Sequential turnover of human immunodeficiency virus type 1 env throughout the course of infection

We examined the rates of variant population turnover of the V1-V2 and V4-V5 hypervariable domains of the human immunodeficiency virus type 1 (HIV-1) gp120 molecule in longitudinal plasma samples from 14 men with chronic HIV-1 infection using heteroduplex tracking assays (HTA). Six men had high rates of CD4+ T-cell loss, and eight men had low rates of CD4+ T-cell loss over 2.5 to 8 years of infection. We found that V1-V2 and V4-V5 env populations changed dramatically over time in all 14 subjects; the changes in these regions were significantly correlated with each another over time. The subjects with rapid CD4 loss had significantly less change in their env populations than the subjects with slow CD4 loss. The two subjects with rapid CD4 loss and sustained low CD4 counts (<150/microl for at least 2 years) showed stabilization of their V1-V2 and V4-V5 populations as reflected by low levels of total change in HTA pattern and low HTA indices (a novel measure of the emergence of new bands and band distribution); this stabilization was not observed in other subjects. The stabilization of env variant populations at low CD4 counts following periods of rapid viral evolution suggests that selective pressure on env, likely from new immune responses, is minimal when CD4 counts drop dramatically and remain low for extended periods of time.     

Independent variation and positive selection in env V1 and V2 domains within maternal-infant strains of human immunodeficiency virus type 1 in vivo.

Multiple targets for immune recognition and cellular tropism are localized to the V1 and V2 hypervariable regions in the amino portion of human immunodeficiency virus type 1 (HIV-1) gp120env. We have assessed genetic diversity in env V1 and V2 hypervariable domains in vivo within epidemiologically related strains of HIV-1. Our strategy was to analyze longitudinal samples from two seropositive mothers and multiple children infected by perinatal transmission. Although the V1 and V2 domains are closely linked in the HIV-1 genome, nucleotide sequences in V1 and in V2 evolved independently in maternal-infant viruses in vivo. A high proportion of the nucleotide substitutions would introduce amino acid diversity in V1 and in V2. A significant excess of nonsynonymous over synonymous substitutions was identified in HIV-1 env V1 and V2 peptides in the mothers and in two older children but was not generally apparent in HIV-1 sequences in infants. An excess of nonsynonymous over synonymous substitutions indicated that there is positive selection for independent genetic variation in the V1 and V2 domains in vivo. It is likely that there are host responses to complex determinants in the V1 or V2 hypervariable domain of HIV-1 gp120.     

Multiple V1/V2 env variants are frequently present during primary infection with human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.     

Turnover of env variable region 1 and 2 genotypes in subjects with late-stage human immunodeficiency virus type 1 infection

The env gene of human immunodeficiency virus type 1 (HIV-1) includes some of the most genetically diverse regions of the viral genome, which are called variable regions 1 through 5 (V1 through V5). We have developed a heteroduplex tracking assay to detect changes in variable regions 1 and 2 of env (V1/V2-HTA). Using sequences from two molecular clones as probes, we have studied the nature of longitudinal virus population changes in a cohort of HIV-1-infected subjects. Viral sequences present in 21 subjects with late-stage HIV-1 infection were initially screened for stability of the virus population by V1/V2-HTA. The virus populations at entry comprised an average of five coexisting V1/V2 genotypic variants (as identified by HTA). Eight of the 21 subjects were examined in detail because of the dynamic behavior of their env variants over an approximately 9-month period. In each of these cases we detected a single discrete transition of V1/V2 genotypes based on monthly sampling. The major V1/V2 genotypes (those present at >10% abundance) from the eight subjects were cloned and sequenced to define the nature of V1/V2 variability associated with a discrete transition. Based on a comparison of V1/V2 genotypic variants present at entry with the newly emerged variants we categorized the newly emerged variants into two groups: variants without length differences and variants with length differences. Variants without length differences had fewer nucleotide substitutions, with the changes biased to either V1 or V2, suggestive of recent evolutionary events. Variants with length differences included ones with larger numbers of changes that were distributed, suggestive of recall of older genotypes. Most length differences were located in domains where the codon motif AVT (V = A, G, C) had become enriched and fixed. Finally, recombination events were detected in two subjects, one of which resulted in the reassortment of V1 and V2 regions. We suggest that turnover in V1/V2 populations was largely driven by selection on either V1 or V2 and that escape was accomplished either through changes focused in the region under selection or by the appearance of a highly divergent variant.     

