HIV-1 V3 domain variation in brain and spleen of children with AIDS: Tissue-specific evolution within host-determined quasispecies

DNA coding for the principal neutralization epitope of HIV-1 (the V3 domain of the envelope glycoprotein gp120) was amplified by polymerase chain reaction from postmortem brain and spleen tissue of three perinatally infected children who died of AIDS with progressive encephalopathy. Sequences obtained directly (without cloning) from this DNA were compared with sequences of 52 molecular clones made from this DNA. Cluster analysis showed that V3 domain sequences from two of the three children were similar to sequences from the American MN/SC isolates, while those from one child were more closely similar to the Caribbean RF isolate. Comparison of sequences obtained directly with consensus sequences derived from cloned DNA showed that V3 sequences are characteristic for an individual host. In one child, the V3 sequence determined directly from brain DNA was very distant from the consensus brain clone sequence and from the spleen sequences, suggesting a diverging quasispecies distribution. Site-directed hybridization demonstrated that brain-specific sequences present in 33% of brain-derived clones were absent from clones derived from spleen. The evidence suggests that brain- and spleen-specific variants evolve independently within each host-delimited quasispecies.     

河南省分离的HIV-1 B-Thai亚型流行株全基因组克隆及序列分析

目的 克隆、鉴定自河南省分离的HIV-1 B-Thai亚型流行株并进行系统发育分析。方法收集河南省1份HIV-1感染者全血后分离的淋巴细胞，在植物血凝素存在下与健康献血员淋巴细胞共培养，从培养的淋巴细胞中提取前病毒DNA。在HIV-1基因的长末端重复序列的保守区设计引物，利用LA Tag长链扩增系统扩增HIV-1全长基因，纯化的PCR产物与pWSK29-T载体连接，成功克隆CNHN24株。对鉴定的3个克隆进行全长测序，利用局部同源法计算同源率，同时采用Phylip软件绘制HIV-1 Env、Gag和Pol基因的进化树。结果 V3V4环序列分析表明：本次克隆的CNHN24株病毒为HIV-1 B-Thai亚型，V3环区氨基酸序列比对发现在9个位点发生氨基酸替换。在含有9010bp，全长HIV-1基因的CNHN24株克隆中未发现明显的缺失、插入和重排现象，Gag、Pol、Vpr和Vif基因与RL42株的同源率达到95．42％～97．08％，进化分析显示，CNHN24株与RL42株的遗传距离最近。结论 HIV-1 CNHN24株为国内实验室完成的HIV-1全长基因克隆，为进一步研究HIV-1流行特征提供了基础。     

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.     

Significance of plasma and peripheral blood mononuclear cell derived HIV-1 sequences in establishing epidemiologic linkage between two individuals multiply exposed to HIV-1

Establishing epidemiologic linkage in individuals multiply exposed to HIV can be a difficult task. To date, only peripheral blood mononuclear cell (PBMC)-derived sequences have been used in studying HIV-1 transmission between individuals. So far, the combined utility of plasma and PBMC-derived HIV-1 sequences has not been assessed in establishing epidemiologic linkage in people involved in transmission of HIV. In this study, both PBMC (DNA) and plasma (RNA) derived viral quasispecies was used in establishing epidemiologic linkage between two infected individuals (B-90 and B-69) multiply exposed to HIV-1 via injecting drug use. A detailed sequence, and phylogenetic analyses of HIV-1V3 region quasispecies derived from these two compartments clearly demonstrated compartmentalization of viral quasispecies between PBMC and plasma. More importantly, these data also demonstrate that in order to establish epidemiologic linkage between individuals multiply exposed to HIV-1, analyses of viral strains from both plasma and PBMC compartments may be necessary. The PBMC compartment alone may not provide sufficient information on epidemiologic linkage, overall diversification of viral quasispecies, replacement of older strains and the emergence of new viral recombinant strains in vivo. These are the first analyses that demonstrate the incremental value of plasma derived sequences, when used in conjunction with PBMC-derived sequences, in establishing the epidemiologic linkage between individuals multiply exposed to HIV parenterally. Further, the plasma derived HIV-1 sequences may prove to be invaluable in predicting a recent transmission between two epidemiologically-linked individuals.     

