIV-1C variants, or chains of HIV transmission, in Mochudi were enumerated, and their sequence signatures were reconstructed. Seven of 20 genotyped seroconverters were found in 7 distinct clusters.

Conclusions

The study provides essential characteristics of the HIV transmission network in a community in Botswana, suggests the importance of high sampling coverage, and highlights the need for broad HIV genotyping to determine the spread of community-unique and community-mixed viral variants circulating in local epidemics. The proposed methodology of cluster analysis enumerates circulating HIV variants and can work well for surveillance of HIV transmission networks. HIV genotyping at the community level can help to optimize and balance HIV prevention strategies in trials and combined intervention packages.     

Full-Length Human Immunodeficiency Virus Type 1 Genomes from Subtype C-Infected Seroconverters in India, with Evidence of Intersubtype Recombination

The development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine is likely to depend on knowledge of circulating variants of genes other than the commonly sequenced gag and env genes. In addition, full-genome data are particularly limited for HIV-1 subtype C, currently the most commonly transmitted subtype in India and worldwide. Likewise, little is known about sequence variation of HIV-1 in India, the country facing the largest burden of HIV worldwide. Therefore, the objective of this study was to clone and characterize the complete genome of HIV-1 from seroconverters infected with subtype C variants in India. Cocultured HIV-1 isolates were obtained from six seroincident individuals from Pune, India, and virtually full-length HIV-1 genomes were amplified, cloned, and sequenced from each. Sequence analysis revealed that five of the six genomes were of subtype C, while one was a mosaic of subtypes A and C, with multiple breakpoints in env, nef, and the 3′ long terminal repeat as determined by both maximal χ² analysis and phylogenetic bootstrapping. Sequences were compared for preservation of known cytotoxic T lymphocyte (CTL) epitopes. Compared with those of the HIV-1_LAI sequence, 38% of well-defined CTL epitopes were identical. The proportion of nonconservative substitutions for Env, at 61%, was higher (P < 0.001) than those for Gag (24%), Pol (18%), and Nef (32%). Therefore, characterized CTL epitopes demonstrated substantial differences from subtype B laboratory strains, which were most pronounced in Env. Because these clones were obtained from Indian seroconverters, they are likely to facilitate vaccine-related efforts in India by providing potential antigens for vaccine candidates as well as for assays of vaccine responsiveness.     

Viral linkage in HIV-1 seroconverters and their partners in an HIV-1 prevention clinical trial

Background

Characterization of viruses in HIV-1 transmission pairs will help identify biological determinants of infectiousness and evaluate candidate interventions to reduce transmission. Although HIV-1 sequencing is frequently used to substantiate linkage between newly HIV-1 infected individuals and their sexual partners in epidemiologic and forensic studies, viral sequencing is seldom applied in HIV-1 prevention trials. The Partners in Prevention HSV/HIV Transmission Study (ClinicalTrials.gov #{"type":"clinical-trial","attrs":{"text":"NCT00194519","term_id":"NCT00194519"}}NCT00194519) was a prospective randomized placebo-controlled trial that enrolled serodiscordant heterosexual couples to determine the efficacy of genital herpes suppression in reducing HIV-1 transmission; as part of the study analysis, HIV-1 sequences were examined for genetic linkage between seroconverters and their enrolled partners.

Methodology/Principal Findings

We obtained partial consensus HIV-1 env and gag sequences from blood plasma for 151 transmission pairs and performed deep sequencing of env in some cases. We analyzed sequences with phylogenetic techniques and developed a Bayesian algorithm to evaluate the probability of linkage. For linkage, we required monophyletic clustering between enrolled partners'' sequences and a Bayesian posterior probability of ≥50%. Adjudicators classified each seroconversion, finding 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) indeterminate transmissions, with linkage determined by consensus env sequencing in 91 (84%). Male seroconverters had a higher frequency of unlinked transmissions than female seroconverters. The likelihood of transmission from the enrolled partner was related to time on study, with increasing numbers of unlinked transmissions occurring after longer observation periods. Finally, baseline viral load was found to be significantly higher among linked transmitters.

Conclusions/Significance

In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage determination process.     