Differential Selection of Specific Human Immunodeficiency Virus Type 1/JC499 Variants after Mucosal and Parenteral Inoculation of Chimpanzees

Regardless of the route of transmission, it is generally accepted that the human immunodeficiency virus type 1 (HIV-1) quasispecies transmitted from an infected individual to an uninfected individual is genetically homogeneous. This finding and the observation that HIV-1 genotypes in recipients are minor variants in the donors suggest strongly that selection for specific variants occurs. However, most analyses have been limited to the V3 region of env. In addition, the exact time at which most new infections occurred was not known, making it almost impossible to analyze virus populations present in donor-recipient pairs at the time of HIV-1 transmission. To circumvent this problem, three chimpanzees were inoculated with a genetically defined stock of cell-free HIV-1/JC499 by one of three routes: intravenously or via the cervical or penile mucosa. PCR products of the C2-to-V5 region of env were amplified from both proviral DNA and virion RNA in blood samples collected soon after infection and were screened by heteroduplex analysis (HDA). Those PCR products with distinct HDA banding patterns were cloned and sequenced. In all three animals, transmitted variants encoded one of two V3-loop populations identified in the inoculum, indicating relative homogeneity in this region. However, different virus populations, defined by combinations of specific V4 and V5 sequences, were found when variants in the animal inoculated intravenously (at least 13 V4-plus-V5 combinations) were compared with those in the two animals inoculated by the mucosal routes (limited to only four V4-plus-V5 combinations). The only V4-plus-V5 population in variants found in all three chimpanzees was the major population in the inoculum, which contained viruses with more than 30 different V4-plus-V5 combinations. That the majority of the V4-plus-V5 genotypes in variants transmitted to all three animals were minor populations in the inoculum indicated that selective transmission defined by the V4-plus-V5 regions had occurred but that distinct populations were transmitted by parenteral versus mucosal routes. These results indicate that the putative homogeneity of HIV-1 variants in a newly infected individual might be an artifact of the region of the env gene evaluated and that regions other than V3 might play a major role in selective transmission.     

Host-specific modulation of the selective constraints driving human immunodeficiency virus type 1 env gene evolution

To address the evolution of human immunodeficiency virus type 1 (HIV-1) within a single host, we analyzed the HIV-1 C2-V5 env regions of both cell-free genomic-RNA- and proviral-DNA-derived clones. Sequential samples were collected over a period of 3 years from six untreated subjects (three typical progressors [TPs] and three slow progressors [SPs], all with a comparable length of infection except one. The evolutionary analysis of the C2-V5 env sequences performed on 506 molecular clones (253 RNA- and 253 DNA-derived sequences) highlighted a series of differences between TPs and SPs. In particular, (i) clonal sequences from SPs (DNA and RNA) showed lower nucleotide similarity than those from TPs (P = 0. 0001), (ii) DNA clones from SPs showed higher intra- and intersample nucleotide divergence than those from TPs (P < 0.05), (iii) higher host-selective pressure was generally detectable in SPs (DNA and RNA sequences), and (iv) the increase in the genetic distance of DNA and RNA sequences over time was paralleled by an increase in both synonymous (Ks) and nonsynonymous (Ka) substitutions in TPs but only in nonsynonymous substitutions in SPs. Several individual peculiarities of the HIV-1 evolutionary dynamics emerged when the V3, V4, and V5 env regions of both TPs and SPs were evaluated separately. These peculiarities, probably reflecting host-specific features of selective constraints and their continuous modulation, are documented by the dynamics of Ka/Ks ratios of hypervariable env domains.     

Determinants of syncytium formation in microglia by human immunodeficiency virus type 1: role of the V1/V2 domains

Microglia are the main reservoir for human immunodeficiency virus type 1 (HIV-1) in the central nervous system (CNS), and multinucleated giant cells, the result of fusion of HIV-1-infected microglia and brain macrophages, are the neuropathologic hallmark of HIV dementia. One potential explanation for the formation of syncytia is viral adaptation for these CD4(+) CNS cells. HIV-1(BORI-15), a virus adapted to growth in microglia by sequential passage in vitro, mediates high levels of fusion and replicates more efficiently in microglia and monocyte-derived-macrophages than its unpassaged parent (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. Gonzalez-Scarano, J. Virol. 70:7654-7662, 1996). Since the interaction between the viral envelope glycoprotein and CD4 and the chemokine receptor mediates fusion and plays a key role in tropism, we have analyzed the HIV-1(BORI-15) env as a fusogen and in recombinant and pseudotyped viruses. Its syncytium-forming phenotype is not the result of a switch in coreceptor use but rather of the HIV-1(BORI-15) envelope-mediated fusion of CD4(+)CCR5(+) cells with greater efficiency than that of its parental strain, either by itself or in the context of a recombinant virus. Genetic analysis indicated that the syncytium-forming phenotype was due to four discrete amino acid differences in V1/V2, with a single-amino-acid change between the parent and the adapted virus (E153G) responsible for the majority of the effect. Additionally, HIV-1(BORI-15) env-pseudotyped viruses were less sensitive to decreases in the levels of CD4 on transfected 293T cells, leading to the hypothesis that the differences in V1/V2 alter the interaction between this envelope and CD4 or CCR5, or both. In sum, the characterization of the envelope of HIV-1(BORI-15), a highly fusogenic glycoprotein with genetic determinants in V1/V2, may lead to a better understanding of the relationship between HIV replication and syncytium formation in the CNS and of the importance of this region of gp120 in the interaction with CD4 and CCR5.     