Greater diversity of HIV-1 quasispecies in HIV-infected individuals with active tuberculosis

OBJECTIVE: A continual increase in intrapatient HIV-1 heterogeneity is thought to contribute to evasion of host immune response and eventual progression to AIDS. Tuberculosis (TB) is diagnosed both early and late during the course of HIV-1 disease and may increase diversity of HIV-1 quasispecies by activating the HIV-1 immune response and increasing HIV-1 replication. We examined whether HIV-1 heterogeneity is altered in HIV-1-infected individuals with TB. METHODS: Blood samples were obtained from 7 HIV-1-infected patients with active TB (HIV/TB patients) and 9 HIV-1-infected patients (HIV patients) in Kampala, Uganda (CD4 counts of 0-650 cells/microl and HIV loads of 700-750,000 RNA copies/ml). The C2-C3 region of the HIV-1 envelope gene (env) was amplified by nested polymerase chain reaction (PCR) from lysed peripheral blood mononuclear cells (PBMCs) of each patient, and then subject to sequencing, clonal-quasispecies analysis and heteroduplex tracking analysis (HTA). RESULTS: HTA of env DNA fragments showed increased heterogeneity in the HIV/TB individuals compared with the HIV group. Further sequence and HTA analysis on ten individual env clones for each patient showed significantly greater HIV mutation frequencies in HIV/TB patients than in HIV patients. CONCLUSION: An increase in HIV-1 heterogeneity may be associated with a TB-mediated increase in HIV-1 replication. However, a diverse HIV-1 quasispecies population in HIV/TB patients as opposed to tight quasispecies clusters in HIV patients suggests a possible dissemination of lung-derived HIV-1 isolates from the TB-affected organ.     

Nef and LTR sequence variation from sequentially derived human immunodeficiency virus type 1 isolates

Variation in HIV-1 nef and LTR DNA sequences was assessed longitudinally during disease progression in four HIV-1-infected subjects. Point mutations were found among quasispecies obtained at a single time point in each individual, with increasing diversity with disease progression in two of three patients for whom sufficient data were available for analysis. Deletions and rearrangements were more common in late than early stages of disease. Continued sequence evolution in HIV-1 quasispecies with nef deletions along with coexistence of nef-bearing quasispecies suggest that nef-deleted quasispecies are capable of replication in vivo, possibly complemented by quasispecies lacking such deletions and/or by adaptation to a specialized niche within the patients.     

Construction and immunogenicity study of a 297-bp humanized HIV V3 DNA of an approximated last common ancestor in mice

DNA immunization represents one of the promising HIV-1 vaccine approaches. To overcome the obstacle of genetic variation, we used the last common ancestor (LCA) or "center-of-the-tree" approach to study a DNA fragment of the HIV-1 envelope surrounding the V3 region. A humanized codon of the 297-bp consensus ancestral sequence of the HIV-1 envelope (codons 291-391) was derived from the 80 most recent HIV-1 isolates from the 8 circulating HIV-1 subtypes worldwide. This 297-bp humanized "multi-clade" V3 DNA was amplified by a PCR-based technique. The PCR product was well expressed in vitro whereas the corresponding non-humanized V3 DNA (subtype A/E) could not be expressed. However, both V3 DNA constructs as well as the full-length HIV-1 envelope construct (A/E) were found to be immunogenic in mice by the footpad-swelling assay. Moreover, intracellular and extracellular interferon-gamma could be detected upon in vitro stimulation of spleen cells although the response was relatively weak. Further improvement of our humanized V3 DNA is needed.     

Truncated forms of human and simian immunodeficiency virus in infected individuals and rhesus macaques are unique or rare quasispecies

Truncated proviruses of variable sizes are present in peripheral blood mononuclear cells (PBMC) of human immunodeficiency virus type 1 (HIV-1)-infected persons and simian immunodeficiency virus (SIV)-infected rhesus macaques. Here, we investigated whether the highly deleted HIV and SIV proviruses are present in infected organisms as multiple copies or whether each truncated provirus is unique. Using end-point dilution, multiple long-distance (LD) DNA PCR assays were run in parallel using DNA extracted from PBMC of seropositive, treatment-naive persons and from lymph nodes of a rhesus monkey inoculated with cloned, full-length SIVmac239 DNA. The PCR products were titrated and mapped. Most truncated proviruses were present in the DNA samples tested as single, nonintegrated molecules that differed from one another in size and/or nucleotide sequence. These results indicate that truncated primate lentiviral sequences found in infected tissues are unique or rare quasispecies that do not replicate significantly.     

Simple determination of human immunodeficiency virus type 1 syncytium-inducing V3 genotype by PCR.

