Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HIV Reverse Transcriptase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,693 records · Page 94Linked to original sources
PubMed2004

Differential virus evolution in blood and genital tract of HIV-infected females: evidence for the involvement of drug and non-drug resistance-associated mutations.

There is increasing evidence that male or female genital tract represent a distinct replication compartment for human immunodeficiency virus type 1 (HIV-1) and that such compartments may serve as a virus reservoir. Forty-four paired plasma and vaginal samples from HIV-infected females undergoing HAART were collected to examine the viral responses to antiretroviral therapy and to assess the possible role of the vaginal tract as a reservoir for drug-resistant variants. Twenty-one females had detectable viral RNA both in plasma and vaginal fluid, whereas 14 females had detectable virus only in plasma. Twelve paired samples were used to analyze HIV-1 pol sequences for the presence of drug resistance-associated mutations. Nine of the twelve paired samples exhibited discordant drug resistance mutation patterns. The other three females showed identical drug resistance-associated mutations. However, further examination of protease and RT showed numerous non-drug-associated mutations that corresponded to predefined CTL epitopes. These non-drug-associated mutations were different between plasma and vaginal viruses, suggesting that evolution of HIV-1 was independent in these two compartments.

Adult↗
PubMed2004

Characterization of human immunodeficiency virus type 1 Pr160 gag-pol mutants with truncations downstream of the protease domain.

We have constructed a series of HIV-1 Gag-pol mutants by progressive deletion of the pol sequence downstream of the viral protease (PR) domain. Effects of the truncation mutations on virus particle production and Gag particle processing were analyzed. Analysis indicated that removal of the integrase (IN) domain had no major effect on the efficiency of particle processing, but resulted in a marked reduction in virus particle budding. Deletion of both the IN and RNase H domains, however, restored the production of virus particles to wild-type level. The proteolytic processing of virus particle was significantly impaired when the p51RT domain was truncated. All of the truncated Gag-pol proteins could be incorporated into virus particles and demonstrated an immunofluorescence staining pattern similar to that of the wild type (wt). Our data are consistent with the proposal that signals for directing the Gag-pol transport and particle incorporation are determined by its N-terminal Gag domain. Truncated Gag-pol retaining an intact p51RT was able to complement a PR-defective mutant to produce infectious pseudotyped virions, with a virus titer 20-70% of that of wt. Pseudotyped virions produced by the Gag-pol lacking an intact p51RT were noninfectious or poorly infectious. This suggests that an intact p51RT domain is required for the Gag-pol to mediate production of mature infectious virus particles in trans.

Cell Line↗
PubMed2004

Inhibition of human immunodeficiency virus type 1 replication by siRNA targeted to the highly conserved primer binding site.

The initiation of HIV-1 reverse transcription occurs at an 18-nucleotide sequence in the viral genome designated as the primer binding site (PBS), which is complementary to the 3' terminal nucleotides of tRNA(Lys,3). Since the PBS is highly conserved among all infectious HIV-1, it represents an attractive target for the development of new therapeutics to inhibit viral replication. In this study, we have evaluated three approaches using small interfering RNA (siRNAs) targeted to the PBS for the capacity to inhibit HIV-1 replication. In the first, transfection of a 21-nucleotide siRNA complementary to the PBS into cells inhibited production of HIV-1 following infection. Control siRNAs of the same length complementary to HIV-1 gag mRNA or to gfp mRNA decreased the production of virus or had no effect on virus replication, respectively. Analysis of the PBS of integrated proviruses derived from viruses that ultimately grew in cultures transfected with siRNA all contained wild-type PBS sequence, demonstrating that HIV-1 did not mutate to escape inhibition by siRNA. In the second approach, hairpin siRNA targeted to the wild-type PBS were expressed using an adeno-associated virus (AAV) vector. HIV-1 replication was inhibited in cells infected with AAV encoding the siRNA to the wild-type PBS, but not in cells infected with AAV encoding an siRNA of the same length targeted to an irrelevant PBS. Finally, studies from this laboratory have shown that alteration of the PBS to be complementary to tRNAHis results in the production of infectious virus that rapidly reverts to utilize tRNALys,3 following in vitro culture. A proviral genome containing a PBS complementary to tRNAHis that encodes an siRNA molecule complementary to the wild-type PBS under control of a U6 promoter within the nef gene was as infectious as the parent HIV-1 genome containing no insert in nef. The virus with the PBS only complementary to tRNAHis reverted to use tRNALys,3, coincident with rapid virus growth, while the virus encoding siRNA grew slower than the virus without siRNA and maintained the PBS complementary to tRNAHis longer in culture. At later times of infection, viruses with the PBS complementary to tRNAHis and the siRNA exhibited a rapid increase in p24 antigen in the culture. Analysis of the PBS revealed that it was now complementary to tRNALys,3. Analysis of the gene encoding the siRNA revealed that the reversion of the PBS coincided with the deletion of the gene encoding siRNA. The results of these studies show that siRNA targeted to the PBS of HIV-1 can inhibit virus replication, supporting the concept that HIV-1 has evolved a strong preference to select tRNALys,3 for high-level replication and establishing the PBS and primer selection as a potential target for new therapeutics.

