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PubMed2003

Efficient initiation of HIV-1 reverse transcription in vitro. Requirement for RNA sequences downstream of the primer binding site abrogated by nucleocapsid protein-dependent primer-template interactions.

Synthesis of HIV-1 (-) strong-stop DNA is initiated following annealing of the 3' 18 nucleotides (nt) of tRNA(3)(Lys) to the primer binding site (PBS) near the 5' terminus of viral RNA. Here, we have investigated whether sequences downstream of the PBS play a role in promoting efficient (-) strong-stop DNA synthesis. Our findings demonstrate a template requirement for at least 24 bases downstream of the PBS when tRNA(3)(Lys) or an 18-nt RNA complementary to the PBS (R18), but not an 18-nt DNA primer, are used. Additional assays using 18-nt DNA-RNA chimeric primers, as well as melting studies and circular dichroism spectra of 18-nt primer:PBS duplexes, suggest that priming efficiency is correlated with duplex conformation and stability. Interestingly, in the presence of nucleocapsid protein (NC), the 24 downstream bases are dispensable for synthesis primed by tRNA(3)(Lys) but not by R18. We present data supporting the conclusion that NC promotes extended interactions between the anticodon stem and variable loop of tRNA(3)(Lys) and a sequence upstream of the A-rich loop in the template. Taken together, this study leads to new insights into the initiation of HIV-1 reverse transcription and the functional role of NC-facilitated tRNA-template interactions in this process.

Base Sequence↗
PubMedDate not supplied

Resistance profiles of (+)2'-deoxy-3'-oxa-4'-thiocytidine and (-)2'-deoxy-3'-oxa-4'-thio-5-fluorocytidine.

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.

Anti-HIV Agents↗
PubMed1991

Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes.

Human immunodeficiency virus type 1 (HIV-1) infection of T lymphocytes requires cellular proliferation and DNA synthesis. Human monocytes were shown to have low DNA synthesis rates, yet the monocytotropic BaL isolate of HIV-1 was able to infect these cells efficiently. Monocytes that were irradiated to assure no DNA synthesis could also be readily infected with HIV-1BaL. Such infections were associated with the integration of HIV-1BaL DNA into the high molecular weight, chromosomal DNA of monocytes. Thus, normal, nonproliferating monocytes differ from T lymphocytes in that a productive HIV-1 infection can occur independently of cellular DNA synthesis. These results suggest that normal nonproliferating mononuclear phagocytes, which are relatively resistant to the destructive effects of this virus, may serve as persistent and productive reservoirs for HIV-1 in vivo.

Cells, Cultured↗
PubMed1996

The interaction of macrophage and non-macrophage tropic isolates of HIV-1 with thymic and tonsillar dendritic cells in vitro.

Dendritic cells isolated from thymus and tonsil were tested for susceptibility to HIV-1 strains that are tropic for macrophages or for T cell lines. DCs were purified by cell sorting and before infection expressed high levels of CD4 and HLA-DR and lacked markers for T, B, NK cells, or macrophages. Viral entry and reverse transcription was found after pulsing with strains of HIV-1 that could infect macrophages. During the first 36 h the PCR signals for gag sequences increased in DCs and macrophages. In contrast little if any viral DNA was found after pulsing macrophages or DCs with HIV-1 that was able to infect T cell lines. DCs pulsed with HIV-1 were able to transmit infection to responding T cells during an allogeneic or superantigen response. Selection for virus able to infect lymphoid DCs and other DCs expressing CD4 and its transfer to T cells during subsequent immune responses may provide a mechanism for the observed predominance of macrophage-tropic HIV-1 after in vivo transmission.

CD4 Antigens↗
PubMed1998

Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton.