Human immunodeficiency virus type 1 (HIV-1) phenotype variability plays an important role in the pathogenesis of AIDS. The presence of syncytium-inducing (SI) HIV-1 isolates in infected individuals is associated with a rapid decline of CD4+ T cells, rapid disease progression, and reduced survival time after AIDS diagnosis. The strong association between the SI capacity of HIV-1 and the presence of positively charged amino acid residues at positions 306 and/or 320 in the third variable domain (V3) of gp120 could here be confirmed in 97% of 402 primary HIV-1 isolates, indicating that the V3 genotype may be useful for prediction of the viral phenotype. The V3 DNA sequences revealed a remarkably limited codon usage for the amino acid residues that are responsible for virus phenotype. On the basis of this limited SI-specific DNA sequence variation, four SI-specific oligonucleotides were designed for selective amplification of V3 from SI but not non-SI HIV-1 isolates. This PCR analysis allowed the prediction of the biological phenotype of HIV-1 isolates on the basis of the V3 genotype and may prove to be useful for monitoring SI capacity of HIV-1 isolates in infected individuals.     

Human immunodeficiency virus type-1 group M quasispecies evolution: diversity and divergence in patients co-infected with active tuberculosis

The evolution of intra-host human immunodeficiency virus type 1 (HIV-1) quasispecies prior and after treating active tuberculosis (TB) with chemotherapy in HIV-1/TB patients was assessed. Two time points HIV-1 quasispecies were evaluated by comparing HIV-1-infected patients with active tuberculosis (HIV-1/TB) and HIV-1-infected patients without tuberculosis (HIV-1/non-TB). Plasma samples were obtained from the Frankfurt HIV cohort, and HIV-1 RNA was isolated. C2V5 env was amplified by PCR and molecular cloning was performed. Eight to twenty-five clones were sequenced from each patient. Various phylogenetic analyses were performed. We found a significant increase in diversity and divergence in HIV-1/TB compared to the HIV-1/non-TB. For HIV-1/TB, the average rate of evolution of C2V5 env was higher than previous reports (2.4 × 10⁻⁴ substitution/site/day). Two groups of HIV-1/TB were observed based on the rate of HIV-1 evolution and coreceptor usage: A fast evolving R5-tropic dominating group and a relatively slowly evolving X4 group. The results demonstrated that active TB has an impact on HIV-1 viral diversity and divergence over time. The influence of active TB on longitudinal evolution of HIV-1 may be predominant for R5 viruses.     

Active HIV-1 redistribution and replication in the brain with HIV encephalitis

Summary.  The central nervous system (CNS) is of particular importance in human immunodeficiency virus type 1 (HIV-1) infection. First, the CNS may be difficult to access for anti-retroviral treatment and may become a sanctuary for residual viruses. Second, HIV-1 infection may lead to AIDS dementia complex (ADC) culminating in HIV-1 encephalitis. In order to examine the pattern of drug resistance and the role of encephalitis in enhancing viral redistribution to the CNS, we compared pol gene quasispecies of the spleen and brain in two patients with and two patients without HIV-1 encephalitis, who had been treated with zidovudine (AZT). Although a variable degree of AZT resistance was noted in both the spleen and brain of all patients, phylogenetic analysis indicated that quasispecies developed rather independently in the systemic circulation (spleen) and CNS (brain) of patients without HIV-1 encephalitis, while similar pol gene sequences were obtained from the two compartments of patients with HIV-1 encephalitis. env gene V3 region of patients with HIV-1 encephalitis showed distinct quasispecies in the spleen and brain. Our results suggest that HIV-1 redistribution to CNS is more active in cases with encephalitis and that HIV-1 distributed late to CNS grow actively under certain selective pressure exerted on the V3 region of the env gene. Received June 12, 1998 Accepted August 15, 1998     

Viral dynamics and in vivo fitness of HIV-1 in the presence and absence of enfuvirtide

OBJECTIVES: To estimate the in vivo fitness cost of enfuvirtide (ENF) resistance, we analyzed dynamic shifts in the HIV-1 quasispecies under changing selective pressure in 3 subjects on failing ENF-based regimens who interrupted ENF while maintaining stable background regimens. Subsequently, ENF was readministered for 4 weeks as "pulse intensification." METHODS: The proportion of plasma virus carrying the V38A mutation in gp41 was quantified by allele-specific real-time polymerase chain reaction in serial samples collected from 3 subjects at 1- to 4-week intervals. Fitness differences were calculated using a method that corrected for time dependence of the viral replication rate. RESULTS: The V38A mutant made up >or=85% of the quasispecies at baseline and decayed to <5% over 12-24 weeks; plasma HIV-1 RNA levels remained stable during this time. Fitness differences for mutant versus wild type ranged from -25% to -65%, providing in vivo evidence for the reduced fitness of ENF-resistant HIV-1. The V38A mutant virus reemerged rapidly during the ENF pulse. CONCLUSIONS: These results demonstrate that the HIV-1 quasispecies undergoes dynamic changes in response to withdrawal and reinitiation of fusion inhibitor therapy. The relative stability of plasma HIV-1 titers during decay of V38A suggests that factors other than viral fitness likely define viral load set-point in patients with advanced disease.     

