Genetic and phenotypic features of blood and genital viral populations of clinically asymptomatic and antiretroviral-treatment-naive clade a human immunodeficiency virus type 1-infected women

In the present study, we assessed whether human immunodeficiency virus type 1 (HIV-1) genetic compartmentalization was associated with phenotypic CCR5 (R5) or CXCR4 (X4) coreceptor usage differences between the systemic and the genital viral populations. Four clinically asymptomatic and treatment-na?ve clade A HIV-1-infected patients were selected from a cohort of 274 African women, because they were free of all the biological cofactors known to modify the kinetics of viral production in the genital tract. HIV RNA envelope sequences (V1 to V3) derived from plasma and cervicovaginal secretions (CVS) were amplified, subcloned, and sequenced. CCR5 or CXCR4 coreceptor usage was determined by production of recombinant viral particles, followed by single-cycle infection assays of indicator cell lines, using the tropism recombinant test. In these four selected patients, CVS-derived sequences appeared to be genetically distinct from blood-derived sequences (P < or = 0.001). Two patients were found to harbor virus populations with only the R5 phenotype in both compartments, whereas viruses using CXCR4 in addition to CCR5 were detected in two other patients. In particular, one woman harbored genital virus populations with mixed R5 and X4 phenotypes associated with peripheral blood populations with only the R5 phenotype. These results demonstrate genetic compartmentalization of HIV between the plasma and genital secretions of clinically asymptomatic, treatment-na?ve, clade A-infected women. Also, for one patient, we report phenotypic coreceptor usage differences between the systemic (R5) and genital (R5/X4) viral populations. These features may be critical for the development of further mucosal vaccines, therapies, or new preventive strategies to block heterosexual transmission.     

Functional complementation of the envelope hypervariable V3 loop of human immunodeficiency virus type 1 subtype B by the subtype E V3 loop.

The hypervariable V3 loop within gp120 of human immunodeficiency virus type 1 (HIV-1) is the major determinant of cell tropism and the entry coreceptor usage of the virus. However, the information obtained thus far has been from only subtype B from North America and Europe, and little is known about other subtypes whose V3 amino acids differ by as much as 50% from subtype B V3. In this study, we examined the functional potential of the V3 element of the HIV-1 subtype E, the most crucial variant causing the AIDS epidemic throughout southeast Asia. A panel of HIV-1LAI recombinants was constructed by the overlap extension method, by which the LAI V3 loop was precisely replaced by that of the subtype E nonsyncytium-inducing (NSI) or syncytium-inducing (SI) variant. All of the recombinant viruses infected peripheral blood mononuclear cells, whereas only those with SI V3 infected MT2 cells, a CD4(+) T cell line. Consistently, the SI V3 recombinants used CXCR4, while the NSI V3 recombinants used CCR5 for infection of HOS-CD4(+) cells. Finally, only the NSI V3 sequence conferred CC-chemokine sensitivity on the parental virus. The data support the notion that the HIV-1 V3 loop consists of a relatively independent domain in gp120 and suggest that the subtype E V3 loop indeed contains the functional element to dictate the cell tropism, coreceptor preference, and chemokine sensitivity of the virus. These findings are of immediate importance in understanding V3 structure-function relationship and for examining phenotypic evolution of HIV-1 subtype E.     

Predicting HIV-1 coreceptor usage with sequence analysis

Bioinformatics approaches are increasingly being used to identify and understand the genetic variation underlying changes in HIV-1 biological phenotype. The variable regions of the viral envelope are the major determinant of virus coreceptor usage and cell tropism. Specifically, amino acids 11 and 25 in the 3rd variable (V3) loop have been found to strongly influence viral syncytium inducing capacity and coreceptor usage. Many additional V3 loop changes, however, as well as changes elsewhere in Env, are thought to contribute to phenotype. In this review we describe several recently developed methods to analyze this variability and their use to predict biological phenotype based on sequence information. These approaches have identified changes in the V3 loop, in addition to the known changes at positions 11 and 25, that affect phenotype and significantly enhance our ability to predict phenotype from genotype. Besides improving phenotype prediction, methods that score V3 sequences on a continuous scale can also assist in the interpretation of evolutionary information about shifts in phenotype, and the relationship between that evolution and pathogenesis. Several examples and potential practical applications of this scoring are discussed. We conclude that advances in computational approaches have enhanced both our ability to predict and to understand HIV-1 biological phenotype evolution. Further development of these methods, by extending analysis to regions outside the V3 loop and to clades beyond subtype B, will extend our understanding of HIV-1 pathogenesis and inform treatment strategies.     