Molecular Evidence for Nosocomial Transmission of Human Immunodeficiency Virus from a Surgeon to One of His Patients

We have investigated the molecular evidence in favor of the transmission of human immunodeficiency virus (HIV) from an HIV-infected surgeon to one of his patients. After PCR amplification, the env and gag sequences from the viral genome were cloned and sequenced. Phylogenetic analysis revealed that the viral sequences derived from the surgeon and his patient are closely related, which strongly suggests that nosocomial transmission occurred. In addition, these viral sequences belong to group M of HIV type 1 but are divergent from the reference sequences of the known subtypes.     

Genetic Variation in a Human Immunodeficiency Virus Type 2 Live-Virus Macaca nemestrina Vaccine Model

Four pigtailed macaques were inoculated with an infectious, apathogenic human immunodeficiency virus type 2 (HIV-2) molecular clone (HIV-2_KR) and subsequently challenged with a highly pathogenic strain, HIV-2₂₈₇, together with two naive control animals. After challenge, two animals inoculated with a high dose of the immunizing strain were protected from CD4 decline and immunodeficiency. To examine the role of genetic heterogeneity in protection, fragments of the env gene were amplified from peripheral blood mononuclear cell DNA and plasma RNA of challenged animals by PCR, examined by using a heteroduplex tracking assay (HTA), and sequenced. By HTA, variation was detected principally within the V1 and V2 regions of envelope. Extent of variation in viral DNA clones as assessed by HTA correlated with inoculum size, as did the degree of variation in sequences of clones derived from viral DNA. Conversely, a rapid reduction in the number of plasma viral RNA variants was noted by HTA at 8 weeks postinfection in protected animals; this reduction was not present in naive or unprotected macaques. Sequences derived from plasma viral RNA were found to be more closely related than corresponding viral DNA sequences, and protection correlated with a significant reduction in variation in plasma RNA sequences in animals given the identical inocula of HIV-2₂₈₇. Nonsynonymous mutations were significantly less prevalent in the protected animals. An additional potential glycosylation site was predicted to be present in the V2 region in all but one clone, and amino acid signatures related to protection were identified in viral DNA and RNA clones within both the V1 and V2 regions. Examination of the role of viral variation in this HIV-2 live-virus vaccine model may provide valuable insights into immunopathogenesis.     

Impaired Replication Capacity of Acute/Early Viruses in Persons Who Become HIV Controllers

Human immunodeficiency virus type 1 (HIV-1) controllers maintain viremia at <2,000 RNA copies/ml without antiretroviral therapy. Viruses from controllers with chronic infection were shown to exhibit impaired replication capacities, in part associated with escape mutations from cytotoxic-T-lymphocyte (CTL) responses. In contrast, little is known about viruses during acute/early infection in individuals who subsequently become HIV controllers. Here, we examine the viral replication capacities, HLA types, and virus sequences from 18 HIV-1 controllers identified during primary infection. gag-protease chimeric viruses constructed using the earliest postinfection samples displayed significantly lower replication capacities than isolates from persons who failed to control viremia (P = 0.0003). Protective HLA class I alleles were not enriched in these early HIV controllers, but viral sequencing revealed a significantly higher prevalence of drug resistance mutations associated with impaired viral fitness in controllers than in noncontrollers (6/15 [40.0%] versus 10/80 [12.5%], P = 0.018). Moreover, of two HLA-B57-positive (B57⁺) controllers identified, both harbored, at the earliest time point tested, signature escape mutations within Gag that likewise impair viral replication capacity. Only five controllers did not express “protective” alleles or harbor viruses with drug resistance mutations; intriguingly, two of them displayed typical B57 signature mutations (T242N), suggesting the acquisition of attenuated viruses from B57⁺ donors. These data indicate that acute/early stage viruses from persons who become controllers have evidence of reduced replication capacity during the initial stages of infection which is likely associated with transmitted or acquired CTL escape mutations or transmitted drug resistance mutations. These data suggest that viral dynamics during acute infection have a major impact on HIV disease outcome.Human immunodeficiency virus type I (HIV-1)-infected individuals who control viremia spontaneously without antiviral therapy have been termed HIV controllers (3, 18, 21, 48, 52). Unraveling the mechanisms associated with this phenotype should provide important insights regarding HIV pathogenesis and could contribute to vaccine development.Host and viral genetics, as well as host innate and adaptive immune responses, influence the rate of disease progression in HIV-1 infection (reviewed in reference 18). Several studies have reported the correlation between in vitro HIV replication capacity and level of plasma virus loads or disease progression in individuals with chronic infection (6, 13, 35, 45, 50, 55). Studies of HIV-1 elite controllers (EC), who control viremia to below the limit of detection in commercial assays, have revealed the presence of replication-competent viruses in these individuals (7), although these viruses appear to be less fit based on studies of envelope (35) and Gag-protease (45). This fitness defect in the chronic phase of infection is due at least in part to fitness-impairing mutations induced by cytotoxic-T-lymphocyte (CTL) responses restricted by “protective” HLA class I alleles (46).In contrast, little is known about viruses obtained from the acute/early phase of infection in persons who subsequently become HIV-1 controllers, largely due to the difficulty in enrolling such people during the acute/early phase of infection. The characterization of acute/early-phase viruses in individuals who subsequently achieve low set-point virus loads is of paramount importance to our understanding of the mechanisms of HIV-1 control.In the present study, we analyzed acute/early-phase plasma HIV RNA sequences from 18 untreated individuals who were diagnosed during the acute/early phase and subsequently became controllers (<2,000 RNA copies/ml). We compared these to sequences from a group of HIV-1 noncontrollers enrolled similarly during acute/early infection. We also generated chimeric viruses carrying patient-derived gag-protease sequences from acute/early-phase infection and compared the viral replication capacities of the chimeric viruses from controllers and from noncontrollers.We observed that the chimeric viruses derived from controllers have significantly reduced replicative capacities compared to those from noncontrollers. Moreover, we observed that at least 80% of these individuals who go on to become controllers featured transmission of attenuated drug-resistant viruses, transmission of HLA-B57-restricted CTL escape variants to HLA-mismatched recipients, selection of attenuated CTL escape variants in HLA-B57-positive (B57⁺) recipients, or combinations of these factors. Taken together, these results indicate that the initial viral dynamics have a major influence on the subsequent course of disease.     