乙型肝炎病毒C基因启动子区准种与变异特点的研究

目的 研究乙型肝炎病毒(HBV)C基因启动子区难种与变异的特点。方法 以中国株HBV基因序列为依据，设计特异性聚合酶链反应(PCR)引物，自3例慢性HBV感染患者外周血血清中扩增HBVCP序列，克隆入pGEM Teasy质粒，随机挑选克隆进行DNA测序以确定病毒的变异程度。结果 测序结果发现HBV CP序列高度保守，但在TATA样盒(1—3)可发生多种突变，其中184nt(T→C)位替换突变员为常见；在直接重复序列(DR)I上游存有一缺失突变高发区33．3％(5／15)。结论 CP区内有一缺失高变区，TATA样盒3的变异可能影响前C蛋白的表达。结果 提示HBV长期携带者体内有HBV准种共存。     

Genetic characterization of HIV type 1 and type 2 from Bissau, Guinea-Bissau (West Africa)

Previous studies from Guinea-Bissau (West Africa) have demonstrated a unique epidemiology with respect to both HIV-1 and HIV-2 infection. In order to evaluate the prevalence and dynamics of HIV-1 and HIV-2 subtypes in Bissau, the capital city of Guinea-Bissau, a cross-sectional study was set up using serological and molecular techniques. Plasma samples from 103 individuals were screened for HIV-1 and HIV-2 antibodies by ELISA and Western-blot. Seropositive results were confirmed by PCR amplification of proviral sequences in primary peripheral blood mononuclear cells (PBMC) with env and LTR primer sets for HIV-2 and env, LTR and pol primers for HIV-1. A total of 38/103 individuals were HIV-seroreactive (four HIV-1, 15 HIV-2, 19 HIV-1/HIV-2). A total of eight out of 19 dually seropositive specimens showed double PCR amplification of HIV-1 and HIV-2 proviral sequences, accounting for 21% of the infected individuals. In the remaining 11 individuals either HIV-2 or HIV-1 sequences were detected, the majority (n=9) amplifying only HIV-2. These screening data demonstrate a high discrepancy between serology and PCR results for dually seroreactive samples, Western-blot giving an overestimation of double infection. Additionally, HIV-1 strains were subtyped by heteroduplex mobility assay (HMA) on the basis of gp120 sequences. Subtyping of HIV-2 was carried out by DNA sequencing and phylogenetic analysis of env V3 molecular clones. For both HIV-1 and HIV-2 strains circulating in Bissau, our results indicate dominance of subtype A.     

Genetic composition of replication competent clonal HIV-1 variants isolated from peripheral blood mononuclear cells (PBMC), HIV-1 proviral DNA from PBMC and HIV-1 RNA in serum in the course of HIV-1 infection

The HIV-1 quasispecies in peripheral blood mononuclear cells (PBMC) is considered to be a mix of actively replicating, latent, and archived viruses and may be genetically distinct from HIV-1 variants in plasma that are considered to be recently produced.Here we analyzed the genetic relationship between gp160 env sequences from replication competent clonal HIV-1 variants that were isolated from PBMC and from contemporaneous HIV-1 RNA in serum and HIV-1 proviral DNA in PBMC of four longitudinally studied therapy naïve HIV-1 infected individuals.Replication competent clonal HIV-1 variants, HIV-1 RNA from serum, and HIV-1 proviral DNA from PBMC formed a single virus population at most time points analyzed. However, an under-representation in serum of HIV-1 sequences with predicted CXCR4 usage was sometimes observed implying that the analysis of viral sequences from different sources may provide a more complete assessment of the viral quasispecies in peripheral blood in vivo.     