Determining human immunodeficiency virus coreceptor use in a clinical setting: degree of correlation between two phenotypic assays and a bioinformatic model

Two recombinant phenotypic assays for human immunodeficiency virus (HIV) coreceptor usage and an HIV envelope genotypic predictor were employed on a set of clinically derived HIV type 1 (HIV-1) samples in order to evaluate the concordance between measures. Previously genotyped HIV-1 samples derived from antiretroviral-na?ve individuals were tested for coreceptor usage using two independent phenotyping methods. Phenotypes were determined by validated recombinant assays that incorporate either an approximately 2,500-bp ("Trofile" assay) or an approximately 900-bp (TRT assay) fragment of the HIV envelope gp120. Population-based HIV envelope V3 loop sequences ( approximately 105 bp) were derived by automated sequence analysis. Genotypic coreceptor predictions were performed using a support vector machine model trained on a separate genotype-Trofile phenotype data set. HIV coreceptor usage was obtained from both phenotypic assays for 74 samples, with an overall 85.1% concordance. There was no evidence of a difference in sensitivity between the two phenotypic assays. A bioinformatic algorithm based on a support vector machine using HIV V3 genotype data was able to achieve 86.5% and 79.7% concordance with the Trofile and TRT assays, respectively, approaching the degree of agreement between the two phenotype assays. In most cases, the phenotype assays and the bioinformatic approach gave similar results. However, in cases where there were differences in the tropism results, it was not clear which of the assays was "correct." X4 (CXCR4-using) minority species in clinically derived samples likely complicate the interpretation of both phenotypic and genotypic assessments of HIV tropism.     

Stable coreceptor usage of HIV in patients with ongoing treatment failure on HAART.

BACKGROUND: Disease progression in HIV infection has been associated with switch of viral coreceptor usage from CCR5 to CXCR4. OBJECTIVES: To investigate the relationship between HIV-coreceptor tropism and clinical and virological outcome in 40 heavily pretreated patients over time. METHODS: Coreceptor phenotype was predicted after sequencing the V3 loop of the HIV glycoprotein 120. RESULTS: Coreceptor use was stable during observation time in 87% of patients, and CCR5 tropism was predominant. Viral mutations in the pol gene and clinical parameters were not predictive for coreceptor switching. CONCLUSIONS: Even in patients with repeated HAART failure, CCR5 antagonists might be a valuable treatment option.     

Relationship between V3 genotype, biologic phenotype, tropism, and coreceptor use for primary isolates of human immunodeficiency virus type 1.

OBJECTIVES: The predictive value of positively charged amino acids at positions 11 and 25 within the V3 loop region of the human immunodeficiency virus type 1 (HIV-1) envelope gene for the syncytium-inducing (SI) phenotype was assessed. STUDY DESIGN/METHODS: Sequencing was performed on DNA extracted from primary peripheral blood mononuclear cells (PBMCs) and complementary DNA (cDNA) prepared from serial viral isolates from 10 HIV-1-seropositive subjects. Proviral DNA sequencing was also performed on biologic clones from most of these subjects. RESULTS: Positive charge at position 11 and/or 25 in 257 isolate cDNA, PBMC DNA, and biologic clone PBMC DNA sequences was compared with 69 phenotypic determinations, of which 62.3% were SI. V3 genotype was 51.2% sensitive and 85.8% specific for the SI phenotype, with positive and negative predictive values of 62.8% and 79.0%, respectively. Cellular tropism failed to correlate with V3 genotype, coreceptor use, or biologic phenotype. Exclusive use of CCR5 was associated with the nonsyncytium-inducing (NSI) phenotype. Overall, V3 loop charge was higher in SI than in NSI isolates (5.01 and 3.78, respectively; p = 0.0211). CONCLUSIONS: The predictive power of SI phenotype from V3 genotype is relatively weak, especially in a low SI prevalence population. The direct measurement of viral phenotype, cellular tropism, and coreceptor use in HIV-1 isolates is essential for accurate biologic characterization.     