After interaction of human immunodeficiency virus type 1 (HIV-1) virions with cell surface receptors, a series of poorly characterized events results in establishment of a viral reverse transcription complex in the host cell cytoplasm. This process is coordinated in such a way that reverse transcription is initiated shortly after formation of the viral reverse transcription complex. However, the mechanism through which virus entry and initiation of reverse transcription are coordinated and how these events are compartmentalized in the infected cell are not known. In this study, we demonstrate that viral reverse transcription complexes associate rapidly with the host cell cytoskeleton during HIV-1 infection and that reverse transcription occurs almost entirely in the cytoskeletal compartment. Interruption of actin polymerization before virus infection reduced association of viral reverse transcription complexes with the cytoskeleton. In addition, efficient reverse transcription was dependent on intact actin microfilaments. The localization of reverse transcription to actin microfilaments was mediated by the interaction of a reverse transcription complex component (gag MA) with actin but not vimentin (intermediate filaments) or tubulin (microtubules). In addition, fusion, but not endocytosis-mediated HIV-1 infectivity, was impaired when actin depolymerizing agents were added to target cells before infection but not when added after infection. These results point to a previously unsuspected role for the host cell cytoskeleton in HIV-1 entry and suggest that components of the cytoskeleton promote establishment of the reverse transcription complex in the host cell and also the process of reverse transcription within this complex.

Cell Line↗
PubMed2003

Determinant of HIV-1 mutational escape from cytotoxic T lymphocytes.

CD8+ class I-restricted cytotoxic T lymphocytes (CTLs) usually incompletely suppress HIV-1 in vivo, and while analogous partial suppression induces antiretroviral drug-resistance mutations, epitope escape mutations are inconsistently observed. However, escape mutation depends on the net balance of selective pressure and mutational fitness costs, which are poorly understood and difficult to study in vivo. Here we used a controlled in vitro system to evaluate the ability of HIV-1 to escape from CTL clones, finding that virus replicating under selective pressure rapidly can develop phenotypic resistance associated with genotypic changes. Escape varied between clones recognizing the same Gag epitope or different Gag and RT epitopes, indicating the influence of the T cell receptor on pressure and fitness costs. Gag and RT escape mutations were monoclonal intra-epitope substitutions, indicating limitation by fitness constraints in structural proteins. In contrast, escape from Nef-specific CTL was more rapid and consistent, marked by a polyclonal mixture of epitope point mutations and upstream frameshifts. We conclude that incomplete viral suppression by CTL can result in rapid emergence of immune escape, but the likelihood is strongly determined by factors influencing the fitness costs of the particular epitope targeted and the ability of responding CTL to recognize specific epitope variants.

Amino Acid Sequence↗
PubMed1999

Functional T cell reconstitution and human immunodeficiency virus-1-specific cell-mediated immunity during highly active antiretroviral therapy.

Lymphoproliferative responses (LPRs) to recall antigens (Ags) and human immunodeficiency virus type 1 (HIV-1) Gag and frequencies of circulating HIV-1-specific cytotoxic T lymphocyte precursors (CTLps) were measured in 12 patients undergoing highly active antiretroviral therapy (HAART) after long-standing HIV-1 infection. LPRs to at least 1 recall Ag became detectable or increased in all patients during HAART. No significant LPRs to Gag-p24 were observed, whereas 4 of 8 patients tested presented with Gag-p17-specific LPRs. HIV-1-specific CTLp frequencies became measurable or increased early during therapy in 6 of 10 patients tested and were maintained or decreased thereafter. Increasing HIV-1-specific CTLp frequencies were seen only in association with partial HAART failure in 1 patient. In conclusion, restoration of CD4+ T lymphocyte responsiveness to recall Ags is achieved during HAART. The data provide evidence for limited HIV-1-specific CD4+ memory T cells during advanced HIV-1 infection and suggest that both CD4+ and CD8+ HIV-1-specific T cells are poorly stimulated when viral load is suppressed.

Adult↗
PubMed2004

Genotypic and phenotypic predictors of the magnitude of response to tenofovir disoproxil fumarate treatment in antiretroviral-experienced patients.