Base Sequence↗
PubMed2005

The role of upstream U3 sequences in HIV-1 replication and CD4+ T cell depletion in human lymphoid tissue ex vivo.

The LTRs of all primate lentiviruses contain long U3 regions overlapping the nef gene. To assess the relevance of the modulatory U3 region for HIV-1 replication, we inactivated the T-rich region, the Polypurine tract and attachment (att) sequences in nef by silent mutations and inserted intact cis-regulatory elements just upstream of the core enhancer. These modifications severely truncated the U3 region and eliminated the nef overlap. The resulting HIV-1 mutants expressed functional Nef, replicated efficiently and caused CD4+ T cell depletion in ex vivo-infected lymphoid tissue suggesting that the modulatory U3 region might not be essential for efficient HIV-1 gene expression and AIDS pathogenesis.

CD4 Lymphocyte Count↗
PubMed2006

Macrophage activation and human immunodeficiency virus infection: HIV replication directs macrophages towards a pro-inflammatory phenotype while previous activation modulates macrophage susceptibility to infection and viral production.

Macrophages are pivotal for the regulation of immune and inflammatory responses, but whether their role in HIV infection is protective or deleterious remains unclear. In this study, we investigated the effect of pro- and anti-inflammatory stimuli on macrophage sensitivity to two different aspects of HIV infection: their susceptibility to infection stricto sensu, which we measured by endpoint titration method, and their ability to support virus spread, which we measured by using an RT activity assay in infection kinetics. We show a partially protective role for pro-inflammatory agents as well as for IL-4. We also illustrate that various different stimuli display differential effects on macrophage susceptibility to HIV and on virus replication that occurs thereafter. On the other hand, HIV replication strongly repressed CD206 and CD163 expression, thus clearly orientating macrophages towards a pro-inflammatory phenotype, but independently of TNF. Taken together, our results emphasize that HIV infection of macrophages sets up inflammation at the cell level but through unexpected mechanisms. This may limit target susceptibility and participate in virus clearance but may also result in tissue damage.

Antigens, CD↗
PubMed2006

Immunoglobulin A antibodies against internal HIV-1 proteins neutralize HIV-1 replication inside epithelial cells.

We show that intraepithelial cell neutralization of HIV by IgA antibodies to internal viral proteins can occur during antibody transcytosis from the basolateral to the apical surface. Polarized epithelial cells expressing the polymeric immunoglobulin receptor (pIgR) were transfected with HIV proviral DNA, and IgA was added to the basolateral side. Transcytosing IgA antibodies against Gag and RT significantly inhibited HIV replication as assessed by infection of HeLa-CD4-LTR/beta-Gal cells and direct p24 assay. Consistent with intracellular neutralization, colocalization of the internal virus proteins and their IgA antibodies was demonstrated by confocal microscopy. Thus, at least in the context of infections of polarized epithelia, antibody-mediated neutralization may not be restricted to viral surface antigens.