Diversity of HIV-1 Subtype B: Implications to the Origin of BF Recombinants

Background

The HIV-1 subtype B epidemic in Brazil is peculiar because of the high frequency of isolates having the GWGR tetramer at V3 loop region. It has been suggested that GWGR is a distinct variant and less pathogenic than other subtype B isolates.

Methodology/Principal Findings

Ninety-four percent of the HIV-1 subtype B worldwide sequences (7689/8131) obtained from the Los Alamos HIV database contain proline at the tetramer of the V3 loop of the env gene (GPGR) and only 0.74% (60/8131) have tryptophan (GWGR). By contrast, 48.4% (161/333) of subtype B isolates from Brazil have proline, 30.6% (102/333) contain tryptophan and 10.5% (35/333) have phenylalanine (F) at the second position of the V3 loop tip. The proportion of tryptophan and phenylalanine in Brazilian isolates is much higher than in worldwide subtype B sequences (chi-square test, p = 0.0001). The combined proportion of proline, tryptophan and phenylalanine (GPGR+GWGR+GFGR) of Brazilian isolates corresponds to 89% of all amino acids in the V3 loop. Phylogenetic analysis revealed that almost all subtype B isolates in Brazil have a common origin regardless of their motif (GWGR, GPGR, GGGR, etc.) at the V3 tetramer. This shared ancestral origin was also observed in CRF28_BF and CRF29_BF in a genome region (free of recombination) derived from parental subtype B. These results imply that tryptophan substitution (e.g., GWGR-to-GxGR), which was previously associated with the change in the coreceptor usage within the host, also occurs at the population level.

Conclusions/Significance

Based on the current findings and previous study showing that tryptophan and phenylalanine in the V3 loop are related with coreceptor usage, we propose that tryptophan and phenylalanine in subtype B isolates in Brazil are kept by selective mechanisms due to the distinct coreceptor preferences in target cells of GWGR, GFGR and GFGR viruses.     

