Performance of Geno2Pheno[coreceptor] to infer coreceptor use in human immunodeficiency virus type 1 (HIV-1) subtype A.

BACKGROUND: Assessment of human immunodeficiency virus type 1 (HIV-1) coreceptor usage is required prior to treatment with the CCR5 antagonist maraviroc to exclude the presence of CXCR4-using (X4) strains. Genotype-based interpretation systems are mostly designed on subtype B and have been reported to be less accurate for subtype A/CRF02_AG. OBJECTIVES: To evaluate the performance of the widely used Geno2Pheno[coreceptor] (G2P[c]) algorithm for prediction of coreceptor usage with subtype A/CRF02_AG vs. subtype B. STUDY DESIGN: Co-receptor tropism of 24 subtype A/CRF02_AG and 24 subtype B viruses was measured phenotypically by a homebrew single-cycle assay and genotypically by using G2P[c]. Samples with discrepant genotype-phenotype results were analyzed by next generation sequencing (NGS) and interpreted by the NGS Geno2Pheno algorithm (G2P[454]). RESULTS: At 10% false positive rate (FPR), the G2P[c]/phenotype discordance rate was 12.5% (n = 3) for subtype A/CRF02_AG and 8.3% (n = 2) for subtype B. Minority X4 species escaping detection by bulk sequencing but documented by NGS explained the two subtype B and possibly one subtype A/CRF02_AG discordant case. The other two subtype A/CRF02_AG miscalled by G2P[c] could be explained by X4 overcalling at borderline FPR and/or by algorithm failure. DISCUSSION: Our study did not demonstrate relevantly higher G2P[c] inaccuracy with subtype A/CRF02_AG with respect to subtype B. Genotype/phenotype discordances can be due to different reasons, including but not limited to, algorithm inaccuracy. Very large genotype/phenotype correlation panels are required to detect and explain the reason for any consistent difference in genotypic tropism prediction for subtype A/CRF02_AG vs. subtype B.

Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions.

UNLABELLED: HIV-1 requires the CD4 receptor and a coreceptor (CCR5 [R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Coreceptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late-stage and 6 early-stage subjects. We also used endpoint dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false-positive rate [FPR] of ≤2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), which was significantly higher than what has been seen previously by using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with an intermediate FPR (2 to 20%) could be either X4/dual-tropic or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of <10%, and these variants carried a distinctive sequence change. Phylogenetic analysis of both the V1/V2 and C2/V3 regions showed evidence of recombination within but very little recombination between the X4 and R5 lineages, suggesting that these populations are genetically isolated. IMPORTANCE: Primer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed by using next-generation sequencing (NGS) and with a greater depth of sampling than single-genome amplification (SGA). Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional information for accurately predicting X4 variants by using V3 sequences. Limited recombination occurs between X4 and R5 lineages, suggesting that X4 and R5 variants are genetically isolated and may be replicating in different cell types or that X4/R5 recombinants have reduced fitness.

Predicted co-receptor tropism and sequence characteristics of China HIV-1 V3 loops: implications for the future usage of CCR5 antagonists and AIDS vaccine development.

BACKGROUND: The co-receptor tropism of any given HIV-1 isolate is closely associated with the progression of AIDS. Understanding the co-receptor tropism and genetic diversity of circulating HIV-1 strains is critical for AIDS treatment and vaccine development. METHODS: All available China HIV-1 V3 sequences with known subtypes/circulating recombinant forms (CRFs) and transmission routes were retrieved from the Los Alamos HIV Sequence Database. HIV-1 co-receptor tropism was predicted using online tool HIV-1 PhenoPred. RESULTS: All C/CRF07_BC/CRF08_BC strains appeared to use CCR5 for cell entry (R5 strains), while 61.1% of subtype B and 38.7% of CRF01_AE were also R5, indicating a higher prevalence of R5 (76.9%) than X4. The prevalence of R5 remained relatively stable over the different sample years regardless of C/CRF07_BC/CRF08_BC, B, or CRF01_AE subtypes. The co-receptor usage of HIV-1 appeared to be associated with the different subtypes, rather than transmission route. Furthermore, the V3 sequences of C/CRF07_BC/CRF08_BC were more genetically homogeneous relative to both subtypes B and CRF01_AE. CONCLUSIONS: The higher prevalence of R5 and higher level of homogeneity of V3 sequences in C/CRF07_BC/CRF08_BC suggest that CCR5 antagonists will be promising drugs for future AIDS treatment in China, and that circulating R5 strains are valuable candidates for AIDS vaccine development.

CD4 independent binding of HIV gp120 to mannose receptor on human spermatozoa.

