Identification and recovery of minor HIV-1 variants using the heteroduplex tracking assay and biotinylated probes

We describe a method to identify and recover minor human immunodeficiency virus type 1 (HIV-1) sequence variants from a complex population. The original heteroduplex tracking assay (HTA) was modified by incorporating a biotin tag into the probe to allow for direct sequence determination of the query strand. We used this approach to recover sequences from minor HIV-1 variants in the V3 region of the env gene, and to identify minor drug-resistant variants in pro. The biotin-HTA targeting of the V3 region of env allowed us to detect minor V3 variants, of which 45% were classified as CXCR4-using viruses. In addition, the biotin-protease HTA was able to detect mixtures of wild-type sequence and drug-resistance mutations in four subjects that were not detected by bulk sequence analysis. The biotin-HTA is a robust assay that first separates genetic variants then allows direct sequence analysis of major and minor variants.     

An orphan G protein-coupled receptor, GPR1, acts as a coreceptor to allow replication of human immunodeficiency virus types 1 and 2 in brain-derived cells

Twelve G protein-coupled receptors, including chemokine receptors, act as coreceptors and determinants for the cell tropisms of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We isolated HIV-1 variants from T-cell-line (T)- and macrophage (M)-tropic (i.e., dualtropic) (R5-R3-X4) HIV-1 strains and also produced six HIV-1 mutants carrying single-point amino acid substitutions at the tip of the V3 region of the Env protein of HIV-1. These variants and three mutants infected brain-derived CD4-positive cells that are resistant to M-, T-, or dualtropic (R5, X4, or R5-X4) HIV-1 strains. However, a factor that determines this cell tropism has not been identified. This study shows that primary brain-derived fibroblast-like cell strains, BT-3 and BT-20/N, as well as a CD4-transduced glioma cell line, U87/CD4, which were susceptible to these HIV-1 variants and mutants and the HIV-2ROD strain, expressed mRNA of an orphan G protein-coupled receptor (GPCR), GPR1. When a CD4-positive cell line which was strictly resistant to infection with diverse HIV-1 and HIV-2 strains was transduced with GPR1, the cell line became susceptible to these HIV-1 variants and mutants and to an HIV-2 strain but not to T- or dualtropic HIV-1 strains, and numerous syncytia formed after infection. These results indicate that GPR1 functions as a coreceptor for the HIV-1 variants and mutants and for the HIV-2ROD strain in vitro.     

Evolutionary variants of the human immunodeficiency virus type 1 V3 region characterized by using a heteroduplex tracking assay.

Syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) are evolutionary variants that are associated with rapid CD4+ cell loss and rapid disease progression. The heteroduplex tracking assay (HTA) was used to detect evolutionary V3 variants by amplifying the V3 sequences from viral RNA derived from 50 samples of patient plasma. For this V3-specific HTA (V3-HTA), heteroduplexes were formed between the patient V3 sequences and a probe with the subtype B consensus V3 sequence. Evolution was then measured by divergence from the consensus. The presence of evolutionary variants was correlated with SI detection data on the same samples from the MT-2 cell culture assay. Evolutionary variants were correlated with the SI phenotype in 88% of the samples, and 96% of the SI samples contained evolutionary variants. In most cases the evolutionary V3 variants represented discrete clonal outgrowths of virus. Sequence analysis of the six discordant samples that did not show this correlation indicated that three non-syncytium-inducing (NSI) samples had V3 sequences that had evolved away from the consensus sequence but not toward an SI genotype. A fourth sample showed little evolution away from the consensus but was SI, which indicates that not all SI variants require basic substitutions in V3. The other two samples had SI-like genotypes and NSI phenotypes, suggesting that V3-HTA was able to detect SI emergence in these samples in the absence of their detection in vitro. V3-HTA was also used to confirm SI variant selection in MT-2 cells and to examine the possibility of variant selection during virus culture in peripheral blood cells.     

Emergence of gp120 V3 Variants Confers Neutralization Resistance in an R5 Simian-Human Immunodeficiency Virus-Infected Macaque Elite Neutralizer That Targets the N332 Glycan of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIV_AD8) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan.     