Stable coreceptor usage of HIV in patients with ongoing treatment failure on HAART

BackgroundDisease progression in HIV infection has been associated with switch of viral coreceptor usage from CCR5 to CXCR4.ObjectivesTo investigate the relationship between HIV-coreceptor tropism and clinical and virological outcome in 40 heavily pretreated patients over time.MethodsCoreceptor phenotype was predicted after sequencing the V3 loop of the HIV glycoprotein 120.ResultsCoreceptor use was stable during observation time in 87% of patients, and CCR5 tropism was predominant. Viral mutations in the pol gene and clinical parameters were not predictive for coreceptor switching.ConclusionsEven in patients with repeated HAART failure, CCR5 antagonists might be a valuable treatment option.     

A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism

Human immunodeficiency virus type 1 (HIV-1) is characterized by sequence variability. The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism) and pathogenesis. This report describes a novel denaturing heteroduplex tracking assay (HTA) to analyze the genetic variation of HIV-1 V3 DNA. It improved upon previous non-denaturing HTA approaches to distinguish HIV-1 CCR5 and CXCR4 tropic viruses in mixed populations. The modifications included the use of a single-stranded fluorescent probe based on the consensus V3 sequence of HIV-1 CCR5 tropic viruses, Locked Nucleic Acid (LNA) "clamps" at both ends of heteroduplex DNA, and denaturing gel electrophoresis using Mutation Detection Enhancement (MDE(?)) as matrix. The analysis demonstrated that the LNA "clamps" increased its melting temperature (T(m)) and the thermal stability of heteroduplex DNA. The partially denaturing gel used a defined concentration of formamide, and significantly induced mobility shifts of heteroduplex DNA that was dependent on the number and patterns of DNA mismatches and insertions/deletions. This new technique successfully detected tropisms of 53 HIV-1 V3 clones of known tropism, and was able to separate and detect multiple V3 DNA variants encoding tropisms for CCR5 or CXCR4 in a mixture. The assay had the sensitivity to detect 0.5% minority species. This method may be useful as a research tool for analysis of viral quasispecies and for genotypic prediction of HIV-1 tropism in clinical specimens.     

Coreceptor usage and RANTES sensitivity of non-syncytium-inducing HIV-1 isolates obtained from patients with AIDS

OBJECTIVES: The biologic phenotype of HIV-1 primary isolates obtained from approximately 50% of patients who progress to AIDS switches from non-syncytium-inducing (NSI) to syncytium-inducing (SI). We evaluated possible associations between virus coreceptor usage, sensitivity to inhibition by beta-chemokines, and disease progression of patients who continue to yield NSI isolates after developing AIDS. STUDY DESIGN/METHODS: Sequential virus isolates were analyzed for biologic phenotype using the MT-2 cell assay, for sensitivity to beta-chemokines using RANTES inhibition, and for coreceptor usage using U87.CD4 and GHOST.CD4 cells expressing different chemokine/orphan receptors or donor peripheral blood mononuclear cells (PBMC) defective in CCR5 expression. In addition, the env V3 region was sequenced and the length of the V2 region determined. RESULTS: All NSI isolates, regardless of patient status at time of isolation, were dependent on CCR5 expression for cell entry. Furthermore, there was no indication of broadened coreceptor usage of NSI isolates obtained from persons with late-stage AIDS. A majority of NSI isolates remained RANTES sensitive; however, virus variants with reduced sensitivity were observed. The V2 lengths and the V3 sequences exhibited no or minor changes at analysis of sequential NSI isolates. CONCLUSIONS: Our data suggest that NSI isolates obtained from AIDS patients remain CCR5 dependent (ie, R5) and, in many cases, also remain sensitive to RANTES inhibition. However, virus variants with decreased sensitivity to RANTES inhibition may evolve during disease progression, not only as a result of a switch from NSI to SI but also in patients who develop AIDS while continuing to maintain R5 isolates.     