Characterization of human immunodeficiency virus type 1 CRF01_AE env genes derived from recently infected Thai individuals

Transmitted/founder virus is responsible for the establishment of human immunodeficiency virus type 1 (HIV-1) infection and induces primary anti-HIV-1 immune responses; therefore, it is important to study the viral population to understand the early events of HIV-1 infection. We amplified HIV-1 env genes from sera derived from recently infected Thai individuals, and established envelope glycoproteins (Env)-recombinant viruses. Generated Env-recombinant viruses were tested for their neutralization susceptibility to neutralizing human monoclonal antibodies (NHMAbs) and entry inhibitors, as well as being subjected to genotypic analysis. Most recombinant viruses were susceptible to neutralization by NHMAbs to Env gp41, whereas approximately one-third of the recombinant viruses were susceptible to a NHMAb against the CD4 binding site of gp120. In addition, all env genes were classified into CRF01_AE genes and showed low genetic divergence. Taken together with our previous studies on CRF01_AE env genes derived from chronically infected Thai individuals, these results suggested that the immunological and genetic characteristics of CRF01_AE Env derived from recently infected Thai individuals were different from those derived from chronically infected individuals.     

Phenotypic and genotypic comparisons of CCR5- and CXCR4-tropic human immunodeficiency virus type 1 biological clones isolated from subtype C-infected individuals

Individuals infected with human immunodeficiency virus type 1 (HIV-1) subtype C infrequently harbour X4 viruses. We studied R5 and X4 biological clones generated from HIV-1 subtype C-infected individuals. All subtype C R5 viruses demonstrated slower profiles of replication on CD4⁺ lymphocytes in comparison to subtype B viruses, whereas subtype C X4 viruses replicated with comparable efficiency to subtype B X4 viruses. No differences were identified in CC or CXC chemokine inhibitions (RANTES and SDF-1α, respectively) between subtype C and subtype B viruses. Immature dendritic cells were shown in coculture experiments to similarly enhance the infection of subtype C and subtype B R5 as well as X4 viruses. By amino acid sequence analysis, we showed that the R5 and X4 subtype C gp120 envelope gene alterations were similar to those for a switching subtype B virus, specifically with respect to the V3 charge and envelope N-linked glycosylation patterns. By phylogenetic analysis, we showed that one patient was infected with HIV-1 C′ and the other was infected with HIV-1 C" and that one of the patients harbored a virus that was a recombinant in the gp120 env gene between an R5 and an X4 virus, with the resultant virus being R5. No differences were identified between the long terminal repeat regions of the subtype C R5 and X4 biological clones. These results indicate that even though R5 subtype C viruses are restrictive for virus replication, the R5-to-X4 phenotype switch can occur and does so in a manner similar to that of subtype B viruses.     

Dendritic cells preferentially transfer CXCR4-using human immunodeficiency virus type 1 variants to CD4+ T lymphocytes in trans

Human immunodeficiency virus type 1 (HIV-1) preferentially utilizes the CCR5 coreceptor for target cell entry in the acute phase of infection, while later in disease progression the virus switches to the CXCR4 coreceptor in approximately 50% of patients. In response to HIV-1 the adaptive immune response is triggered, and antibody (Ab) production is elicited to block HIV-1 entry. We recently determined that dendritic cells (DCs) can efficiently capture Ab-neutralized HIV-1, restore infectivity, and transmit infectious virus to target cells. Here, we tested the effect of Abs on trans transmission of CCR5 or CXCR4 HIV-1 variants. We observed that transmission of HIV-1 by immature as well as mature DCs was significantly higher for CXCR4- than CCR5-tropic viral strains. Additionally, neutralizing Abs directed against either the gp41 or gp120 region of the envelope such as 2F5, 4E10, and V3-directed Abs inhibited transmission of CCR5-tropic HIV-1, whereas Ab-treated CXCR4-tropic virus demonstrated unaltered or increased transmission. To further study the effects of coreceptor usage we tested molecularly cloned HIV-1 variants with modifications in the envelope that were based on longitudinal gp120 V1 and V3 variable loop sequences from a patient progressing to AIDS. We observed that DCs preferentially facilitated infection of CD4⁺ T lymphocytes of viral strains with an envelope phenotype found late in disease. Taken together, our results illustrate that DCs transmit CXCR4-tropic HIV-1 much more efficiently than CCR5 strains; we hypothesize that this discrimination could contribute to the in vivo coreceptor switch after seroconversion and could be responsible for the increase in viral load.     

