Coreceptor Tropism Research Articles

Improvement of HIV-1 coreceptor tropism prediction by employing selected nucleotide positions of the env gene in a Bayesian network classifier

This study aimed to develop a genotypic method to predict HIV-1 coreceptor usage by employing the nucleotide sequence of the env gene in a tree-augmented naive Bayes (TAN) classifier, and to evaluate its accuracy in prediction compared with other available tools. A wrapper data-mining strategy interleaved with a TAN algorithm was employed to evaluate the predictor value of every single-nucleotide position throughout the HIV-1 env gene. Based on these results, different nucleotide positions were selected to develop a TAN classifier, which was employed to predict the coreceptor tropism of all the full-length env gene sequences with information on coreceptor tropism currently available at the Los Alamos HIV Sequence Database. Employing 26 nucleotide positions in the TAN classifier, an accuracy of 95.6%, a specificity (identification of CCR5-tropic viruses) of 99.4% and a sensitivity (identification of CXCR4/dual-tropic viruses) of 80.5% were achieved for the in silico cross-validation. Compared with the phenotypic determination of coreceptor usage, the TAN algorithm achieved more accurate predictions than WebPSSM and Geno2pheno [coreceptor] (P<0.05). The use of the methodology presented in this work constitutes a robust strategy to identify genetic patterns throughout the HIV-1 env gene differently present in CCR5-tropic and CXCR4/dual-tropic viruses. Moreover, the TAN classifier can be used as a genotypic tool to predict the coreceptor usage of HIV-1 isolates reaching more accurate predictions than with other widely used genotypic tools. The use of this algorithm could improve the correct prescribing of CCR5 antagonist drugs to HIV-1-infected patients.

Sensitive Cell-Based Assay for Determination of Human Immunodeficiency Virus Type 1 Coreceptor Tropism

CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.

Longitudinal HIV-1 gp120-C2V3C3 phylogenetic surveillance and tropism evolution in patients under HAART

This 8-year longitudinal study was aimed to analyze the HIV-1 gp120-C2V3C3 sequence dynamics, their phylogenetic relationships and tropism evolution in patients under HAART. Such viral analysis comprised two compartments: plasma and PBMC. Fifty gp120-C2V3C3 genomic sequences were characterized from 16 patients: 41 from plasma when viremia was measurable and 9 from PBMCs if plasma viral load was undetectable. The vast majority of HIV isolates (43 out of 50) were ascribed to BF subtype, irrespective of the compartment (plasma or mononuclear-cells) analyzed. A statistically well-supported clustering phenomenon was observed for each patient sampling data. Each cluster comprised viral sequences from both compartments analyzed. In the vast majority of cases, the viral sequences obtained along active production periods were intermingled with those identified from proviral sequences. A substantial stability of co-receptor tropism for the HIV BF subtype was observed over an 8-year followup.

High prevalence of CXCR4 usage among treatment-naive CRF01_AE and CRF51_01B-infected HIV-1 subjects in Singapore

BackgroundRecent studies suggest HIV-1 inter-subtype differences in co-receptor usage. We examined the correlation between HIV-1 subtype and co-receptor usage among treatment-naïve HIV-1 subjects in Singapore. Additionally, we investigated whether the subtype co-receptor association was influenced by stage of infection.MethodsV3 sequences of HIV-1 envelope protein gp120 were obtained from 110 HIV treatment-naïve patients and genotypic co-receptor tropism determination was performed using Geno2pheno. Two false-positive rate (FPR) cut-offs, 10% and 5.75% were selected for tropism testing.ResultsSubtype assignment of viral strains from 110 HIV-infected individuals based on partial sequencing of HIV-1 pol, gp120 and gp41 were as follows: 27 subtype B, 64 CRF01_AE, 10 CRF51_01B, and 9 other subtypes. At FPR=10%, 10 (100%) CRF51_01B-infected subjects and 26 (40.6%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 7 (25.9%) subtype B subjects and 1 (11.1%) CRF33_01B-infected subject (P < 0.001). At FPR=5.75%, 10 (100%) CRF51_01B-infected subjects and 20 (31.3%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 4 (14.8%) subtype B and 1 (11.1%) CRF33_01B-infected subjects (P < 0.001). Among those with evidence of seroconversion within 2 years prior to study enrolment, 100% of CRF51_01B-infected subjects had CXCR4-using virus, independent of Geno2pheno FPR.ConclusionCRF51_01B and CRF01_AE-infected individuals have higher prevalence of CXCR4-usage compared to subtype B infected individuals. Further studies examining these differences could help optimise the use of CCR5-antagonist in populations with these subtypes, and increase our understanding of HIV-1 biology.

