HIV-1 reverse transcriptase mutations that confer decreased in vitro susceptibility to anti-RT DNA aptamer RT1t49 confer cross resistance to other anti-RT aptamers but not to standard RT inhibitors

RNA and DNA aptamers specific for HIV-1 reverse transcriptase (RT) can inhibit reverse transcription in vitro. RNA aptamers have been shown to potently block HIV-1 replication in culture. We previously reported mutants of HIV-1 RT with substitutions N255D or N265D that display resistance to the DNA aptamer RT1t49. Variant viruses bearing these mutations singly or in combination were compromised for replication. In order to address the wider applicability of such aptamers, HIV-1 RT variants containing the N255D, N265D or both (Dbl) were tested for the extent of their cross-resistance to other DNA/RNA aptamers as well as to other RT inhibitors. Both N265D and Dbl RTs were resistant to most aptamers tested. N255D mutant displayed mild resistance to two of the DNA aptamers, little change in sensitivity to three and hypersensitivity to one. Although all mutants displayed wild type-like ribonuclease H activity, their activity was compromised under conditions that prevent re-binding. This suggests that the processivity defect caused by these mutations can also affect RNase H function thus contributing further to the replication defect in mutant viruses. These results indicate that mutants conferring resistance to anti-RT aptamers significantly affect many HIV-1 RT enzymatic activities, which could contribute to preventing the development of resistance in vivo. If such mutations were to arise in vivo, our results suggest that variant viruses should remain susceptible to many existing anti-RT inhibitors. This result was tempered by the observation that NRTI-resistance mutations such as K65R can confer resistance to some anti-RT aptamers

Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain

A plasmid construct expressing the p66 version of the human immunodeficiency virus reverse transcriptase as a bacterial fusion protein was subjected to in vitro mutagenesis, and the resulting variant proteins were assayed to define the locations of the two major enzymatic activities. The DNA polymerase activity was localized to the N-terminal portion of the protein; mutations altering or eliminating the C-terminal portion had little or no effect on that activity. The results suggest that, in contrast with previous reports, the p51 subunit found in virions should exhibit DNA polymerase activity. Mutations in many parts of the protein eliminated RNase H activity, suggesting that several areas are needed for proper folding and generation of that activity

The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, contains conserved motifs common to retroviral reverse transcriptases and telomerases. Within the C motif of hTERT is the Leu866-Val867-Asp868-Asp869 tetrapeptide that includes a catalytically essential aspartate dyad. Site-directed mutagenesis of Tyr183 and Met184 residues in HIV-1 RT, residues analogous to Leu866 and Val867, revealed that they are key determinants of nucleotide binding, processivity and fidelity. In this study, we show that substitutions at Val867 lead to significant changes in overall enzyme activity and telomere repeat extension rate, but have little effect on polymerase processivity. All Val867 substitutions examined (Ala, Met, Thr) led to reduced repeat extension rates, ranging from ∼20 to 50% of the wild-type rate. Reconstitution of V867M hTERT and telomerase RNAs (TRs) with mutated template sequences revealed the effect on extension rate was associated with a template copying defect specific to template A residues. Furthermore, the Val867 hTERT mutants also displayed increased nucleotide incorporation fidelity, implicating Val867 as a determinant of telomerase fidelity. These findings suggest that by evolving to have a valine at position 867, the wild-type hTERT protein may have partially compromised polymerase fidelity for optimal and rapid repeat synthesis

New clues to understanding HIV nonprogressors: Low cholesterol blocks HIV trans infection

A small percentage of HIV-infected subjects (2 to 15%) are able to control disease progression for many years without antiretroviral therapy. Years of intense studies of virologic and immunologic mechanisms of disease control in such individuals yielded a number of possible host genes that could be responsible for the preservation of immune functions, from immune surveillance genes, chemokines, or their receptors to anti-HIV restriction factors. A recent mBiopaper by Rappocciolo et al. (G. Rappocciolo, M. Jais, P. Piazza, T. A. Reinhart, S. J. Berendam, L. Garcia-Exposito, P. Gupta, and C. R. Rinaldo, mBio 5:e01031-13, 2014) describes another potential factor controlling disease progression: cholesterol levels in antigen-presenting cells. In this commentary, we provide a brief background of the role of cholesterol in HIV infection, discuss the results of the study by Rappocciolo et al., and present the implications of their findings

Isolation and characterization of a dideoxyguanosine triphosphate-resistant mutant of human immunodeficiency virus reverse transcriptase

The appearance of drug-resistant strains of viral pathogens is a major difficulty confounding current efforts to block viral infections. The identification and analysis of mutations responsible for drug resistance can provide important clues helpful in understanding the mechanisms of resistance and in the eventual development of better therapies. We have used a direct screening method to scan libraries of mutagenized genes encoding the reverse transcriptase of human immunodeficiency virus type 1, and have recovered a variant enzyme that is resistant to the chain-terminator inhibitor 2',3'-dideoxyguanosine triphosphate. The single substitution mutation in this variant conferred broad crossresistance to a variety of other antiviral compounds currently in clinical trials. Virus carrying the mutation was fully infectious in cultured human lymphocytes. The replication of the mutant virus was highly resistant to phosphonoformic acid but did not show increased resistance to the prodrug dideoxyguanosine

HIV Exploits Antiviral Host Innate GCN2-ATF4 Signaling for Establishing Viral Replication Early in Infection.

