Covariation of amino acid positions in HIV-1 protease

We have examined patterns of sequence variability for evidence of linked sequence changes in HIV-1 subtype B protease using translated sequences from protease inhibitor (PI) treated and untreated subjects downloaded from the Stanford HIV RT and Protease Sequence Database (http://hivdb.stanford.edu). The final data set size was 648 sequences from untreated subjects (notx) and 531 for PI-treated subjects (tx). Each subject was uniquely represented by a single sequence. Mutual information was calculated for all pairwise comparisons of positions with nonconsensus amino acids in at least 5% of sequences; significance of pairwise association was assessed using permutation tests. In addition pairs of positions were assessed for linkage by comparing the observed occurrences of amino acid combinations to expected values. The mutual information statistic indicated linkage between nine pairs of sites in the untreated data set (10:93, 12:19, 35:38, 37:41, 62:71, 63:64, 71:77, 71:93, 77:93). Strong statistical support for linkage in the treated data set was seen for 32 pairs, eight involving position 10:7 involving position 71, with the rest being 12:19, 15:77, 20:36, 30:88, 35:36, 35:37, 36:62, 36:77, 46:82, 46:84, 48:54, 48:82, 54:82, 63:64, 63:90, 73:90, 77:93, and 84:90. Most associations were positive, although negative associations were seen for five pairs of interactions. Structural proximity suggests that numerous pairs may interact within a local environment. These interactions include two distinct clusters around 36/77 and 71/93. While some of these interactions may reflect fortuitous linkage in heavily treated subjects with many resistance mutations, others will likely represent important cooperative interactions that are amenable to experimental validation.     

Ancestral and consensus envelope immunogens for HIV-1 subtype C

Immunogens based on "centralized" (ancestral or consensus) HIV-1 sequences minimize the genetic distance between vaccine strains and contemporary viruses and should thus elicit immune responses that recognize a broader spectrum of viral variants. However, the biologic, antigenic and immunogenic properties of such inferred gene products have to be validated experimentally. Here, we report the construction and characterization of the first full-length ancestral (AncC) and consensus (ConC) env genes of HIV-1 (group M) subtype C. The codon-usage-optimized genes expressed high levels of envelope glycoproteins that were incorporated into HIV-1 virions, mediated infection via the CCR5 co-receptor and retained neutralizing epitopes as recognized by plasma from patients with chronic HIV-1 subtype C infection. Guinea pigs immunized with AncC and ConC env DNA developed high titer binding, but no appreciable homologous or heterologous neutralizing antibodies. When tested by immunoblot analysis, sera from AncC and ConC env immunized guinea pigs recognized a greater number of primary subtype C envelope glycoproteins than sera from guinea pigs immunized with a contemporary subtype C env control. Mice immunized with AncC and ConC env DNA developed gamma interferon T cell responses that recognized overlapping peptides from the cognate ConC and a heterologous subtype C Env control. Thus, both AncC and ConC env genes expressed functional envelope glycoproteins that were immunogenic in laboratory animals and elicited humoral and cellular immune responses of comparable breadth and magnitude. These results establish the utility of centralized HIV-1 subtype C Env immunogens and warrant their continued evaluation as potential components of future AIDS vaccines.     

Phylogenetic structure in African HIV-1 subtype C revealed by selective sequential pruning

Subtype C is the most prevalent clade in the HIV-1 pandemic. Previous studies suggested that African HIV-1 subtype C lacks a well-defined phylogenetic structure. Here we show that, by sequential pruning of ambiguously positioned taxa, a well-defined intrasubtype C phylogenetic structure becomes apparent, with 52% African HIV-1 subtype C isolates analyzed in envelope sequences branching within 11 clusters, also supported in a tree of full-length genomes, and all with geographical associations. Among 46 viruses recently transmitted in South Africa, 70% branched within 7 clusters (41% in the largest one) and 15% additional isolates were intercluster recombinants. Choice of the outgroup sequence and inclusion of intrasubtype recombinant viruses in the analyses could greatly affect support of clusters. The identification of clusters comprising a large proportion of African HIV-1 subtype C viruses may have implications for the design of vaccines intended for use in areas where subtype C is prevalent.     

Subtype-specific conservation of isoleucine 309 in the envelope V3 domain is linked to immune evasion in subtype C HIV-1 infection

The V3 region of the HIV-1 envelope (Env) glycoprotein gp120 is a key functional domain yet it exhibits distinct mutational patterns across subtypes. Here an invariant residue (Ile 309) was replaced with Leu in 7 subtype C patient-derived Envs from recent infection and 4 related neutralizing antibody escape variants that emerged later. For these 11 Envs, I309L did not alter replication in primary CD4 T cells; however, replication in monocyte-derived macrophages was enhanced. Infection of cell lines with low CD4 or CCR5 revealed that I309L enhanced utilization of CD4 but did not affect the ability to use CCR5. This CD4-enhanced phenotype tracked with sensitivity to sCD4, indicating increased exposure of the CD4 binding site. The results suggest that Ile 309 preserves a V3-mediated masking function that occludes the CD4 binding site. The findings point to an immune evasion strategy in subtype C Env to protect this vulnerable immune target.     

Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 subtype C infected patients in Northern India

BACKGROUND: Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients. OBJECTIVES: The objective of this study was to develop and validate an affordable one step real-time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients. RESULTS: We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients. CONCLUSIONS: This RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India.     

High replication fitness and transmission efficiency of HIV-1 subtype C from India: Implications for subtype C predominance

HIV-1 subtype C has been the predominant subtype throughout the course of the HIV-1 epidemic in India regardless of the geographic region of the country. In an effort to understand the mechanism of subtype C predominance in this country, we have investigated the in vitro replication fitness and transmission efficiency of HIV-1 subtypes A and C from India. Using a dual infection growth competition assay, we found that primary HIV-1 subtype C isolates had higher overall relative fitness in PBMC than subtype A primary isolates. Moreover, in an ex vivo cervical tissue derived organ culture, subtype C isolates displayed higher transmission efficiency across cervical mucosa than subtype A isolates. We found that higher fitness of subtype C was not due to a trans effect exerted by subtype C infected PBMC. A half genome A/C recombinant clone in which the 3′ half of the viral genome of subtype A was replaced with the corresponding subtype C3′ half, had similar replicative fitness as the parental subtype A. These results suggest that the higher replication fitness and transmission efficiency of subtype C virus compared to subtype A virus from India is most probably not due to the envelope gene alone and may be due to genes present within the 5′ half of the viral genome or to a more complex interaction between the genes located within the two halves of the viral genome. These data provide a model to explain the asymmetric distribution of subtype C over other subtypes in India.     

Sensitive detection of the K103N non-nucleoside reverse transcriptase inhibitor resistance mutation in treatment-naïve HIV-1 infected individuals by rolling circle amplification

Primary or transmitted antiretroviral drug resistance mutations pose a significant obstacle for optimizing antiviral treatment. When present at low-levels, resistance mutations are less likely to be detected by standard genotyping assays. This study utilizes a novel rolling circle amplification (RCA) method using padlock probes to achieve the sensitive, specific and low-level detection of the NNRTI resistance K103N from 59 HIV+ treatment-naïve patients from Beijing, China. Using standard genotyping methods, primary drug resistance mutations to either protease or RT inhibitors were found in 25% (15/59) of patients attending hospital clinics in Beijing. Among these 15 patients with antiretroviral (ARV) resistance mutations, standard sequence-based genotyping revealed that most (10/15) had the 103N. Using a highly sensitive RCA assay, 5 more patients among the 59 treatment-naïve cohort were found to have the 103N, but at low-levels, leading to an overall rate of 103N at 25.4% (15/59) in this population. The high prevalence of the 103N suggests that baseline resistance testing should be performed before treatment in this population. Importantly, the new RCA technology allows large-scale, sensitive detection of drug resistance mutations, including detection of minority populations with minimal equipment requirement.     

HIV-1 C亚型密码子优化gp120负载人DC疫苗的构建及体外功能

目的构建HIV-1C亚型gp120负载人树突状细胞(dentriti ccell,DC)疫苗,并对其体外功能进行初步检测。方法利用Amaxa细胞核转染技术将pcDNA3.1-gp120质粒转染至人成熟DC,以Western blot检测gp120的表达。通过流式细胞仪检测DC表面共刺激分子的变化、混合淋巴细胞反应、CD8+T细胞表面活化分子CD25的表达及其分泌IFN-γ的变化。结果通过Western blot检测,gp120在DC中得到了正确表达。经流式细胞仪检测,DC表面分子CD80表达率由刺激前的33.34%上升至43.20%,CD86表达率由刺激前的60.08%上升至90.34%;负载gp120DC刺激淋巴细胞增殖率为86.72%;CD8+T细胞表面分子CD25表达率由刺激前的5.27%上升至74.21%,IFN-γ的表达率达37%。结论负载了HIV-1gp120的人树突状细胞能够显著刺激淋巴细胞的增殖、增强CD8+T细胞表面活化分子CD25表达以及促进CD8+T细胞分泌IFN-γ,为下一步DC治疗性疫苗的体内研究奠定基础。     

HIV-1 subtype C in vitro growth and coreceptor utilization

Human immunodeficiency virus type 1 subtype C (HIV-1C) accounts for about 50% of all HIV infections in the pandemic and is the predominant subtype in the heavily burdened region of southern Africa. HIV-1C possesses unique genetic and phenotypic features that might be associated with biological differences compared to other subtypes. Here, we generated virus isolates from individuals at different stages of HIV-1C infection and investigated the chemokine receptor repertoire that the derived HIV-1C isolates may utilize for entry. Our results show that the R5 phenotype predominates among viruses in Botswana, with a lesser contribution of viruses showing the dualtropic X4R5 phenotype. No viruses of pure X4 phenotype were found, which suggests no discernable evolution of HIV-1C to a monotropic X4 phenotype as the epidemic ages in Botswana. Usage of other coreceptors was rare and apparently insignificant. These results enhance our understanding of HIV-1C biology, with implications for designing and testing therapeutic and prophylactic agents.     