Detección de mutaciones de resistencia en ADN proviral en infección por VIH-1

Analysis of proviral DNA in HIV infection may have useful applications if techniques that are sufficiently sensitive to detect minor variants, such as single genome sequencing (SGS), are used. This technology, which is performed in DNA from whole blood, improves determination of co-receptor tropism and the detection of both circulating and archived resistance mutations (RM) that are undetectable in plasma. Many of these RM have clinical relevance and their identification could be useful in specific situations, such as the start of antiretroviral therapy to exclude the transmission of resistant strains, as information to be weighed in simplification strategies in patients with undetectable viral loads, and in antiretroviralexperienced patients with limited availability of clinical reports. Clonal analysis using SGS has yielded useful information on the pathogenesis and diagnosis of HIV-1 infection but, in its current design, is inappropriate for the clinical laboratory. The new generation of sequencing technology, which allows the simultaneous analysis of a large number of sequences, has begun to be applied in the analysis of the diversity of plasma HIV-1. The use of these highly sensitive, automated techniques in the analysis of HIV-1 cellular reservoirs will potentially allow all variants recorded during the history of the disease to become available. This information would be highly useful for the clinical management of HIV-1 infection at the individual and population level.

Characterization of a dual-tropic Human immunodeficiency virus (HIV-1) strain derived from the prototypical X4 isolate HXBc2

Human immunodeficiency virus type 1 (HIV-1) coreceptor usage and tropism can be modulated by the V3 loop sequence of the gp120 exterior envelope glycoprotein. For coreceptors, R5 viruses use CCR5, X4 viruses use CXCR4, and dual-tropic (R5X4) viruses use either CCR5 or CXCR4. To understand the requirements for dual tropism, we derived and analyzed a dual-tropic variant of an X4 virus. Changes in the V3 base, which allow gp120 to interact with the tyrosine-sulfated CCR5 N-terminus, and deletion of residues 310/311 in the V3 tip were necessary for efficient CCR5 binding and utilization. Thus, both sets of V3 changes allowed CCR5 utilization with retention of the ability to use CXCR4. We also found that the stable association of gp120 with the trimeric envelope glycoprotein complex in R5X4 viruses, as in X4 viruses, is less sensitive to V3 loop changes than gp120-trimer association in R5 viruses.

Concordance between HIV-2 genotypic coreceptor tropism predictions based on plasma RNA and proviral DNA

In this study, assessing HIV-2 tropism among 43 paired plasma/peripheral blood mononuclear cell specimens, the concordance between proviral DNA and plasma RNA genotypic tropism prediction was 74%. All the discordances were attributable to the prediction of R5 in RNA and X4/dual-mixed in DNA. HIV-2 genotypic tropism test based on proviral DNA is a suitable tool for tropism determination in HIV-2-infected patients with low or undetectable viral load.

Human immunodeficiency virus type 1 gp120 envelope characteristics associated with disease progression differ in family members infected with genetically similar viruses

The human immunodeficiency virus type 1 (HIV-1) envelope protein provides the primary contact between the virus and host, and is the main target of the adaptive humoral immune response. The length of gp120 variable loops and the number of N-linked glycosylation events are key determinants for virus infectivity and immune escape, while the V3 loop overall positive charge is known to affect co-receptor tropism. We selected two families in which both parents and two children had been infected with HIV-1 for nearly 10 years, but who demonstrated variable parameters of disease progression. We analysed the gp120 envelope sequence and compared individuals that progressed to those that did not in order to decipher evolutionary alterations that are associated with disease progression when individuals are infected with genetically related virus strains. The analysis of the V3-positive charge demonstrated an association between higher V3-positive charges with disease progression. The ratio between the amino acid length and the number of potential N-linked glycosylation sites was also shown to be associated with disease progression with the healthier family members having a lower ratio. In conclusion in individuals initially infected with genetically linked virus strains the V3-positive charges and N-linked glycosylation are associated with HIV-1 disease progression and follow varied evolutionary paths for individuals with varied disease progression.