Prevalence of CCR5-tropic HIV-1 Among Treatment-Experienced Individuals in Spain

Abstract

Objective: To determine the prevalence of CCR5-tropic HIV-1 among treatment-experienced patients in Spain. Design: Epidemiologic, cross-sectional, and non-interventional study between January and June 2008 in HIV-1–infected patients in Spain. Methods: A total of 485 treatment-experienced patients from across Spainand with a plasma viral load of >1000 copies/mL were studied. Viral tropism, CD4+ cell count, plasma viral load, stage of disease, and current treatment strategies were determined. Univariate and multivariate logistic regression analyses were used to determine association of coreceptor use with patients’ characteristics. Results: Coreceptor usage was determined by viral tropism assays: 290 (68.9%) patients had CCR5-tropic HIV-1 virus, and 131 (31.1%) had dual-tropic/mixed or CXCR4 virus variants. Mean CD4+ cell counts in the R5 group (319.4 cells/mm3) were higher than in the non-R5 group (237.9 cells/mm3) (p = .0005). There was an inverse relationship between CD4+ cell counts and plasma viral load, but regression analyses on covariates associated with CCR5 tropism showed that only a higher CD4+ cell number was significantly associated with CCR5 coreceptor usage. Conclusions: The prevalence of CCR5-tropic HIV-1 among treatment-experienced patients in Spain is higher than previously found in other geographical settings. We did not find independent markers predictive of coreceptor usage other than a relationship with CD4+ levels.     

M嗜性HIV-1毒株一过性感染T细胞系的研究

目的了解早期分离毒株一过性感染Ｔ细胞的分子机制。方法将毒株在Ｔ细胞系培养后,在观察其生物学性状的同时,对外膜基因进行序列分析,确定毒株的基因变异,并结合异源双链泳动分析,了解病毒群的复杂度。结果序列分析显示这些病毒都是Ｍ嗜性、非介导融合型（ＮＳＩ）,与其生物学性状一致;在Ｔ细胞系传代过程中发生较大变异,都在不同程度上表现出偏离Ｍ嗜性、ＮＳＩ的序列特征;尽管有一毒株（编号：ＣＮ９７００１）在末代序列中已有介导融合型（ＳＩ）的特征,但却未能显示ＳＩ的表型。异源双链泳动分析显示,病毒群复杂度在传代刚开始时低,以后一直较高。结论结果提示,Ｍ嗜性、ＮＳＩ毒株为适应Ｔ细胞系,向不同变异方向作了尝试,但都未成功。虽然ＨＩＶ外膜基因与毒株的细胞嗜性、受体使用,与介导融合等特性有关,但这些表型的体现与否,还与毒株的整个基因组背景有关。在早期感染的Ｍ嗜性、ＮＳＩ毒株群中并没有Ｔ嗜性／ＳＩ毒株存在,是后来在体内随感染延续而产生的。     