Antiviral innate host defenses against acute viral infections include suppression of host protein synthesis to restrict viral protein production. Less is known about mechanisms by which viral pathogens subvert host antiviral innate responses for establishing their replication and dissemination. We investigated early innate defense against human immunodeficiency virus (HIV) infection and viral evasion by utilizing human CD4+ T cell cultures in vitro and a simian immunodeficiency virus (SIV) model of AIDS in vivo Our data showed that early host innate defense against the viral infection involves GCN2-ATF4 signaling-mediated suppression of global protein synthesis, which is exploited by the virus for supporting its own replication during early viral infection and dissemination in the gut mucosa. Suppression of protein synthesis and induction of protein kinase GCN2-ATF4 signaling were detected in the gut during acute SIV infection. These changes diminished during chronic viral infection. HIV replication induced by serum deprivation in CD4+ T cells was linked to the induction of ATF4 that was recruited to the HIV long terminal repeat (LTR) to promote viral transcription. Experimental inhibition of GCN2-ATF4 signaling either by a specific inhibitor or by amino acid supplementation suppressed the induction of HIV expression. Enhancing ATF4 expression through selenium administration resulted in reactivation of latent HIV in vitro as well as ex vivo in the primary CD4+ T cells isolated from patients receiving suppressive antiretroviral therapy (ART). In summary, HIV/SIV exploits the early host antiviral response through GCN2-ATF4 signaling by utilizing ATF4 for activating the viral LTR transcription to establish initial viral replication and is a potential target for HIV prevention and therapy.IMPORTANCE Understanding how HIV overcomes host antiviral innate defense response in order to establish infection and dissemination is critical for developing prevention and treatment strategies. Most investigations focused on the viral pathogenic mechanisms leading to immune dysfunction following robust viral infection and dissemination. Less is known about mechanisms that enable HIV to establish its presence despite rapid onset of host antiviral innate response. Our novel findings provide insights into the viral strategy that hijacks the host innate response of the suppression of protein biosynthesis to restrict the virus production. The virus leverages transcription factor ATF4 expression during the GCN2-ATF4 signaling response and utilizes it to activate viral transcription through the LTR to support viral transcription and production in both HIV and SIV infections. This unique viral strategy is exploiting the innate response and is distinct from the mechanisms of immune dysfunction after the critical mass of viral loads is generated

Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter

<p>Abstract</p> <p>Background</p> <p>Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.</p> <p>Results</p> <p>A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4⁺cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.</p> <p>Conclusion</p> <p>The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.</p

Phylogenetic Characterization of Six Full-Length HIV-1 Subtype C Molecular Clones from Three Patients: Identification of Rare Subtype C Strains Containing Two NF-κB Motifs in the Long Terminal Repeat

Molecular surveillance is the backbone of HIV-1 vaccinology. Full-length HIV-1 sequences are useful tools that can provide a better understanding of the epidemiology in a given region. A limited number of full-length HIV-1 sequences are available from India, where >95% of the HIV infections are due to HIV-1 subtype C (HIV-1C), which is distinct from the prototype African HIV-1C. In this study, we sequenced six full-length clones isolated from three patients. Extensive phylogenetic analyses of the full-length viral sequences using bioinformatic tools identified a separate cluster of Indian strains, thus confirming the distinct phylogenetic identity of the Indian HIV-1C. Notably, the long terminal repeat (LTR) of two of the six molecular clones contained only two NF-κB binding sites. The sequences also displayed features characteristic of HIV-1C including a Tat dicysteine motif, a shortened Rev open reading frame, and a predicted CCR5 coreceptor tropism for gp120 of three of the proviral sequences

Multivariable analysis to determine if HIV-1 Tat dicysteine motif is associated with neurodevelopmental delay in HIV-infected children in Malawi

Abstract Background HIV-1 Tat protein is implicated in HIV-neuropathogenesis. Tat C31S polymorphism (TatCS) has been associated with milder neuropathology in vitro and in animal models but this has not been addressed in a cohort of HIV-infected adults or children. Methods HIV viral load (VL) in plasma and cerebrospinal fluid (CSF) were determined and plasma HIV tat gene was sequenced. Neurodevelopmental assessment was performed using Bayley Scales of Infant Development III (BSID-III), with scores standardized to Malawian norms. The association between TatCS and BSID-III scores was evaluated using multivariate linear regression. Results Neurodevelopmental assessment and HIV tat genotyping were available for 33 children. Mean age was 19.4 (SD 7.1) months, mean log VL was 5.9 copies/mL (SD 0.1) in plasma and 3.9 copies/mL (SD 0.9) in CSF. The prevalence of TatCC was 27 %. Z-scores for BSID-III subtests ranged from −1.3 to −3.9. TatCC was not associated with higher BSID-III z-scores. Conclusions The hypothesis of milder neuropathology in individuals infected with HIV TatCS was not confirmed in this small cohort of Malawian children. Future studies of tat genotype and neurocognitive disorder should be performed using larger sample sizes and investigate if this finding is due to differences in HIV neuropathogenesis between children and adults