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.     

Natural infection of chimpanzees with new lentiviruses related to HIV-1/SIVcpz

To determine newly identified lentiviruses, termed simian immunodeficiency virus (SIV)cpz97CG4 and SIVcpz97CG6, from two wild-captured juvenile brother chimpanzees in the Republic of Congo, subgenomic pol (integrase, 288 bp), 5'tat/rev-env Cl (including vpu, 354 bp) and env (C2-C4, 544 bp) gene fragments were amplified and sequenced. The analysis revealed significantly discordant phylogenetic positions of SIVcpz97CG in each genomic region. In the trees derived from partial env sequences (V3), both SIVcpz strains clustered in human immunodeficiency virus type 1 (HIV-1) subtype A. However, in the trees derived from partial pol (integrase) and 5'tat/rev-env C1 (including vpu) sequences, they clustered independently from any of the known HIV-1 subtypes. Especially, in the 5'tat/rev-vpu tree, they branched before the root of HIV-1 group M. These findings suggest that these Congolese SIVcpz genomes are mosaic, probably due to a recombinational event in the recent past, and it provides evidence for a rather recently occurring cross-species transmission between humans and chimpanzees.     

Evidence for a functional interaction between the V1/V2 and C4 domains of human immunodeficiency virus type 1 envelope glycoprotein gp120.

The domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein that are required for envelope function have been partially characterized. Little is known, however, about the nature of the interactions between these domains. To identify regions of the HIV-1 envelope glycoprotein that are involved in interactions necessary for proper envelope function, we constructed a series of 14 envelope recombinants between the env genes of two HIV-1 isolates. The envelope chimeras were examined for their ability to induce syncytia, to be proteolytically processed, and to function during a spreading viral infection. Our results demonstrate that the exchange between the two isolates of the first and second hypervariable regions (V1/V2) of gp120 results in defects in envelope glycoprotein processing, syncytium formation, and infectivity. Long-term passage of cultures infected with virus bearing a V1/V2 chimeric envelope glycoprotein leads to the emergence of a revertant virus with replication characteristics comparable to those of the wild type. Analysis of the revertant indicated that an Ile-->Met change in the C4 region of gp120 (between hypervariable regions V4 and V5) is responsible for the revertant phenotype. This single amino acid change restores infectivity without significantly affecting gp160 processing, CD4 binding, or the levels of virion-associated gp120. While the Ile-->Met change in C4 greatly enhances the fusogenic potential of the V1/V2 chimeric envelope glycoprotein, it has a detrimental effect on syncytium formation when analyzed in the context of the wild-type envelope. These results suggest that an interaction required for proper envelope glycoprotein function occurs between the V1/V2 and C4 regions of gp120.     

Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1alpha

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.     

Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by the virus envelope binding to CD4 and the conformationally altered envelope subsequently binding to one of two chemokine receptors. HIV-1 envelope glycoprotein (gp120) has five variable loops, of which three (V1/V2 and V3) influence the binding of either CCR5 or CXCR4, the two primary coreceptors for virus entry. Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4 in some HIV-1 isolates, but more commonly additional mutations in V1/V2 are observed during coreceptor switching. We have modeled coreceptor switching by introducing most possible combinations of mutations in the variable loops that distinguish a previously identified group of CCR5- and CXCR4-using viruses. We found that V3 mutations entail high risk, ranging from major loss of entry fitness to lethality. Mutations in or near V1/V2 were able to compensate for the deleterious V3 mutations and may need to precede V3 mutations to permit virus survival. V1/V2 mutations in the absence of V3 mutations often increased the capacity of virus to utilize CCR5 but were unable to confer CXCR4 use. V3 mutations were thus necessary but not sufficient for coreceptor switching, and V1/V2 mutations were necessary for virus survival. HIV-1 envelope sequence evolution from CCR5 to CXCR4 use is constrained by relatively frequent lethal mutations, deep fitness valleys, and requirements to make the right amino acid substitution in the right place at the right time.     

Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein.