Effects of sequence alterations on results from genotypic tropism testing

Backgroundgeno2pheno_[coreceptor] is a bioinformatic method for genotypic tropism determination (GTD) which has been extensively validated.ObjectivesGTD can be affected by sequencing/base-calling variability and unreliable representation of minority populations in Sanger bulk sequencing. This study aims at quantifying the robustness of geno2pheno_[coreceptor] with respect to these issues. GTD with a single amplification or in triplicate (henceforth singleton/triplicate) is considered.Study DesignFrom a dataset containing 67,997HIV-1 V3 nucleotide sequences, two datasets simulating sequencing variability were created. Further two datasets were created to simulate unreliable representation of minority variants. After interpretation of all sequences with geno2pheno_[coreceptor], probabilities of change of predicted tropism were calculated.Resultsgeno2pheno_[coreceptor] tends to report reduced false-positive rates (FPRs) when sequence alterations are present. Triplicate FPRs tend to be lower than singleton FPRs, resulting in a bias towards classifying viruses as X4-capable. Alterations introduced into nucleotide sequences by simulation change singleton predicted tropism with a probability ≤ 2%. Triplicate prediction lowers this probability for predicted X4 tropism, but raises it for predicted R5 tropism ≤ 6%. Simulated limited detection of minority variants in X4 sequences resulted in unchanged predicted tropism with probability above 90% as compared to probability above 98% with triplicate FPRs.Conclusionsgeno2pheno_[coreceptor] proved to be robust when sequence alterations are present and when detectable minorities are missed by bulk sequencing. Changes in tropism prediction due to sequence alterations as well as triplicate prediction are much more likely to result in false X4-capable predictions than in false R5 predictions.     

HIV-1 co-receptor usage based on V3 loop sequence analysis: preferential suppression of CXCR4 virus post HAART?

Disease progression during human immunodeficiency virus type 1 (HIV-1) infection has been associated with a switch of viral coreceptor usage from CCR5 to CXCR4. The current study investigates the effect of anti retroviral therapy (ART) on the viral tropism in a group of patients based on the V3 loop sequence, in ART na?ve patients prior to and 24 weeks after ART. Genomic DNA was extracted from the PBMCs of these patients, and the C2-V5 region of the HIV-1 env genes were cloned and sequenced. The coreceptor usage was predicated based on V3 loop amino acid sequences using Geno2pheno and PSSM programs. Our results indicate that following ART, the plasma viral loads of both CXCR4 and CCR5 viruses were significantly decreased. We observed a relatively higher ratio of R5 than X4 virus after 24 weeks of ART and both the positive charges and the net charges of the V3 regions were decreased significantly (p < 0.05) after ART. We conclude that ART significantly, reduced both X4 and R5 viruses with a preferential suppression of X4 virus. These data will help improve prognostic outcomes and help clinicians determine the course of treatment in patients who exhibit virologic failure while taking a CCR5 antagonist.     

HIV-1 CRF01_AE coreceptor usage prediction using kernel methods based logistic model trees

The determination of HIV-1 coreceptor usage plays a major role in HIV treatment. Since Maraviroc has been used in a treatment for patients those exclusively harbor R5-tropic strains, the efficient performance of classifying HIV-1 coreceptor usage can help choose the most advantaged HIV treatment. In general, HIV-1 variants are classified as R5-tropic and X4-tropic or dual/mixed tropic based on their coreceptor usages. The classification of the coreceptor usage has been developed by using the various computational methods or genotypic algorithms based on V3 amino acid sequences. Most genotypic tools have been designed based on a data set of the HIV-1 subtype B that seemed to be reliable only for this subtype. However, the performance of these tools decreases in non-B subtypes. In this study, the support vector machine (SVM) method has been used to classify the HIV-1 coreceptor. To develop an efficient SVM classifier, we present a feature selector using the logistic model tree (LMT) method to select the most relevant positions from the V3 amino acid sequences. Our approach achieves as high as 97.8% accuracy, 97.7% specificity, and 97.9% sensitivity measured by ten-fold cross-validation on 273 sequences.     