Epithelial Cells↗
PubMed2006

In vitro analysis of the effects of mutations in the G-tract of the human immunodeficiency virus type 1 polypurine tract on RNase H cleavage specificity.

The recognition and precise cleavage of the polypurine tract (PPT) of the human immunodeficiency virus type 1 (HIV-1) is an essential step in HIV-1 reverse transcription. The accurate cleavage, and the subsequent removal, of the PPT by the RNase H activity of HIV-1 RT defines the left end of the double-stranded viral DNA genome, the substrate for integration into the host genome. Previous analyses have shown that mutations in the 3'-end (G-tract) of the PPT cause alterations in RNase H cleavage specificity. In particular, mutations at positions 2 and 5 in the G-tract increased the frequency of retention of PPT sequences in the 2-LTR circle junction. To better understand why these mutations affected PPT cleavage in vivo, we analyzed the cleavage of PPT substrates in vitro that contained altered sequences and unusual base substitutions. Our results, herein, confirm that mutations at positions 2 and 5 of the G-tract do significantly alter the cleavage specificity at the PPT/U3 junction, and further suggest that the miscleavages observed in vivo were due to an improper generation of the PPT primer, as opposed to its improper removal. Finally, our results point to the structure of the PPT, rather than the base-specific contacts between the PPT and HIV-1 RT, as the primary determinants of RNase H cleavage specificity at the PPT/U3 junction.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type 1.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I-RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1.

Adenosine Diphosphate↗
PubMed2002

Selective inhibition of the activities of both eukaryotic DNA polymerases and DNA topoisomerases by elenic acid.

(R)-(-)-Elenic acid (R-2,4-dimethyl-22-(p-hydroxyphenyl)-docos-3(E)-enoic acid) (EA) is a DNA topoisomerase II inhibitor found in an Indonesian sponge, Plakinastrella sp. We found and report here that it is a potent inhibitor of calf DNA polymerase alpha (IC(50)=7.7 microM) and rat DNA polymerase beta (IC(50)=12.9 microM). EA did not bind to DNA directly. EA did not influence the activities of DNA polymerases such as plant DNA polymerases I and II and prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I, or other DNA metabolic enzymes such as human immunodeficiency virus (HIV) reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. Interestingly, EA was also an inhibitor of DNA topoisomerases I and II, although the enzymatic characteristics including modes of action, amino acid sequences and three-dimensional structures were markedly different from those of DNA polymerases. EA could prevent the growth of NUGC-3 cancer cells, and the LD(50) value was 22.5 microM. The cells were halted at G1 and G2/M phase in the cell cycle. From these results, the action mode of EA is discussed.

Animals↗
PubMed2000

Apelin peptides block the entry of human immunodeficiency virus (HIV).

The orphan G protein-coupled receptor APJ has been shown to be a coreceptor for human and simian immunodeficiency virus (HIV and SIV) strains. We have determined that some HIV and SIV strains use APJ as a coreceptor to infect the brain-derived NP-2/CD4 cells. Because apelin is an endogenous ligand for the APJ receptor, we examined the inhibitory effects of apelin peptides on HIV infection, and found that the apelin peptides inhibit the entry of some HIV-1 and HIV-2 into the NP-2/CD4 cells expressing APJ. The inhibitory efficiency has been found to be in the order of apelin-36>apelin-17>apelin-13>apelin-12.

Amino Acid Sequence↗
PubMed1999

New substrates of DNA polymerases.

Bis-(2'-deoxynucleoside) 5',5'-tetraphosphates and bis-(2'-deoxynucleoside) 5',5'-triphosphates were shown to be a new type of substrate for several DNA polymerases of human, bacterial and viral origin. Their substrate properties depend both on their structure and on the nature of the enzyme. They are incorporated by both termini in correspondence with the template nucleotide program in the active center. The results obtained support the mechanism of their direct incorporation rather than prior hydrolysis to dNTP. The highest activity of these compounds was observed for HIV reverse transcriptase. The probable biological significance of the reaction is discussed.

Bacteria↗
PubMed2003

Memory in retroviral quasispecies: experimental evidence and theoretical model for human immunodeficiency virus.