淮安市经异性性传播的HIV感染者中HIV-1病毒基因亚型特征分析

目的 了解淮安市经异性性传播HIV感染者中HIV-1病毒基因亚型分布及其流行特征.方法 从60名异性性传播HIV-1感染者血液中提取DNA或RNA,用巢式PCR或RT-PCR方法扩增gag、env基因区片段,测定序列并分析.结果 60份标本中,确定了48份标本的基因亚型,发现有4种HIV-1亚型和重组型.其中CRF01_AE亚型占62.5％,CRF07_BC亚型占22.9％,CRF08_BC亚型占6.3％,B亚型占6.3％.结论淮安市异性性传播感染HIV者中,流行着CRF01_AE、CRF07_BC、CRF08_BC、B亚型这4种亚型病毒株,CRF01_AE为主要流行亚型.     

Molecular determinants of HIV-1 subtype C coreceptor transition from R5 to R5X4

The molecular mechanism(s) underlying transition from CCR5 to CXCR4 usage of subtype C viruses remain largely unknown. We previously identified a subtype C HIV-1 infected child whose virus demonstrated CXCR4 usage along with CCR5 upon longitudinal follow-up. Here we delineated the molecular determinants of Env involved in expanded coreceptor usage. Residue changes in three positions of Env V3 domain are critical for the dual tropic phenotype. These include: substitution of arginine at position 11, MG or LG insertion between positions 13 and 14, and substitution of threonine at the position immediately downstream of the GPGQ crown. Introducing these mutations into V3 region of a heterologous R5 virus also conferred dual tropism. Molecular modeling of V3 revealed a possible structural basis for the dual tropic phenotype. Determining what defines a subtype C X4 virus will lead to a better understanding of subtype C HIV-1 pathogenesis, and will provide important information relevant to anti-retroviral therapy.     

HIV-1 黑龙江省分离株CHNHLJ03009包膜克隆及分析

目的 分析黑龙江省Ⅰ型人免疫缺陷病毒（human irnmunodeficiency virus type 1，HIV-1）原代分离株包膜糖蛋白的变异性及表型特征。方法从一名HIV阳性但未发病的感染者外周血单个核细胞提取DNA，采用保守引物进行HIV包膜直接克隆，进行序列分析及系统树分析。构建了一株带该包膜蛋白的伪病毒并利用表达CXCR4和CCR5的靶细胞进行了感染实验，观察该病毒包膜利用辅助受体的表型特征。结果共获得2个有功能全长env克隆，分别命名为CHNHIJ03009c34（GenBank序列号为AY905493）和CHNHIA03009c33。用全长env氨基酸序列与国内外分离株进行同源性比较分析发现，与分离株CHNHLJ03009c34的包膜同源性最高的病毒为云南的HIV-1 B’亚型分离株RIA2，同源性为91．52％。系统发育分析结果表明，该株为泰国B’亚型，与云南分离株RIA2的遗传距离最近。对包膜蛋白结构分析结果显示，分离株CHNHLJ03009c34包膜在抗原性和亲水性上与RIA2没有明显差别。感染性检测结果显示该病毒只能感染U87．CD4．CCR5细胞，不能感染U87．CD4．CX-CR4细胞。结论本研究从黑龙江省一名HIV-1阳性者克隆到HIV-1CHNHLJ03009病毒的包膜基因，该病毒属于B’亚型（泰国亚型），为R5亲嗜性毒株。该包膜基因克隆为首次报道的黑龙江分离株。     

Interactive association of proviral load and IFN-gamma-secreting T cell responses in HIV-1C infection

We investigated the interactive relationship between proviral DNA load and virus-specific IFN-gamma-secreting T cell responses in HIV-1C infection. The presence or absence of correlation, and inverse or direct type of correlation, if any, were dependent on targeted viral gene product. Responses to Gag p24 or to Pol were associated with lower proviral DNA load. Associations between proviral DNA load and T cell responses did not necessarily mirror relationships between plasma RNA load and T cell responses. An interaction analysis showed a synergy in that lower proviral DNA and lower plasma RNA load were associated with high Gag p24-specific IFN-gamma-secreting T cell response (interaction test P = 0.0003). Our findings support the idea that HIV proteins have differential value for vaccine design, and suggest that, for HIV-1C, Gag p24 may be one of the most attractive regions to include in vaccine designs to control both plasma RNA load and cell-associated proviral DNA load.     