Prevalence of Transmitted HIV-1 Drug Resistance Mutations in Children and Adolescents in São Paulo, Brazil

HIV drug resistance genotype testing, performed on 39 HIV-infected treatment-naive children from 2000 to 2011, identified 5 children (12.8%) with drug resistance mutations: 5.3% to nucleoside reverse transcriptase inhibitors, 5.3% to nonnucleoside reverse transcriptase inhibitors and 7.7% to protease inhibitors. There was a trend for increasing prevalence of drug resistance mutations during the 11-year study period.

Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

Genotypic HIV‐coreceptor tropism prediction with geno2pheno [coreceptor]: differences depending on HIV‐1‐subtype

Purpose of the studyDetermination of HIV‐1 coreceptor tropism is a major prerequisite before starting treatment with a CCR5‐antagonist. While most of the patients currently under treatment with maraviroc are probably infected with HIV‐1 subtype B viruses, recently published data show differences in the distribution of coreceptor tropism in different HIV‐1 subtypes.MethodsIn a Germany‐wide project within the HIV‐GRADE society, V3‐loop sequences of 2466 isolates were analysed with geno2pheno for coreceptor tropism using a FPR cut‐off of 10%. HIV‐1 subtype was determined by using the COMET HIV subtyping tool. Sequences consisted of at least the V3 loop fragment. The ratio of CCR5 vs CXCR4 tropic viruses was calculated for each subtype. A normalized mean for all analyzed subtypes was calculated to extrapolate the overall ratio of coreceptor usage distribution. From this the expected distribution in the particular subtype was calculated and compared to the observed one. Statistical analysis was performed using the chi2 test.Summary of ResultsMost samples were classified as HIV‐1 subtype B (79%, n=1952). Other subtypes present in at least 23 samples were A1 (9.5%, n=234), C (4.8%, n=118), CRF01_AE (2.2%, n=55), G (1.6%, n=39), D (1.1%, n=27), F (0.9%, n=23). The calculated normalized mean distribution over all subtypes was 71% CCR5‐ vs. 29% CXCR4‐tropic viruses. No significant difference compared to the mean distribution could be observed for HIV‐1 subtypes B (71/29%), C (76/24%) and F (70/30%). Higher rates of CXCR4 tropic virus were detected in subtypes D (52/48%, p=0.01) and CRF01_AE (49/51%, p=0.001), while in HIV‐1 subtypes A1 (22/78%, p=0.02) and G (13/87%, p=0.02), a higher rate of CCR5‐tropic virus was observed.ConclusionsOur analysis shows a different distribution of CCR5 and CXCR4 tropic virus in some subtypes. In contrast to other publications, we could not observe a statistically significant difference in subtype C compared to the overall mean distribution, while we could confirm a higher rate of CXCR4‐tropic virus in subtype D, as previously described. Without further data on treatment success of patients with non‐B subtypes under treatment with maraviroc, it remains unclear if subtype‐specific differences in the distribution of tropism are biased by differences in clinical variables before test or if there is a bias in the tropism interpretation system. In the latter case, individual interpretation cut‐offs for different subtypes may be necessary.

No significant association between patient self‐reported non‐adherence to antiretrovirals and HIV‐tropism: a preliminary analysis