Two bases are deleted from the termini of HIV-1 linear DNA during integrative recombination

Proviral integration is a required step in the retrovirus life cycle. The mechanism of integration involves specific modification of the ends of linear viral DNA and subsequent recombination with host sequences. Integration results in the limited loss of sequence information at the termini of the viral genome. The composition of the intact linear DNA termini were inferred by sequencing the 2-long terminal repeat (2-LTR) circle junction that is formed when the linear molecule undergoes intramolecular, blunt-end ligation. The junction sequence contained the nucleotides GTAC that were not present at the ends of the integrated provirus. Comparison with the sequence of the LAV-1 strain of HIV-1 demonstrated that the GT dinucleotide derived from the right-hand terminus (U5) of the linear viral DNA and the AC dinucleotide came from the left-hand terminus (U3). Therefore, the corrected size of the LAV-1 LTR is 637 bp. This conclusion was confirmed independently by assessing the structure of linear viral DNA in acutely infected T cells. A portion of the population of linear HIV-1 DNA molecules were specifically deleted at their 3' ends; the extent of this deletion was 2 bases. This result is consistent with the activity of viral integrase protein on linear viral DNA and it accounts for the structure of integrated HIV-1 proviruses.     

Human immunodeficiency virus type 1 genetic evolution in patients with prolonged suppression of plasma viremia

Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with combination drug regimens results in a reduction of plasma viral load to levels below the limit of detection. To investigate the genomic fluctuations in HIV-1 populations from long-term responders to antiviral therapies we analyzed the viral sequence evolution of env and pol genes from sequential peripheral blood mononuclear cell (PBMC) DNA samples of three infected patients. Analyses of sequences covering the V3 and flanking env regions obtained from blood samples at the beginning of the therapy and at 14 or 24 months from baseline revealed that HIV-1 quasispecies continue to evolve in the three patients following combination antiretroviral therapy. Minor drug-resistant mutant subpopulations were also searched for and found in one patient. Interestingly, no minor resistant subpopulations were found in the other two patients despite the fact that they showed evidence of ongoing viral replication. Finally, the genetic analysis of the env gene shows a reduction in PBMC env viral population diversity after long-term response to the therapy in all the patients analyzed.     

Sequence analysis of selected regions of theenv (V 3 loop and gp 41) andgag (p 7) reading frames of Ethiopian human immunodeficiency virus type 1 strains

Summary Amplified polymerase chain reaction (PCR) products, corresponding to the V3 loop and gp 41 of theenv, and p 7 of thegag region, from proviral DNA of several Ethiopian and Swedish HIV-1 strains were sequenced. Of the six amino acids (GPGRAF) that constitute the principal neutralizing determinant (PND) within the V 3 loop, the Ethiopian isolates all showed two amino acid changes (GPGQTF). Four to five other substitutions were found in the amino acids flanking the PND. Substitution of alanine (A) for threonine (T) should result in a change in the predicted secondary structure, i.e., disappearance of a coil structure. Percentage similarity data on a stretch of 22 amino acids within the V 3 loop showed a concordance of the Ethiopian HIV-1 isolates with the sequences of published macrophage-T-cell tropic HIV isolates. Additionally derived protein sequences in two other regions showed two common substitutions in p 7 and one to two substitutions in gp 41 compared to a recent consensus sequence. These changes are hitherto unique for the Ethiopian strains, and suggest the presence of a clustering of a divergent HIV-1 strain in Addis Ababa, Ethiopia.     

Population-based sequencing of the V3 region of env for predicting the coreceptor usage of human immunodeficiency virus type 1 quasispecies

Genotypic population-based methods could be faster and less expensive than phenotypic recombinant assays for determining human immunodeficiency virus type 1 (HIV-1) coreceptor usage in patient samples, but their clinical use requires good genotype-phenotype correlation and concordance with clonal analyses. We have assessed these requirements by clonal analysis of the V1 to V3 env PCR products of 26 patients infected with subtype B HIV-1. We used the resulting set of molecular clones, all sequenced and characterized using a single-cycle recombinant virus phenotypic entry assay, to reevaluate genotype-phenotype correlations. Combining the previously described 11/25 and net charge rules for the V3 genotype improved the prediction of HIV-1 coreceptor usage. We also evaluated the concordance of population-based and clonal analyses for predicting the coreceptor usage of HIV-1 quasispecies. Our population-based recombinant phenotypic assay and direct sequencing of V3 were similarly sensitive for detecting the presence of minor species in the virus population, and both correlated well with clonal analysis. The improved genotype-phenotype correlation obtained by combining two simple genotypic rules and the good concordance with clonal analyses suggest that direct sequencing of V3 is a valuable alternative to population-based recombinant phenotypic assays.