Persistence of simian immunodeficiency virus Mne variants upon transmission.

In macaques infected with a clone of simian immunodeficiency virus (SIV) Mne, viral variants consistently evolve multiple new potential glycosylation sites in the first variable region (V1) prior to the development of AIDS. In the present study, we asked whether viruses with these glycosylation sites persist when they are transmitted to a naive macaque. Variants that evolved after transmission to a recipient macaque were compared with virus that evolved in the donor, which had been infected by cloned SIV Mne. Upon transmission, the specific serine/threonine-rich motifs potentially encoding novel O-linked glycosylation site(s) in V1 were conserved in virus isolated from lymph node, spleen, and liver tissue from the recipient. There was some accumulation of changes in V3 of envelope in virus from the recipient, whereas changes in this region were not observed in virus from the donor macaque. Some variants detected in the tissue of the recipient at necropsy were most closely related to viruses present in the donor inoculum even though these particular variants were not detected early after infection in the recipient's peripheral blood mononuclear cells. Overall, virus with the predominant V1 sequences associated with progression to disease are transmitted to and persist in the recipient animal.     

A single mutation within the V3 envelope neutralization domain of feline immunodeficiency virus determines its tropism for CRFK cells.

Feline immunodeficiency virus (FIV) isolates differ in the ability to replicate in Crandell feline kidney (CRFK) cells. The difference in tropism between two variants of the Dutch isolate FIV-UT113 was studied by using molecular clones which contained the envelope genes of the variants in a background of the FIV-14 Petaluma sequence. Virus produced from clone pPET-113Th replicated in thymocytes, whereas virus from pPET-113Cr propagated in both thymocytes and CRFK cells, thereby reflecting the phenotypes of the parental variants. Exchange of envelope gene fragments showed that a 464-bp surface protein (SU)-encoding fragment encompassing the third variable region (V3) determines CRFK cell tropism. Sequence analysis of the exchanged fragments demonstrated two amino acid changes that led to an increase of the overall charge of the V3 domain: a G-->R transition at position 397 and a E-->K change at position 407. Mutational analysis of these residues revealed that the E-->K shift was responsible for the change in tropism, while the G-->R mutation improved the replication kinetics in CRFK cells. Mapping of a tropism determinant for FIV to a region which is also a major neutralization domain is reminiscent of human immunodeficiency virus type 1, in which a similar colocation was found.     

A single amino acid substitution in the V1 loop of human immunodeficiency virus type 1 gp120 alters cellular tropism.

Specific point mutations which affect viral tropism have been identified in both the V3 loop and in the CD4-binding region of the human immunodeficiency virus type 1 surface glycoprotein gp120. Here we report that a single point mutation in the first variable region (V1) of human immunodeficiency virus type 1 strain JRCSF is responsible for a change in viral tropism.     

A putative G protein-coupled receptor, RDC1, is a novel coreceptor for human and simian immunodeficiency viruses

More than 10 G protein-coupled receptors (GPCRs) have been shown to act as coreceptors for infection of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We have isolated HIV-1 variants infectious to primary brain-derived CD4-positive cells (BT-3 and BT-20/N) and U87/CD4 glioma cells that are resistant to T-cell line-tropic (T-tropic), macrophage-tropic (M-tropic), and T- and M-tropic (dualtropic) (X4, R5, and R5X4) HIV-1 strains. These primary brain-derived cells were also highly susceptible to HIV-2(ROD), HIV-2(SBL6669), and SIV(mndGB-1). A factor or coreceptor that determines the susceptibility of these brain-derived cells to these HIV and SIV strains has not been fully identified. To identify this coreceptor, we examined amino acid sequences of all known HIV and SIV coreceptors and noticed that tyrosine residues are well conserved in their extracellular amino-terminal domains. By this criterion, we selected 18 GPCRs as candidates of coreceptors for HIV and SIV strains infectious to these brain-derived cells. mRNA expression of an orphan GPCR, RDC1, was detected in the brain-derived cells, the C8166 T-cell line, and peripheral blood lymphocytes, all of which are susceptible to HIV-1 variants, but not in macrophages, which are resistant to them. When a CD4-expressing cell line, NP-2/CD4, which shows strict resistance to infection not only with HIV-1 but also with HIV-2 or SIV, was transduced with the RDC1 gene, the cells became highly susceptible to HIV-2 and SIV(mnd) strains but to neither M- nor T-tropic HIV-1 strains. The cells also acquired a low susceptibility to the HIV-1 variants. These findings indicate that RDC1 is a novel coreceptor for several HIV-1, HIV-2, and SIV strains which infect brain-derived cells.     