OBJECTIVE: To characterize the CD4-independent HIV-binding protein of 160 kDa on human spermatozoa. METHODS: The N-terminal amino acid sequence of the 160 kDa protein and its peptide obtained by tryptic digestion were determined. Polymerase chain reaction amplification of human testicular cDNA was performed using degenerate primers corresponding to peptide sequences of the 160 kDa protein. Localization of 160 kDa protein on sperm was performed using fluorescently labeled gp120, followed by inhibition experiments using antagonists to determine the specificity. RESULTS: The partial cDNA sequence of the 160 kDa protein demonstrated 99% identity with human macrophage mannose receptor. Sequence of testicular mannose receptor was obtained and exhibited 99% identity with that of macrophage mannose receptor. Furthermore, mannose receptor protein from sperm extract was found to have a molecular weight of 160 kDa, congruent with that of 160 kDa HIV-binding protein. gp120 binding and mannose receptor expression were localized to the equatorial segment in 10% of ejaculated sperm, which increased after capacitation. Mannan at molar excess concentrations completely inhibited gp120 binding to sperm. CONCLUSIONS: The 160 kDa, CD4-independent HIV-binding sperm protein has been identified as the human mannose receptor protein. The role of mannose receptor in HIV transmission and association with risk of sexual transmission merit further investigation.

A formylpeptide receptor, FPRL1, acts as an efficient coreceptor for primary isolates of human immunodeficiency virus.

BACKGROUND: More than 10 members of seven-transmembrane G protein-coupled receptors (GPCRs) have been shown to work as coreceptors for human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), and simian immunodeficiency viruses (SIVs). As a common feature of HIV/SIV coreceptors, tyrosine residues are present with asparagines, aspartic acids or glutamic acids in the amino-terminal extracellular regions (NTRs). We noticed that a receptor for N-formylpeptides, FPRL1, also contains two tyrosine residues accompanied by glutamic acids in its NTR. It was reported that monocytes expressing CCR5 and FPRL1 in addition to CD4 are activated by treatment with ligands or agonists of FPRL1. Activated monocytes down-modulate CCR5 and become resistant to infection by HIV-1 strains. Thus, FPRL1 plays important roles in protection of monocyptes against HIV-1 infection. However, its own coreceptor activity has not been elucidated yet. In this study, we examined coreceptor activities of FPRL1 for HIV/SIV strains including primary HIV-1 isolates. RESULTS: A CD4-transduced human glioma cell line, NP-2/CD4, is strictly resistant to HIV/SIV infection. We have reported that when NP-2/CD4 cells are transduced with a GPCR having coreceptor activity, the cells become susceptible to HIV/SIV strains. When NP-2/CD4 cells were transduced with FPRL1, the resultant NP-2/CD4/FPRL1 cells became markedly susceptible to some laboratory-adapted HIV/SIV strains. We found that FPRL1 is also efficiently used as a coreceptor by primary HIV-1 isolates as well as CCR5 or CXCR4. Amino acid sequences linked to the FPRL1 use could not be detected in the V3 loop of the HIV-1 Env protein. Coreceptor activities of FPRL1 were partially blocked by the forymyl-Met-Leu-Phe (fMLF) peptide. CONCLUSION: We conclude that FPRL1 is a novel and efficient coreceptor for HIV/SIV strains. FPRL1 works as a bifunctional factor in HIV-1 infection. Namely, the role of FPRL1 in HIV-1 infection is protective and/or promotive in different conditions. FPRL1 has been reported to be abundantly expressed in the lung, spleen, testis, and neutrophils. We detected mRNA expression of FPRL1 in 293T (embryonal kidney cell line), C8166 (T cell line), HOS (osteosarcoma cell line), Molt4#8 (T cell line), U251MG (astrocytoma cell line), U87/CD4 (CD4-transduced glioma cell line), and peripheral blood lymphocytes. Roles of FPRL1 in HIV-1 infection in vivo should be further investigated.

Frequent intrapatient recombination between human immunodeficiency virus type 1 R5 and X4 envelopes: implications for coreceptor switch.

Emergence of human immunodeficiency virus type 1 (HIV-1) populations that switch or broaden coreceptor usage from CCR5 to CXCR4 is intimately coupled to CD4+ cell depletion and disease progression toward AIDS. To better understand the molecular mechanisms involved in the coreceptor switch, we determined the nucleotide sequences of 253 V1 to V3 env clones from 27 sequential HIV-1 subtype B isolates from four patients with virus populations that switch coreceptor usage. Coreceptor usage of clones from dualtropic R5X4 isolates was characterized experimentally. Sequence analysis revealed that 9% of the clones from CXCR4-using isolates had originated by recombination events between R5 and X4 viruses. The majority (73%) of the recombinants used CXCR4. Furthermore, coreceptor usage of the recombinants was determined by a small region of the envelope, including V3. This is the first report demonstrating that intrapatient recombination between viruses with distinct coreceptor usage occurs frequently. It has been proposed that X4 viruses are more easily suppressed by the immune system than R5 viruses. We hypothesize that recombination between circulating R5 viruses and X4 viruses can result in chimeric viruses with the potential to both evade the immune system and infect CXCR4-expressing cells. The broadening in cell tropism of the viral population to include CXCR4-expressing cells would gradually impair the immune system and eventually allow the X4 population to expand. In conclusion, intrapatient recombination between viruses with distinct coreceptor usage may contribute to the emergence of X4 viruses in later stages of infection.