Results from 2 placebo-controlled intensification trials of tenofovir disoproxil fumarate (DF) in treatment-experienced human immunodeficiency type 1 (HIV-1)-infected patients (n=332) were integrated to determine the effects of resistance at baseline on HIV-1 RNA response. In these trials, there was a high prevalence of HIV-1 resistance mutations, with 94% of patients having nucleoside-associated mutations and 71% having thymidine analogue-associated mutations (TAMs). Statistically significant HIV-1 RNA reductions associated with tenofovir DF treatment, relative to placebo (P<.001), were observed for patients without TAMs (n=97) or for patients with 1-2 (n=88) or >or=3 TAMs (n=147). Response to tenofovir DF was reduced among patients with HIV-1 with >or=3 TAMs inclusive of either the M41L or L210W mutation (n=86) or patients who had a preexisting K65R mutation (n=6). Slightly increased treatment responses were observed when the M184V mutation was present. Phenotypic cutoffs were established at 1.4-fold and 4-fold, respectively, for the beginning of reduced response to tenofovir DF and for a strongly reduced response. The results from these controlled clinical trials provide guidance for the use of tenofovir DF for treatment-experienced patients.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Detection of drug-resistant minority variants of HIV-1 during virologic failure of indinavir, lamivudine, and zidovudine.

We evaluated zidovudine-experienced patients for whom treatment with indinavir, lamivudine, and zidovudine failed, for indinavir-resistant minority variants. Of 10 patients with plasma human immunodeficiency virus type 1 RNA suppression and subsequent rebound, 6 without primary indinavir-resistance mutations underwent clonal analysis. One had evidence of V82A in 9 of 30 clones at week 24, with no increase at week 40. The dominant week-40 82V-M184V clones had changes at protease codons 62-64, compared with all clones at week 24 and minority clones at week 40. Resistance to indinavir can emerge during treatment failure in nucleoside-experienced patients but may be missed by routine sequence analysis. Selection for indinavir-resistant variants on treatment with indinavir, lamivudine, and zidovudine may occur slowly, depending on the genetic context in which they arise.

Acquired Immunodeficiency Syndrome↗
PubMed2004

HIV-1 drug resistance in subjects with advanced HIV-1 infection in whom antiretroviral combination therapy is failing: a substudy of AIDS Clinical Trials Group Protocol 388.

We evaluated phenotypic and genotypic markers of drug resistance in human immunodeficiency virus type 1 (HIV-1) at the time of virologic failure (VF) in subjects in the AIDS Clinical Trials Group Protocol 388 (ACTG 388) who received lamivudine-zidovudine (or lamivudine-stavudine) and either indinavir, efavirenz-indinavir, or nelfinavir-indinavir. At VF, phenotypically susceptible HIV-1 was found in 55% of subjects in the nelfinavir-indinavir arm, compared with 22% in the indinavir arm (P=.006). Phenotypic resistance to lamivudine was less common in the efavirenz-indinavir arm (33% of subjects; P=.002) and the nelfinavir-indinavir arm (43%; P=.003), compared with the indinavir arm (78%). Isolated phenotypic resistance to efavirenz at VF occurred in HIV-1 recovered from 33% of subjects in the efavirenz-indinavir arm; 24% of the subjects had HIV-1 with both efavirenz and lamivudine resistance. Results of genotypic tests were similar. The lower frequency of resistance in the nelfinavir-indinavir arm likely reflects decreased drug exposure that is due to intolerance, which is consistent with the lower potency and tolerability of this combination in ACTG 388. The lower frequency of lamivudine resistance in the efavirenz-indinavir arm is consistent with reports in other studies of potent regimens. Thus, although dual resistance to efavirenz and lamivudine occurred at VF in the efavirenz-indinavir arm, this risk was relatively low when evaluated in the context of the potency and tolerability of this regimen.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Rationale and uses of a public HIV drug-resistance database.

Knowledge regarding the drug resistance of human immunodeficiency virus (HIV) is critical for surveillance of drug resistance, development of antiretroviral drugs, and management of infections with drug-resistant viruses. Such knowledge is derived from studies that correlate genetic variation in the targets of therapy with the antiretroviral treatments received by persons from whom the variant was obtained (genotype-treatment), with drug-susceptibility data on genetic variants (genotype-phenotype), and with virological and clinical response to a new treatment regimen (genotype-outcome). An HIV drug-resistance database is required to represent, store, and analyze the diverse forms of data underlying our knowledge of drug resistance and to make these data available to the broad community of researchers studying drug resistance in HIV and clinicians using HIV drug-resistance tests. Such genotype-treatment, genotype-phenotype, and genotype-outcome correlations are contained in the Stanford HIV RT and Protease Sequence Database and have specific usefulness.