Nef enhances human immunodeficiency virus type 1 infectivity and replication independently of viral coreceptor tropism

We investigated the infectivities and replicative capacities of a large panel of variants of the molecular human immunodeficiency virus type 1 (HIV-1) NL4-3 clone that differ exclusively in the V3 region of the viral envelope glycoprotein and the nef gene. Our results demonstrate that Nef enhances virion infectivity and HIV-1 replication independently of the viral coreceptor tropism.     

Molecular Evidence for Mother-to-Child Transmission of Multiple Variants by Analysis of RNA and DNA Sequences of Human Immunodeficiency Virus Type 1

We have examined the viral selection that may occur during transmission by studying the env gene sequences from four cases of mother-to-child transmission of human immunodeficiency virus type 1. The V3 region sequences were directly amplified from both plasma viral RNA and peripheral blood mononuclear cells containing proviral DNA from mothers at delivery and at the time of diagnosis for children. Transmission occurred perinatally in three cases. The similarity of the viral sequences in each infant sample contrasted with the heterogeneous viral populations in the mothers. Phylogenetic analysis indicated the transmission of one or a few closely related maternal minor virus variants. In contrast, the child virus population in the fourth case was as heterogeneous as that of his mother, and phylogenetic analysis strongly suggested the transmission of multiple maternal variants. This case of multiple transmission was confirmed by analyzing sequences obtained at three times after delivery. Strains with sequences corresponding to the syncytium-inducing phenotype were also transmitted in this fourth case, and this was associated with the rapid development of disease in the child. There was no evidence for transmission of particular viral variants from mother to infant. We have thus described a particular case of vertical human immunodeficiency virus type 1 transmission with the transmission of multiple maternal variants to the infant and a rapid, fatal outcome in the child.     

Human immunodeficiency virus type 1 envelope-mediated neuronal death: uncoupling of viral replication and neurotoxicity

Although brain tissue from patients with human immunodeficiency virus (HIV) and/or AIDS is consistently infected by HIV type 1 (HIV-1), only 20 to 30% of patients exhibit clinical or neuropathological evidence of brain injury. Extensive HIV-1 sequence diversity is present in the brain, which may account in part for the variability in the occurrence of HIV-induced brain disease. Neurological injury caused by HIV-1 is mediated directly by neurotoxic viral proteins or indirectly through excess production of host molecules by infected or activated glial cells. To elucidate the relationship between HIV-1 infection and neuronal death, we examined the neurotoxic effects of supernatants from human 293T cells or macrophages expressing recombinant HIV-1 virions or gp120 proteins containing the V1V3 or C2V3 envelope region from non-clade B, brain-derived HIV-1 sequences. Neurotoxicity was measured separately as apoptosis or total neuronal death, with apoptosis representing 30 to 80% of the total neuron death observed, depending on the individual virus. In addition, neurotoxicity was dependent on expression of HIV-1 gp120 and could be blocked by anti-gp120 antibodies, as well as by antibodies to the human CCR5 and CXCR4 chemokine receptors. Despite extensive sequence diversity in the recombinant envelope region (V1V3 or C2V3), there was limited variation in the neurotoxicity induced by supernatants from transfected 293T cells. Conversely, supernatants from infected macrophages caused a broader range of neurotoxicity levels that depended on each virus and was independent of the replicative ability of the virus. These findings underscore the importance of HIV-1 envelope protein expression in neurotoxic pathways associated with HIV-induced brain disease and highlight the envelope as a target for neuroprotective therapeutic interventions.     