Evolutionary gamut of in vivo Gag substitutions during early HIV-1 subtype C infection

Two analyses of HIV-1 subtype C Gag quasispecies were performed in a prospective cohort of 42 acutely and recently infected individuals by SGA on viral RNA/proviral DNA templates. First, in vivo Gag substitutions were assessed in relation to the HIV-1C consensus sequence, which revealed that 29.3% of detected amino acid substitutions can be classified as reversions to subtype consensus, 61.3% as forward substitutions from subtype consensus, and 9.3% as polymorphisms not associated with the subtype consensus sequence. Second, the proportion, dynamics, and relationships within individual pools of viral quasispecies were analyzed. Among reverse substitutions, 16.1% were minor, 11.0% transient, 13.6% dominant, and 59.2% fixed. In contrast, 31.6% of forward substitutions were minor, 59.3% transient, 3.8% dominant, and 5.3% fixed. The distinct patterns in the spectrum and dynamics of reverse and forward Gag substitutions suggest that these differences should be considered in HIV-1 evolutionary studies and analyses of viral mutational pathways.     

Cloning and analysis of the envelope protein clone of HIV-1, CHNHLJ03009c34 from an infected individual in Heilongjiang province

Envelopeglycoprotein(Env)ofHIV1isacom plexoftwononcovalentlyassociatedsubunits,Gp120andGp41.Gp120isanexternalsubunit thatbindsthecellularreceptorCD4andachemo kinereceptor,suchasCXCR4orCCR5.Gp41isatransmembranesubunit,responsibleforthere ceptor mediatedmembranefusion[1,2].Because ofthehighvariabilityofHIV1,theaminoacid sequenceaswellasthestructureofviralenvelope canvaryandresultinthechangeofviraltropism withtimeextensionanddiseaseprogressionafter infection.DuringtheearlyphaseofHIVinfec…     

Co-evolution of nelfinavir-resistant HIV-1 protease and the p1-p6 substrate

The selective pressure of the competitive protease inhibitors causes both HIV-1 protease and occasionally its substrates to evolve drug resistance. We hypothesize that this occurs particularly in substrates that protrude beyond the substrate envelope and contact residues that mutate in response to a particular protease inhibitor. To validate this hypothesis, we analyzed substrate and protease sequences for covariation. Using the chi2 test, we show a positive correlation between the nelfinavir-resistant D30N/N88D protease mutations and mutations at the p1-p6 cleavage site as compared to the other cleavage sites. Both nelfinavir and the substrate p1-p6 protrude beyond the substrate envelope and contact residue 30, thus possibly making the p1-p6 cleavage site more vulnerable to co-evolution.     

Modulation of the severe CD4+ T-cell loss caused by a pathogenic simian-human immunodeficiency virus by replacement of the subtype B vpu with the vpu from a subtype C HIV-1 clinical isolate

Previously, we showed that the Vpu protein from subtype C human immunodeficiency virus type 1 (HIV-1) was efficiently targeted to the cell surface, suggesting that this protein has biological properties that differ from the well-studied subtype B Vpu protein. In this study, we have further analyzed the biological properties of the subtype C Vpu protein. Flow cytometric analysis revealed that the subtype B Vpu (strain HXB2) was more efficient at down-regulating CD4 surface expression than the Vpu proteins from four subtype C clinical isolates. We constructed a simian-human immunodeficiency virus virus, designated as SHIV(SCVpu), in which the subtype B vpu gene from the pathogenic SHIV(KU-1bMC33) was substituted with the vpu from a clinical isolate of subtype C HIV-1 (strain C.96.BW16B01). Cell culture studies revealed that SHIV(SCVpu) replicated with slightly reduced kinetics when compared with the parental SHIV(KU-1bMC33) and that the viral Env and Gag precursor proteins were synthesized and processed similarly compared to the parental SHIV(KU-1bMC33). To determine if substitution of the subtype C Vpu protein affected the pathogenesis of the virus, three pig-tailed macaques were inoculated with SHIV(SCVpu) and circulating CD4+ T-cell levels and viral loads were monitored for up to 44 weeks. Our results show that SHIV(SCVpu) caused a more gradual decline in the rate of CD4+ T cells in pig-tailed macaques compared to those inoculated with parental subtype B SHIV(KU-1bMC33). These results show for the first time that different Vpu proteins of HIV-1 can influence the rate at which CD4+ T-cell loss occurs in the SHIV/pig-tailed macaque model.