Nonadherence to antiretroviral therapy (ART) may cause virologic failure and disease progression has been associated with switch of viral coreceptor usage from CCR5 to CXCR4. We aimed to assess the association between patient-reported non-adherence and HIV tropism. This is a cross-sectional analysis. HIV-tropism was performed within routine clinical practice either at start of ART or at virological failure. Adherence questionnaire includes: how many times ART has been taken during the last month, missed doses in the last week, timing deviation, refill interruption, drug holidays. Demographics, epidemiological data, HIV and ART history, CD4 and HIVRNA were collected. To assess co-receptor tropism, env V3 genotyping from viremic plasma HIVRNA was performed. For the analysis, dual/mixed viruses were considered as X4. We included 102 individuals: 76% males; median age 42 y (IQR, 37–46); transmission was heterosexual 37%, homosexual 31%, intravenous drug use 29%. Median nadir of CD4 154/mmc (IQR, 53–274), median zenith of HIVRNA 5.26 (4.72–5.70), 46% had AIDS. 124 tropism tests were: 78% R5, 17% X4, 5% dual/mixed. In cases with previous ART, mono/dual ART was found in 26%, median number of regimens was 5 (IQR, 2–10), median time on triple-ART was 54 months (IQR, 0–123) with median time of HIVRNA <50 c/ml of 16 months (IQR, 6.5–34.9). At HIV-tropism, median CD4 and HIV RNA were 321/mmc (IQR, 210–436) and 2.65 (IQR, 2.65–4.91), respectively. Median time between adherence questionnaire and HIV-tropism was 68 days (IQR, 23–116). At adherence questionnaire, median percentage of ART taken during the last month was 100% (IQR, 90–100), 39% reported missed doses in the last week, 40% timing deviation, 7% refill interruption, 17% drug holidays. At univariate analysis, no statistically significant association between non-adherence and dual/mixed-X4 viruses was found (p>0.1). Also gender, age, HIV transmission, AIDS, CD4 nadir, HIVRNA zenith, mono/dual ART, and number of ART regimens were not associated with type of tropism. Only longer time with undetectable HIVRNA before tropism test showed a lower probability of dual/mixed-X4 viruses (OR for each month 0.95; 95% CI 0.90–1.00; p=0.06). No significant association between adherence and HIV-tropism was found in this preliminary analysis. It is possible that patient self-reported adherence is not able to capture nonadherence behaviors that underlie more pronounced viral replication which may be necessary for tropism switch.

The MOTIVATE trials: maraviroc therapy in antiretroviral treatment-experienced HIV-1-infected patients

Although the use of combination antiretroviral therapy has resulted in spectacular improvements in morbidity and mortality of HIV-1 infected patients, a need for the development of antiretroviral compounds with new mechanisms of action remains. Maraviroc (Celsentri®; ViiV Healthcare, Middlesex, UK) is the only drug of the class of chemokine (C-C motif) receptor 5 antagonists registered for treatment for HIV-1-infected antiretroviral therapy-experienced patients. Registration was based on the MOTIVATE-1 and -2 studies, which compared the efficacy and tolerability of maraviroc in combination with optimized background therapy with placebo. The aim of this paper is to review the MOTIVATE studies and to discuss issues related to maraviroc therapy in clinical practice such as assessment of HIV-1 coreceptor tropism.

Natural evolution of CD4+ cell count in patients with CD4 &gt;350 or &gt;500 cells/mm3 at the time of diagnosis according to HIV‐1 coreceptor tropism

The HIV-1 coreceptor usage may play a critical role in AIDS pathogenesis and the X4-using viruses are considered to be more pathogenic than the R5-tropic viruses. These observations may influence the therapeutic decisions by asking for an earlier antiretroviral (ARV) treatment for the patients infected by the X4-tropic viruses compared with those infected by the R5-tropic viruses. The natural evolution of CD4+ cell count for 109 non-treated patients infected by the R5- or X4-tropic HIV-1 viruses with CD4+ >350 and >500 cells/mm(3) at time of diagnosis was compared until the initiation of an ARV regimen. The coreceptor usage was determined from the V3 env region sequence by Geno2Pheno (false positive rate 10%). A mixed linear regression model to analyse the CD4+ data with tropism as fixed effect in the model was used. Overall, 93 (85.3%) and 16 (14.7%) were infected by R5- and X4-tropic viruses, respectively. The median age, baseline CD4+ cell count, and viral load were 34 years (IQR: 30-42), 523 cells/mm(3) (IQR: 420-604), and 4.5 log(10) copies/ml (IQR: 3.9-5.0), respectively. There was no statistical difference in time to progression between the patients harboring R5- or X4-tropic viruses. The same results were observed for the sub-group of patients with CD4+ cell count >500 cells/mm(3). The virus tropism has no impact on the CD4+ cell count evolution in these HIV-1 patients diagnosed with CD4+ >350 or >500 cells/mm(3) suggesting that the tropism determination at time of diagnosis does not seem to be a useful tool to predict the clinical progression.