The sequence variability of viral structure polypeptides has been associated with immune escape mechanisms. The V1 region of simian immunodeficiency virus (SIV) is a highly variable region of the SIVmac env gene. Here, we describe the V1 region as a linear neutralizing epitope. V1 region-specific neutralizing antibodies (NAb) were first demonstrated in a rabbit infected with a recombinant vaccinia virus carrying the env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben). Since we detected in this animal V1 region-specific NAb that were able to neutralize not only human immunodeficiency virus type 2 but also SIVmac32H, we investigated whether a similar immune response is evoked in macaques (Macaca mulatta) either infected with SIVmac or immunized with the external glycoprotein (gp130) of the same virus. Distinctly lower NAb titers were found in the SIVmac-infected animals than in the gp130-immunized macaques. Since the NAb titers in both groups were high enough for competition experiments, we used five overlapping peptides encompassing the whole V1 region for a detailed identification of the epitope. In each of the 12 macaques investigated, we detected a high level of NAb reacting with at least one peptide located in the central part of the V1 region. The relatively high degree of divergence, especially within the central part of the V1 region, which characterized the evolution of the retroviral sequences from the original inoculum in the infected macaques suggests the development of escape mutants. Furthermore, 3 of 12 animals developed NAb directed against the amino-terminal end of the V1 region epitope. Sequence analysis, however, revealed relatively low levels of genetic drift and genetic variability within this part of the V1 region. The induction of V1 env-specific NAb not only in gp130-immunized macaques but also in SIVmac-infected animals in combination with the increased genetic variability of this region in vivo indicates a marked biological significance of this epitope for the virus.     

Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain.

A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed when the HXB2 V1/V2 fusion glycoprotein was expressed without most of the gp70 carrier protein, and thus, this appeared to be an intrinsic property of the V1/V2 domain. Heterogeneity in expression of conformational and glycan-dependent epitopes was also observed for the natural viral env precursor, gPr160, but not for gp120. These results suggested that the closely spaced glycosylation sites 156 and 160 are often alternatively utilized and that the pattern of glycosylation at these positions affects the formation of the conformational structures needed for both expression of native epitopes in this region and processing of gPr160 to mature env products.     

Impact of the HIV-1 env genetic context outside HR1-HR2 on resistance to the fusion inhibitor enfuvirtide and viral infectivity in clinical isolates

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1-HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1-HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1-HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1-HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy.     

Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops

The gp120 envelope glycoprotein of primary human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and the CCR5 chemokine receptor on the target cell. Previously, we adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for CD4-independent replication were limited to the V2 loop-V1/V2 stem. Here we show that elimination of a single glycosylation site at asparagine 197 in the V1/V2 stem is sufficient for CD4-independent gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells expressing CCR5. Deletion of the V1/V2 loops also allowed CD4-independent viral entry and gp120 binding to CCR5. The binding of the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4 degrees C than at 37 degrees C. In the absence of the V1/V2 loops, neither removal of the N-linked carbohydrate at asparagine 197 nor lowering of the temperature increased the CD4-independent phenotypes. A CCR5-binding conformation of gp120, achieved by CD4 interaction or by modification of temperature, glycosylation, or variable loops, was preferentially recognized by the monoclonal antibody 48d. These results suggest that the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4 binding, or an N-linked glycan in the V1/V2 stem.     

Compartmentalization of human immunodeficiency virus type 1 between blood monocytes and CD4+ T cells during infection

Distinct sequences of human immunodeficiency virus type 1 (HIV-1) have been found between different tissue compartments or subcompartments within a given tissue. Whether such compartmentalization of HIV-1 occurs between different cell populations is still unknown. Here we address this issue by comparing HIV-1 sequences in the second constant region through the fifth hypervariable region (C2 to V5) of the surface envelope glycoprotein (Env) between viruses in purified blood CD14(+) monocytes and CD4(+) T cells obtained longitudinally from five infected patients over a time period ranging from 117 to 3,409 days postseroconversion. Viral populations in both cell types at early infection time points appeared relatively homogeneous. However, later in infections, all five patients showed heterogeneous populations in both CD14(+) monocytes and CD4(+) T cells. Three of the five patients had CD14(+) monocyte populations with significantly more genetic diversity than the CD4(+) T-cell population, while the other two patients had more genetic diversity in CD4(+) T cells. The cellular compartmentalization of HIV-1 between CD14(+) monocytes and CD4(+) T cells was not seen early during infections but was evident at the later time points for all five patients, indicating an association of viral compartmentalization with the time course of HIV-1 infection. The majority of HIV-1 V3 sequences indicated a macrophage-tropic phenotype, while a V3 sequence-predicted T-cell tropic virus was found in the CD4(+) T cells and CD14(+) monocytes of two patients. These findings suggest that HIV-1 in CD14(+) monocytes could disseminate and evolve independently from that in CD4(+) T cells over the course of HIV-1 infection, which may have implications on the development of new therapeutic strategies.