Viral quasispecies may possess a molecular memory of their past evolutionary history, imprinted on minority components of the mutant spectrum. Here we report experimental evidence and a theoretical model for memory in retroviral quasispecies in vivo. Apart from replicative memory associated with quasispecies dynamics, retroviruses may harbour a "cellular" or "anatomical" memory derived from their integrative cycle and the presence of viral reservoirs in body compartments. Three independent sets of data exemplify the two kinds of memory in human immunodeficiency virus type 1 (HIV-1). The data provide evidence of re-emergence of sequences that were hidden in cellular or anatomical compartments for extended periods of infection, and recovery of a quasispecies from pre-existing genomes. We develop a three-component model that incorporates the essential features of the quasispecies dynamics of retroviruses exposed to selective pressures. Significantly, a numerical study based on this model is in agreement with the experimental data, further supporting the existence of both replicative and reservoir memory in retroviral quasispecies.

Adaptation, Physiological↗
PubMed2003

Suppression of an intrinsic strand transfer activity of HIV-1 Tat protein by its second-exon sequences.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) has been shown to restrict premature reverse transcription at late stages of virus infection and to thus ensure the integrity of the viral RNA genome for packaging. To gain further insights into the roles of Tat in HIV-1 reverse transcription, we have assessed its effects on the first-strand transfer during the synthesis of minus-strand DNA through use of a reconstituted cell-free system. The results demonstrated that a form of Tat, containing only the first exon (Tat72), was able to enhance the first-strand transfer as efficiently as did the viral nucleocapsid protein. Coincidentally, this form of Tat was unable to inhibit the production of minus-strand strong-stop DNA. Further studies with various mutated forms of Tat showed that its Cys-rich region, rather than the core and Arg-rich domains, was essential for this strand transfer activity. Moreover, this activity of Tat is largely independent of the TAR RNA structure. Although full-length Tat protein (Tat86) was also able to promote strand transfer, this activity was limited by a strong overall inhibition of reverse transcription because of the presence of the second Tat exon. Other nucleic-acid-binding proteins (e.g., single-strand DNA-binding protein) were employed as negative controls and were unable to promote strand transfer in these assays. We propose that Tat possesses nucleic acid chaperone activity and can promote the first-strand transfer during HIV-1 reverse transcription; however, these activities are restricted by the second Tat exon, and the roles of these Tat activities in viral replication remain to be elucidated.

Base Sequence↗
PubMed2003

Identification of constrained peptides that bind to and preferentially inhibit the activity of the hepatitis C viral RNA-dependent RNA polymerase.

A class of disulfide constrained peptides containing a core motif FPWG was identified from a screen of phage displayed library using the HCV RNA-dependent RNA polymerase (NS5B) as a bait. Surface plasmon resonance studies showed that three highly purified synthetic constrained peptides bound to immobilized NS5B with estimated K(d) values ranging from 30 to 60 microM. In addition, these peptides inhibited the NS5B activity in vitro with IC(50) ranging from 6 to 48 microM, whereas in contrast they had no inhibitory effect on the enzymatic activities of calf thymus polymerase alpha, human polymerase beta, RSV polymerase, and HIV reverse transcriptase in vitro. Two peptides demonstrated conformation-dependent inhibition since their synthetic linear versions were not inhibitory in the NS5B assay. A constrained peptide with the minimum core motif FPWG retained selective inhibition of NS5B activity with an IC(50) of 50 microM. Alanine scan analyses of a representative constrained peptide, FPWGNTW, indicated that residues F1 and W7 were critical for the inhibitory effect of this peptide, although residues P2 and N5 had some measurable inhibitory effect as well. Further analyses of the mechanism of inhibition indicated that these peptides inhibited the formation of preelongation complexes required for the elongation reaction. However, once the preelongation complex was formed, its activity was refractory to peptide inhibition. Furthermore, the constrained peptide FPWGNTW inhibited de novo initiated RNA synthesis by NS5B from a poly(rC) template. These data indicate that the peptides confer selective inhibition of NS5B activity by binding to the enzyme and perturbing an early step preceding the processive elongation step of RNA synthesis.

Disulfides↗