Extensive envelope heterogeneity of simian immunodeficiency virus in tissues from infected macaques.

The extent of virus genetic variation within tissues and peripheral blood mononuclear cells (PBMC) from two simian immunodeficiency virus (SIV)-infected macaques was analyzed. The products of PCR amplification of two regions, region 1 (SIV V1 region) and region 2 (region corresponding to the human immunodeficiency virus V3 cysteine loop and part of the C3 region immediately downstream), of the SIV envelope were examined for single-stranded conformation polymorphism followed by sequence analysis of selected clones. The V1 region of the SIV envelope of viruses present within lymphoid tissues displayed extensive heterogeneity, while viral populations within the PBMC and brain appeared to be less variable. Region 2 heterogeneity in both animals was generally confined to three residues in a tissue-specific manner. In addition, virus from the brains of both animals appeared to be distinct compared with viruses present in other tissues and PBMC of the same animal, both in the pattern of PCR-single-stranded conformation polymorphism SCP and in the sequence of region 2. These studies revealed that the tissues of SIV-infected macaques were a reservoir for viral variants distinct from those seen in PBMC.     

Recombinant CD4-selected human immunodeficiency virus type 1 variants with reduced gp120 affinity for CD4 and increased cell fusion capacity.

Variants of molecularly cloned human immunodeficiency virus type 1 (HIV-1) were analyzed following selection for the ability to replicate after exposure to soluble, recombinant CD4 protein (rCD4). Two variants, 4/1 and 16/2, show 8-fold and 16-fold reduced sensitivity to rCD4 neutralization yet remain as sensitive as the parental wild-type (wt) virus to neutralization by rCD4-immunoglobulin G (IgG) chimeric molecules and to inhibition of cellular infection by anti-CD4 antibody. The 4/1 variant is more cytopathic, with faster cell fusion and replication kinetics than the wt virus. The gp120s derived from the 4/1 and 16/2 variants have 3-fold and 30-fold reduced binding affinities to rCD4, respectively. The 4/1 variant exhibits diminished shedding of virion gp120 induced by rCD4. The binding of and neutralization by V3 loop antibodies and other anti-gp120 antibodies is reduced for 4/1 but not for 16/2. Sequence analysis revealed a codon change at amino acid residue 435 in the C4 region of the gp120 of 16/2. This accounts for its rCD4 insensitivity, since the insertion of this mutation in the wt gp120 yields the same phenotype. The 4/1 variant has a codon change in the V3 region of gp120 (amino acid 311), which accounts for its reduced sensitivity to some neutralizing antibodies but not to rCD4. The ready selection of rCD4-resistant variants has obvious relevance for rCD4-based therapeutic stratagems.     

Replication-competent variants of human immunodeficiency virus type 2 lacking the V3 loop exhibit resistance to chemokine receptor antagonists

Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.     

Both R5 and X4 human immunodeficiency virus type 1 variants persist during prolonged therapy with five antiretroviral drugs

A viral reservoir of human immunodeficiency virus type 1 (HIV-1)-infected, resting CD4(+) T cells persists despite suppression of plasma viremia by combination antiretroviral therapy. In a longitudinal analysis of three patients treated with a five-drug regimen, both R5 and X4 HIV-1 variants persisted in the cellular reservoir for up to 3 years.     

A group of V3 sequences from human immunodeficiency virus type 1 subtype E non-syncytium-inducing, CCR5-using variants are resistant to positive selection pressure