HIV-1 coreceptor preference is distinct from target cell tropism: a dual-parameter nomenclature to define viral phenotypes.

HIV-1 infection of cells is mediated by engagement between viral envelope glycoproteins (Env) and a receptor complex comprising CD4 and one of two chemokine receptors, CCR5 and CXCR4, expressed on the surface of target cells. Most CD4+-transformed T cell lines express only CXCR4, but primary lymphocytes and macrophages, the main cellular targets for infection in vivo, express both coreceptors. Cell- and viral strain-specific utilization of these coreceptor pathways, rather than coreceptor expression per se, regulates lymphocyte and macrophage entry and tropism. Virus use of coreceptor[s] (R5, X4, or R5 and X4) and its target cell tropism (lymphocytes, macrophages, and/or transformed T cell lines) are related but distinct characteristics of Envs. A comprehensive classification schema of HIV-1 Env phenotypes that addresses both tropism and coreceptor use is proposed. Defining Env phenotype based on both parameters is important in the development of entry inhibitors and vaccines, for understanding changes in Env that evolve over time in vivo, and for discerning differences among viral species that underlie aspects of pathogenesis and transmission. Recognizing how tropism is related to, yet differs from, coreceptor selectivity is critical for understanding the mechanisms by which these viral characteristics impact pathogenesis.

Non-cytotoxic inhibition of HIV-1 infection by unstimulated CD8+ T lymphocytes from HIV-exposed-uninfected individuals.

OBJECTIVES: Some individuals remain uninfected despite repeated exposure to HIV-1 [exposed-uninfected (EU)]. In addition to genetic factors, acquired immune responses elicited by repeated exposure to HIV antigens may contribute to protection. We investigated the ability of unstimulated CD8+ T lymphocytes from EU individuals to inhibit HIV-1 infection. METHODS: Peripheral blood CD8+ T lymphocytes from a well-characterized cohort of 16 HIV-1-discordant monogamous heterosexual couples were tested for their suppressive activity against HIV-1 strains displaying different coreceptor usage (R5, X4, X4R5). To evaluate the in vivo functional competence of CD8+ T cells, no ex vivo activatory stimuli were used prior to cocultivation with infected CD4+ T cells. In some experiments, a semi-permeable membrane was used to separate CD4+ and CD8+ T cells. RESULTS: Unstimulated CD8+ T cells from all but one of the EU individuals analysed effectively inhibited the growth of all HIV-1 strains, regardless of their coreceptor usage, with a mean potency similar to that of asymptomatic HIV-infected patients. The HIV-inhibitory activity persisted for a long time after ceasing high-risk sexual behaviour, although a moderate decline was observed starting 4 years after the last risk episode. Transwell culture experiments showed that soluble factors are involved in CD8-mediated viral suppression, although the activity was higher when cell-to-cell contact was allowed. CONCLUSIONS: These data demonstrate that CD8+ T cells from EU individuals exert a strong, broad-spectrum HIV-suppressive activity, suggesting a role of non-cytotoxic antiviral mechanisms in resistance to HIV-1 infection.

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.

Chemokine receptors in HIV-1 and SIV infection.

Seven transmembrane segment (7TMS) receptors for chemokines and related molecules have been demonstrated to be essential, in addition to CD4, for HIV and SIV infection. The beta-chemokine receptor CCR5 is the primary, perhaps sole, coreceptor for HIV-1 during the early and chronic phases of infection, and supports infection by most primary HIV-1 and many SIV isolates. Late-stage primary and laboratory-adapted HIV-1, HIV-2, and SIV isolates can use other 7TMS receptors. CXCR4 appears especially important in late-stage HIV infection; several related receptors can also be used. The specificity of SIV viruses is similar. Commonalities among these receptors, combined with analyses of mutated molecules, indicate that discrete, conformationally-dependent sites on the chemokine receptors determine their association with the third variable and conserved regions of viral envelope glycoproteins. These studies are useful for elucidating the mechanism and molecular determinants of HIV-1 entry, and of inhibitors to that entry.