A Diagnostic HIV-1 Tropism System Based on Sequence Relatedness

Key clinical studies for HIV coreceptor antagonists have used the phenotyping-based Trofile test. Meanwhile various simpler-to-do genotypic tests have become available that are compatible with standard laboratory equipment and Web-based interpretation tools. However, these systems typically analyze only the most prominent virus sequence in a specimen. We present a new diagnostic HIV tropism test not needing DNA sequencing. The system, XTrack, uses physical properties of DNA duplexes after hybridization of single-stranded HIV-1 env V3 loop probes to the clinical specimen. Resulting “heteroduplexes” possess unique properties driven by sequence relatedness to the reference and resulting in a discrete electrophoretic mobility. A detailed optimization process identified diagnostic probe candidates relating best to a large number of HIV-1 sequences with known tropism. From over 500 V3 sequences representing all main HIV-1 subtypes (Los Alamos database), we obtained a small set of probes to determine the tropism in clinical samples. We found a high concordance with the commercial TrofileES test (84.9%) and the Web-based tool Geno2Pheno (83.0%). Moreover, the new system reveals mixed virus populations, and it was successful on specimens with low virus loads or on provirus from leukocytes. A replicative phenotyping system was used for validation. Our data show that the XTrack test is favorably suitable for routine diagnostics. It detects and dissects mixed virus populations and viral minorities; samples with viral loads (VL) of <200 copies/ml are successfully analyzed. We further expect that the principles of the platform can be adapted also to other sequence-divergent pathogens, such as hepatitis B and C viruses.     

Infection of macaques with an R5-tropic SHIV bearing a chimeric envelope carrying subtype E V3 loop among subtype B framework

Summary.  To establish simian/human immunodeficiency virus (SHIV) clones bearing a chimeric envelope carrying subtype E V3 loop among subtype B envelope, four subtype E V3 sequences were substituted into SHIV_MD14, a SHIV clone bearing an envelope derived from a CXCR4 (X4)/CCR5 (R5)-dual tropic subtype B HIV-1 strain. SHIV-TH09V3, an only V3-chimera clone capable of replicating in human and macaque peripheral blood mononuclear cells (PBMCs), was propagated in pig-tailed macaque PBMCs and in cynomolgus macaque splenic mononuclear cells. The propagated virus stocks were intravenously inoculated into respective macaque species. SHIV-TH09V3 infected both macaque species as shown by plasma RNA viremia, isolated viruses from PBMCs and plasma, and antibody production against viral proteins. To assess how the substituted V3 sequence affected coreceptor usage, SHIV-TH09V3 stocks propagated in vitro and after isolation from macaques were verified for their corecepor usage by GHOST cells assay. SHIV-TH09V3 maintained R5-tropic phenotype both in vitro and after isolation from macaques, in contrast to the X4/R5-dual tropic SHIV_MD14. This indicates the substituted V3 sequence among the backbone of SHIV_MD14 governs coreceptor usage. Future study of infecting macaques with SHIV-TH09V3 and SHIV_MD14 will focus on differences of the outcome caused by the different V3 sequences in connection with coreceptor usage. Received July 30, 2002; accepted November 13, 2002 Published online March 21, 2003     

Analysis of the functional relationship between V3 loop and gp120 context with regard to human immunodeficiency virus coreceptor usage using naturally selected sequences and different viral backbones

The human immunodeficiency virus type 1 (HIV-1) gp120 V3 loop plays a predominant role in chemokine receptor usage; however, other linear and nonlinear gp120 domains are involved in this step of the HIV-1 replication cycle. At present, the functional relationship between V3 and these domains with regard to coreceptor usage is unclear. To gain insights into the nature of this relationship in naturally selected viral variants, we developed a recombinant strategy based on two different gp120 backbones derived from CXCR4 (X4)- and CCR5 (R5)-tropic viral strains, respectively. Using this recombinant model system, we evaluated the phenotype patterns conferred to chimeric viruses by exogenous V3 loops from reference molecular clones and samples from infected subjects. In 13 of 17 recombinants (76%), a comparable phenotype was observed independently of the gp120 backbone, whereas in a minority of the recombinant viruses (4/17, 24%) viral infectivity depended on the gp120 context. No case of differential tropism using identical V3 sequence in the two gp120 contexts was observed. Site-directed mutagenesis experiments were performed to evaluate the phenotypic impact of specific V3 motifs. The data indicate that while the interaction of HIV-1 with chemokine receptors is driven by V3 loop and influenced by its evolutionary potential, the gp120 context plays a role in influencing the replication competence of the variants, suggesting that compensatory mutations occurring at sites other than V3 are necessary in some cases.     