In a human immunodeficiency virus type 1 (HIV-1)-infected individual, immune-pressure-mediated positive selection operates to maintain the antigenic polymorphism on the gp120 third variable (V3) loop. Recently, we suggested on the basis of sequencing C2/V3 segments from an HIV-1 subtype E-infected family that a V3 sequence lineage group of the non-syncytium-inducing (NSI) variants (group 1) was relatively resistant to positive selection pressure (35). To better understand the relationship between the intensity of positive selection pressure and cell tropism of the virus, we determined the linkage between each V3 genotype and its function of directing coreceptor preference and MT2 cell tropism. The biological characterization of a panel of V3 recombinant viruses showed that all of the group 1 V3 sequences could confer an NSI/CCR5-using (NSI/R5) phenotype on HIV-1(LAI), whereas the group 2 V3 sequence, which was more positively charged than the group 1 sequence, dictated mainly a syncytium-inducing, CXCR4-using (SI/X4) phenotype. Phylogenetic analysis of C2/V3 sequences encoding group 1 or 2 V3 suggested that the variants carrying group 1 V3 are the ancestors of the intrafamilial infection and persisted in the family, while the variants carrying group 2 V3 evolved convergently from the group 1 V3 variants during disease progression in the individuals. Finally, a statistical test showed that the V3 sequence that could dictate an NSI/R5 phenotype had a synonymous substitution rate significantly higher than the nonsynonymous substitution rate. These data suggest that V3 sequences of the subtype E NSI/R5 variants are more resistant to positive selection pressure than those of the SI/X4 variants.     

Genetic differences between blood- and brain-derived viral sequences from human immunodeficiency virus type 1-infected patients: evidence of conserved elements in the V3 region of the envelope protein of brain-derived sequences.

Human immunodeficiency virus type 1 (HIV-1) sequences were generated from blood and from brain tissue obtained by stereotactic biopsy from six patients undergoing a diagnostic neurosurgical procedure. Proviral DNA was directly amplified by nested PCR, and 8 to 36 clones from each sample were sequenced. Phylogenetic analysis of intrapatient envelope V3-V5 region HIV-1 DNA sequence sets revealed that brain viral sequences were clustered relative to the blood viral sequences, suggestive of tissue-specific compartmentalization of the virus in four of the six cases. In the other two cases, the blood and brain virus sequences were intermingled in the phylogenetic analyses, suggesting trafficking of virus between the two tissues. Slide-based PCR-driven in situ hybridization of two of the patients' brain biopsy samples confirmed our interpretation of the intrapatient phylogenetic analyses. Interpatient V3 region brain-derived sequence distances were significantly less than blood-derived sequence distances. Relative to the tip of the loop, the set of brain-derived viral sequences had a tendency towards negative or neutral charge compared with the set of blood-derived viral sequences. Entropy calculations were used as a measure of the variability at each position in alignments of blood and brain viral sequences. A relatively conserved set of positions were found, with a significantly lower entropy in the brain-than in the blood-derived viral sequences. These sites constitute a brain "signature pattern," or a noncontiguous set of amino acids in the V3 region conserved in viral sequences derived from brain tissue. This brain-derived signature pattern was also well preserved among isolates previously characterized in vitro as macrophage tropic. Macrophage-monocyte tropism may be the biological constraint that results in the conservation of the viral brain signature pattern.     

R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.     

Length polymorphism within the second variable region of the human immunodeficiency virus type 1 envelope glycoprotein affects accessibility of the receptor binding site.

Sequential mutations were introduced into the V2 region of human immunodeficiency virus (HIV) type 1 HXB2, affecting the length, charge, and number of potential glycosylation sites. The insertions had no effect on cytopathicity or on the ability of virus to replicate in peripheral blood mononuclear cells and established T-cell lines. However, deletion of amino acids 186 to 188, encoding a conserved glycosylation site, resulted in a nonviable virus, suggesting a minimal length requirement of 40 amino acids for a functional V2 loop. However, all amino acid insertions affected the sensitivity of the variants to neutralization by soluble CD4 and monoclonal antibodies specific for epitopes in the V3 and CD4 binding site regions. Furthermore, these mutant viruses showed resistance to neutralization by HIV-positive human sera. Soluble gp120 mutant glycoproteins showed increased affinities for soluble CD4 and monoclonal antibodies specific for a number of epitopes overlapping the CD4 binding site, confirming that length increases in V2 affect exposure of the CD4 binding site. In summary, these data demonstrate that differences in V2 length modulate immunoreactivity of the envelope glycoprotein and support an association between the V2 and CD4 binding site regions.