Appraising the performance of genotyping tools in the prediction of coreceptor tropism in HIV-1 subtype C viruses

BackgroundIn human immunodeficiency virus type 1 (HIV-1) infection, transmitted viruses generally use the CCR5 chemokine receptor as a coreceptor for host cell entry. In more than 50% of subtype B infections, a switch in coreceptor tropism from CCR5- to CXCR4-use occurs during disease progression. Phenotypic or genotypic approaches can be used to test for the presence of CXCR4-using viral variants in an individual’s viral population that would result in resistance to treatment with CCR5-antagonists. While genotyping approaches for coreceptor-tropism prediction in subtype B are well established and verified, they are less so for subtype C.MethodsHere, using a dataset comprising V3 loop sequences from 349 CCR5-using and 56 CXCR4-using HIV-1 subtype C viruses we perform a comparative analysis of the predictive ability of 11 genotypic algorithms in their prediction of coreceptor tropism in subtype C. We calculate the sensitivity and specificity of each of the approaches as well as determining their overall accuracy. By separating the CXCR4-using viruses into CXCR4-exclusive (25 sequences) and dual-tropic (31 sequences) we evaluate the effect of the possible conflicting signal from dual-tropic viruses on the ability of a of the approaches to correctly predict coreceptor phenotype.ResultsWe determined that geno2pheno with a false positive rate of 5% is the best approach for predicting CXCR4-usage in subtype C sequences with an accuracy of 94% (89% sensitivity and 99% specificity). Contrary to what has been reported for subtype B, the optimal approaches for prediction of CXCR4-usage in sequence from viruses that use CXCR4 exclusively, also perform best at predicting CXCR4-use in dual-tropic viral variants.ConclusionsThe accuracy of genotyping approaches at correctly predicting the coreceptor usage of V3 sequences from subtype C viruses is very high. We suggest that genotyping approaches can be used to test for coreceptor tropism in HIV-1 group M subtype C with a high degree of confidence that they will identify CXCR4-usage in both CXCR4-exclusive and dual tropic variants.

Correlation of coreceptor usage and disease progression

Primary HIV-1 infection is usually initiated by viruses with an exclusive affinity for the C-C chemokine receptor type 5 (CCR5) coreceptor. Viral variants that are also able to bind the C-X-C chemokine receptor type 4 (CXCR4) coreceptor arise during the course of the disease in about 50% of the infected individuals and their emergence is associated with a faster disease progression. In this article we provide a historical overview of the events that led to the discovery of the relationship between viral phenotype, coreceptor tropism and pathogenesis. The prevalence of CCR5 and CXCR4-using viruses differs from study to study, but overall percentages of CXCR4 use fluctuate between 2.0 and 63.0%. The association between coreceptor use and disease stage is recognized, with the lowest X4 prevalence in seroconverters and the highest in the final stage of the disease. Up to date there are insufficient arguments to support an impact of coreceptor tropism on response to combined antiretroviral therapy (cART) or an impact of cART on coreceptor tropism evolution. This review provides an overview of available data on coreceptor use in the different stages of the HIV-1 infection process. Although it is clear that CXCR4-using viruses emerge during the course of infection, the driving forces and mechanisms behind coreceptor switch remain largely unknown.