Functional Characterization of the V1V2 Region of Human Immunodeficiency Virus Type I

The level of proviral DNA sequence variation in the V1V2 region was monitored over time in six HIV-1-infected individuals. Substitutional and length variation was observed, where the majority of length changes, ranging from 28 to 49 amino acids, was located within the V1 region. Evidence for convergent evolution in the V2 region was found. The functional significance of this variation was assessed by cloning the V1V2 sequences into an infectious molecular clone, HXB2. The majority of chimeras replicated, demonstrating that the sequences, though genetically distinct, were capable of conferring a viable phenotype. Chimeras expressing closely related sequences in a constant genetic background displayed different biological phenotypes, with respect to both cytopathicity and cell tropism. However, no association between primary V1V2 amino acid sequence and viability or cytopathicity of the chimeric virus was observed, suggesting that predictions of virus phenotype based on sequences alone may be incorrect. The effect of V1V2 variation on the overall gp120 conformation was measured by expressing the gp120 from a number of viable and nonviable clones. No differences were observed, suggesting that misfolding of the chimeric gp120 protein was not an explanation for the nonviability of some virus clones. Several chimeras were noncytopathic and only able to replicate in PBMC cultures, demonstrating that the V1V2 region, independent of the V3 sequence, is capable of defining both tropism and cytopathicity.     

Basic amino acid residues in the V3 loop of simian immunodeficiency virus envelope alter viral coreceptor tropism and infectivity but do not allow efficient utilization of CXCR4 as entry cofactor

In contrast to human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively), simian immunodeficiency virus (SIVmac) rarely uses CXCR4 (X4) for efficient entry into target cells. Basic amino acid residues in the V3 loop of HIV Env allow efficient coreceptor utilization of X4. Therefore, we investigated if similar changes in the SIVmac Env protein also mediate a coreceptor switch from CCR5 (R5) to X4. Functional analysis revealed that none of eight SIVmac variants, containing V3 regions with an overall charge between +4 and +10, efficiently utilized X4 as entry cofactor. Nonetheless, these alterations had differential effects on SIV coreceptor tropism and on Env expression levels. A single amino acid substitution of L328R, located near the tip of the V3 loop, resulted in grossly reduced Env expression levels and impaired viral infectivity. Notably, additional basic residues restored efficient Env expression and virion incorporation but not infectivity. In comparison to the L328R mutation, changes of P334K and D337K had little disruptive effects on SIVmac entry and replication. Interestingly, mutation of L320K and P321R disrupted coreceptor usage of GPR15 but not R5. These changes also impaired SIVmac replication in peripheral blood mononuclear cells (PBMC) derived from a Delta32/Delta32 donor but not in R5-expressing human or simian PBMC. Our results show that positively charged amino acid residues in the V3 loop affect SIVmac coreceptor tropism and infectivity but do not allow efficient utilization of X4.     

V3 sequence analysis and biological characterization of HIV-1 isolates from asymptomatic and early symptomatic Tanzanian individuals

The aim of this study was to determine HIV-1 V3 sequences, in vitro biological characteristics and co-receptor usage of virus isolates from Tanzania. Virus was isolated from 14 of 17 samples investigated. Four of the isolates induced syncytia in MT-2 cells and used the CXCR4 co-receptor, while the remaining 10 isolates used the CCR5 co-receptor characteristic of non-MT-2 tropic viruses. One of the four MT-2 tropic isolates also used the CCR5 and CCR3 co-receptors. Proviral DNA was detected in all 14 isolates and PCR products were subjected to DNA sequencing. Unambiguous V3 amino acid sequences were obtained from 11 amplificates. Phylogenetic analysis indicated that these sequences were divergent and clustered in HIV-1 subtypes A, C or D. Sequences from the viruses that induced syncytia in MT-2 cells presented characteristic V3 phenotype-associated amino acids. Results of co-receptor analysis are in concordance with the isolate phenotype as determined by replication and induction of syncytia in MT-2 cells. The considerable diversity illustrated by a limited number of isolates from Tanzania is in accordance with reports from other regions of Africa.