The Unique Envelope Gene of the Subgroup J Avian Leukosis Virus Derives from ev/J Proviruses, a Novel Family of Avian Endogenous Viruses

A new subgroup of avian leukosis virus (ALV), designated subgroup J, was identified recently. Viruses of this subgroup do not cross-interfere with viruses of the avian A, B, C, D, and E subgroups, are not neutralized by antisera raised against the other virus subgroups, and have a broader host range than the A to E subgroups. Sequence comparisons reveal that while the subgroup J envelope gene includes some regions that are related to those found in env genes of the A to E subgroups, the majority of the subgroup J gene is composed of sequences either that are more similar to those of a member (E51) of the ancient endogenous avian virus (EAV) family of proviruses or that appear unique to subgroup J viruses. These data led to the suggestion that the ALV-J env gene might have arisen by multiple recombination events between one or more endogenous and exogenous viruses. We initiated studies to investigate the origin of the subgroup J envelope gene and in particular to determine the identity of endogenous sequences that may have contributed to its generation. Here we report the identification of a novel family of avian endogenous viruses that include env coding sequences that are over 95% identical to both the gp85 and gp37 coding regions of subgroup J viruses. We call these viruses the ev/J family. We also report the isolation of ev/J-encoded cDNAs, indicating that at least some members of this family are expressed. These data support the hypothesis that the subgroup J envelope gene was acquired by recombination with expressed endogenous sequences and are consistent with acquisition of this gene by only one recombination event.     

Genetic recombination between human immunodeficiency virus type 1 (HIV-1) and HIV-2, two distinct human lentiviruses

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are genetically distinct viruses that each can cause AIDS. Approximately 1 million people are infected with both HIV-1 and HIV-2. Additionally, these two viruses use the same receptor and coreceptors and can therefore infect the same target cell populations. To explore potential genetic interactions, we first examined whether RNAs from HIV-1 and HIV-2 can be copackaged into the same virion. We used modified near-full-length viruses that each contained a green fluorescent protein gene (gfp) with a different inactivating mutation. Thus, a functional gfp could be reconstituted via recombination, which was used to detect the copackaging of HIV-1 and HIV-2 RNAs. The GFP-positive (GFP⁺) phenotype was detected in approximately 0.2% of the infection events, which was 35-fold lower than the intrasubtype HIV-1 rates. We isolated and characterized 54 GFP⁺ single-cell clones and determined that all of them contained proviruses with reconstituted gfp. We then mapped the general structures of the recombinant viruses and characterized the recombination junctions by DNA sequencing. We observed several different recombination patterns, including those that had crossovers only in gfp. The most common hybrid genomes had heterologous long terminal repeats. Although infrequent, crossovers in the viral sequences were also identified. Taken together, our study demonstrates that HIV-1 and HIV-2 can recombine, albeit at low frequencies. These observations indicate that multiple factors are likely to restrict the generation of viable hybrid HIV-1 and HIV-2 viruses. However, considering the large coinfected human population and the high viral load in patients, these rare events could provide the basis for the generation of novel human immunodeficiency viruses.     

Impact of HIV-1 Backbone on Neutralization Sensitivity: Neutralization Profiles of Heterologous Envelope Glycoproteins Expressed in Native Subtype C and CRF01_AE Backbone

Standardized assays to assess vaccine and antiviral drug efficacy are critical for the development of protective HIV-1 vaccines and drugs. These immune assays will be advanced by the development of standardized viral stocks, such as HIV-1 infectious molecular clones (IMC), that i) express a reporter gene, ii) are representative of globally diverse subtypes and iii) are engineered to easily exchange envelope (env) genes for expression of sequences of interest. Thus far, a subtype B IMC backbone expressing Renilla luciferase (LucR), and into which the ectodomain of heterologous env coding sequences can be expressed has been successfully developed but as execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), non-subtype B HIV-1 reagents are needed to support vaccine development. Here we describe two IMCs derived from subtypes C and CRF01_AE HIV-1 primary isolates expressing LucR (IMC.LucR) that were engineered to express heterologous gp160 Envs. 18 constructs expressing various subtypes C and CRF01_AE Envs, mostly acute, in subtype-matched and –unmatched HIV backbones were tested for functionality and neutralization sensitivity. Our results suggest a possible effect of non-env HIV-1 genes on the interaction of Env and neutralizing antibodies and highlight the need to generate a library of IMCs representative of the HIV-1 subtype spectrum to be used as standardized neutralization assay reagents for assessing HIV-1 vaccine efficacy.