HIV population genotypic tropism testing and its clinical significance

HIV co-receptor tropism testing is recommended before therapy when the C-C chemokine receptor type 5 antagonist maraviroc is initiated. This review addresses the use of population genotypic tropism testing in relation to clinical practice. Genotypic tropism tests predict viral co-receptor tropism based on the sequence of the V3 loop of the viral envelope. HIV occurs in a swarm of variants in the patient's body, called quasispecies. As next-generation sequencing techniques are not generally accessible to date, triplicate testing is often performed to improve sensitivity of population-based approaches, but no prospective studies assessing the performance of single and triplicate procedures related to clinical outcome have been performed yet. For interpretation of the genotype several web-based algorithms have been developed. Varying the cut-off of the commonly used geno2pheno[co-receptor] algorithm changes the sensitivity and specificity of the tropism prediction. In retrospective analyses of clinical studies and cohorts genotypic population tropism testing demonstrated equal correlation with clinical outcome on maraviroc compared with phenotypic assays.In patients with suppressed viraemia, proviral DNA testing is a well tolerated alternative to HIV-RNA testing. Population genotypic methods have greater accessibility, lower cost, and faster turnaround time than other methods. Despite limited sensitivity for minority variants HIV genotypic population tropism testing showed good correlation with clinical outcome.

HIV co-receptor tropism

HIV co-receptor tropism

An expanded model of HIV cell entry phenotype based on multi-parameter single-cell data

BackgroundEntry of human immunodeficiency virus type 1 (HIV-1) into the host cell involves interactions between the viral envelope glycoproteins (Env) and the cellular receptor CD4 as well as a coreceptor molecule (most importantly CCR5 or CXCR4). Viral preference for a specific coreceptor (tropism) is in particular determined by the third variable loop (V3) of the Env glycoprotein gp120. The approval and use of a coreceptor antagonist for antiretroviral therapy make detailed understanding of tropism and its accurate prediction from patient derived virus isolates essential. The aim of the present study is the development of an extended description of the HIV entry phenotype reflecting its co-dependence on several key determinants as the basis for a more accurate prediction of HIV-1 entry phenotype from genotypic data.ResultsHere, we established a new protocol of quantitation and computational analysis of the dependence of HIV entry efficiency on receptor and coreceptor cell surface levels as well as viral V3 loop sequence and the presence of two prototypic coreceptor antagonists in varying concentrations. Based on data collected at the single-cell level, we constructed regression models of the HIV-1 entry phenotype integrating the measured determinants. We developed a multivariate phenotype descriptor, termed phenotype vector, which facilitates a more detailed characterization of HIV entry phenotypes than currently used binary tropism classifications. For some of the tested virus variants, the multivariant phenotype vector revealed substantial divergences from existing tropism predictions. We also developed methods for computational prediction of the entry phenotypes based on the V3 sequence and performed an extrapolating calculation of the effectiveness of this computational procedure.ConclusionsOur study of the HIV cell entry phenotype and the novel multivariate representation developed here contributes to a more detailed understanding of this phenotype and offers potential for future application in the effective administration of entry inhibitors in antiretroviral therapies.

Higher Prevalence of Predicted X4-Tropic Strains in Perinatally Infected Older Children With HIV-1 Subtype C in India

Coreceptor switch from CCR5 to CXCR4 is considered to be less common in HIV-1 subtype C even in advanced stages of infection. In this study, we have examined viral genotypic coreceptor tropism and its clinical, virological, and host genetic determinants among perinatally infected children in India. Genotypic coreceptor tropism analysis was conducted on env V3 sequences using Geno2pheno with a threshold of 10% false-positive rate. A total of 473 sequences were obtained from 72 isolates amplified from children aged 2-17 years. Factors associated with viral tropism in subtype C infections were studied using logistic regression. Among the samples, 98.6% (71 of 72) were HIV-1 subtype C. Coreceptor tropism analysis determined 81.7% (58 of 71) as R5 tropic, 9.9% (7 of 71) as X4 tropic, and 8.5% (6 of 71) as R5/X4 tropic or dual-tropic HIV-1 strains. Children with X4 or R5/X4 strains were more likely to be older than those with R5-tropic strains (P < 0.05), have lower CD4 counts (P < 0.05), and have viral populations with greater intrapopulation viral divergence (P < 0.01). Older age was a significant independent predictor for X4 or R5/X4 tropism in these children (P < 0.05). None were identified as being heterozygous or homozygous for the CCR5[INCREMENT]32 deletion. The high prevalence of X4 and R5/X4 tropic strains among older perinatally infected children with HIV-1 subtype C in India indicate that this phenomenon is not uncommon as previously thought and suggest that coreceptor transition can occur with longer duration of infection and greater disease progression in this population of perinatally infected children living with HIV-1 subtype C.