Anti-HIV Agents↗
PubMed2006

Chemoprophylaxis with tenofovir disoproxil fumarate provided partial protection against infection with simian human immunodeficiency virus in macaques given multiple virus challenges.

We examined the efficacy of tenofovir disoproxil fumarate (TDF) in blocking simian human immunodeficiency virus (SHIV) infection in Chinese rhesus macaques. Once weekly for 14 weeks or until a macaque became infected, 12 male macaques were inoculated intrarectally with amounts of SHIV(SF162P3) (10 median tissue culture infective doses; 3.8 x 10(5) virus particles) that were approximately 5-fold higher than the human immunodeficiency virus type 1 RNA levels noted in human semen during an acute infection. Of the 12 macaques, 4 received oral TDF daily, 4 received oral TDF once weekly, and 4 (control animals) received no TDF. The control animals became infected after receiving a median of 1.5 virus inoculations; macaques receiving TDF daily (1 macaque remained uninfected after 14 inoculations) and those receiving TDF weekly became infected after a median duration of 6.0 and 7.0 weeks, respectively. Although infection was delayed in treated macaques, compared with control macaques, the differences were not statistically significant (P=.315); however, the study was limited by the small numbers of animals evaluated and the variability in blood levels of TDF that resulted from oral dosing. These data demonstrate that treatment with oral TDF provided partial protection against SHIV infection but ultimately did not protect all TDF treated animals against multiple virus challenges.

Adenine↗
PubMed2007

HIV-1 drug resistance in variants from the female genital tract and plasma.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) drug-resistance mutations may arise in a fraction of viral variants, and these variants may differ between compartments, including the genital tract and blood. METHODS: We studied 14 women with detectable HIV-1 in both the genital tract and plasma despite antiretroviral treatment. We obtained HIV-1 RNA sequences from 280 unique viral variants and then determined the resistance genotype and the predicted phenotype (Virtual Phenotype; Virco BVBA) of each variant. RESULTS: Eight patients (57%) displayed mutations conferring high-level HIV-1 drug resistance. Although we observed differences in specific mutations among viral variants, 13 of the 14 women showed highly concordant HIV-1 genotypic and predicted phenotypic resistance patterns in the 2 compartments. In 1 patient, resistance mutations appeared only in plasma; all variants in her genital tract, which displayed a low viral load, were susceptible. CONCLUSIONS: These data suggest that, for the majority of women, determination of HIV-1 drug resistance in the plasma will approximate the drug-resistance pattern in the genital tract. Analysis of individual variants enabled us to identify minority species bearing distinctive linked mutations, which may serve as a source of novel resistance genotypes. These data are relevant to clinical management and the evolution of drug resistance.

Adult↗
PubMed1997

Induction of human immunodeficiency virus type 1 expression in monocytic cells by Cryptococcus neoformans and Candida albicans.

Because candidiasis and cryptococcosis are common in human immunodeficiency virus (HIV)-infected persons, the effect of Cryptococcus neoformans and Candida albicans on HIV expression in monocytic cells was examined. Stimulation of the latently HIV-infected myelomonocytic cell line OM-10.1 with C. neoformans and C. albicans in the presence of pooled human serum caused a ratio-dependent increase in HIV production. Induction of HIV by C. neoformans was enhanced by anti-capsular antibody, while induction by both organisms was inhibited by anti-TNF-alpha antibody. In THP-1 cells transfected with HIV plasmid constructs, both organisms induced transcription from the HIV long terminal repeat that was dependent on intact NF-kappaB binding sequences. Thus, C. neoformans and C. albicans enhance HIV expression in monocytic cells through a TNF-alpha- and NF-kappaB-dependent mechanism. In HIV-infected patients, such enhancement may further impair host immunity and could accelerate the course of HIV disease.

Binding Sites↗
PubMed2002

Differential processing of HLA A2-restricted HIV type 1 cytotoxic T lymphocyte epitopes.

Cytotoxic T lymphocytes (CTLs) play a key role in the control of persistent viral infections. Differences in the quality of this cellular immune response influence the long-term outcome of such infections, but the factors that determine which virus-derived peptide epitopes are targeted by CTLs remain poorly understood. Here, we examine the antigen-processing requirements of three human leukocyte antigen (HLA) A*0201-restricted HIV-1 CTL epitopes. Each of these three peptides appears to be generated by a distinct proteolytic pathway, despite presentation on the cell surface in association with the same HLA class I molecule. Presentation of the commonly immunodominant SLYNTVATL (HIV-1 p17 Gag; residues 77-85) epitope was unaffected by inhibition of the proteasome with lactacystin, but was dependent on the presence of the beta-subunit LMP7. These findings are consistent with emerging data on the complexity of peptide epitope generation, and suggest that differences in antigen processing might contribute to patterns of CTL recognition in vivo.

Animals↗
PubMed2001

Genetic analysis of hiv type 1 strains in bujumbura (burundi): predominance of subtype c variant.

In order to characterize the HIV-1 strains circulating in Burundi, 18 blood samples from nontreated patients were collected in Bujumbura and viral DNA and RNA were sequenced in the env and pol genes, respectively. The phylogenetic analysis of the V3 coding region of HIV-1 gp120 revealed that 83% (15/18) of the isolates belonged to the C subtype. The RT and protease coding regions of the pol gene also clustered with subtype C. A potential A/C recombinant between the protease (subtype A) and the RT and V3 coding regions (both subtype C) was identified. Drug resistance mutations were not detected in the RT gene. However, mutation M36I, associated with resistance to ritonavir and nelfinavir, was found in 17 of 18 Burundi isolates. In conclusion, this first characterization of HIV-1 strains circulating in Burundi confirms the dramatic emergence of subtype C in East Africa.

Amino Acid Sequence↗
PubMed2001

HIV type 1 genetic diversity and genotypic drug susceptibility in the Republic of Moldova.

HIV-1 genetic diversity and, for the first time, genotypic drug susceptibility was investigated for strains circulating in the Republic of Moldova (of the former Soviet Union). Eighty-three samples from adults recently infected by intravenous drug use (IDU) (n = 60), heterosexual contact (n = 8), and from blood donors (n = 15) that tested positive from 1997 to 1998, and originating from different regions of Moldova were serotyped. By group-specific and subtype-specific peptide ELISA, patients were infected by serotype A (n = 65), serotype B (n = 1), or were nontypable (n = 17). Heteroduplex mobility assay (HMA) confirmed 11 subtype A and the one subtype B infection. Analyses of pol and env sequences for six of the IDUs confirmed that they were infected with subtype A strain. These strains clustered tightly with subtype A strains isolated from the former Soviet Union in phylogenetic analysis. No mutations associated with drug resistance were detected. The Republic of Moldova is culturally more closely related to Romania (where subtype F dominates the epidemic), but depends economically on Russia (where subtype A is established among IDUs). Thus, our results suggest that the spread of HIV in this region is driven by drug networks rather than being due to cultural similarities.

Adolescent↗
PubMed2002

Mother-to-child HIV type 1 transmission in Argentina: BF recombinants have predominated in infected children since the mid-1980s.

The molecular epidemiology of HIV-1 in Argentina is more complex than was previously appreciated. One circulating recombinant form, CRF12_BF, and many related BF recombinant forms predominate in the capital city, Buenos Aires. This study of HIV-1 subtypes acquired perinatally between 1984 and 2000 has permitted, for the first time, a reconstruction of the history of BF recombination in Argentina. Sequencing of a partial genome region from the beginning of vpu to the beginning of env(gp120), which spans a breakpoint common in most contemporary Argentine BF recombinants, enabled samples to be rapidly screened. Among 23 children born between 1984 and 2000, 15 including 1 child born in 1986, harbored a BF recombinant. Thirteen of the 15 recombinants shared a common breakpoint at the 5' end of env(gp120). Full genome sequencing of two viruses, from 1986 and 1987, respectively, revealed them to be genetically related but not identical to CRF12_BF. Both contained more subtype B sequence than did CRF12_BF. BF recombinants related to CRF12_BF have been in circulation in Buenos Aires since 1986 and continue to predominate in perinatal transmissions.

Adolescent↗