Preferences for the selection of unique tRNA primers revealed from analysis of HIV-1 replication in peripheral blood mononuclear cells

BACKGROUND: All human immunodeficiency virus (HIV-1) uses a host tRNA(Lys,3 )as the primer for reverse transcription. The tRNA(Lys,3 )is bound to a region on the HIV-1 genome, the primer-binding site (PBS), that is complementary to the 18 terminal nucleotides of tRNA(Lys,3). How HIV-1 selects the tRNA from the intracellular milieu is unresolved. RESULTS: HIV-1 tRNA primer selection has been investigated using viruses in which the primer-binding site (PBS) and a sequence within U5 were altered so as to be complementary to tRNA(Met), tRNA(Pro )or tRNA(Ile). Analysis of the replication of these viruses in human peripheral blood mononuclear cells (PBMC) revealed preferences for the selection of certain tRNAs. HIV-1 with the PBS altered to be complementary to tRNA(Met), with and without the additional mutation in U5 to be complementary to the anticodon of tRNA(Met), stably maintains the PBS complementary to tRNA(Met )following extended in vitro culture in PBMC. In contrast, viruses with either the PBS or PBS and U5 mutated to be complementary to tRNA(Ile )were unstable during in vitro replication in PBMC and reverted to utilize tRNA(Lys,3). Viruses with the PBS altered to be complementary to tRNA(Pro )replicated in PBMC but reverted to use tRNA(Lys,3); viruses with mutations in both the U5 and PBS complementary to tRNA(Pro )maintained this PBS, yet replicated poorly in PBMC. CONCLUSION: The results of these studies demonstrate that HIV-1 has preferences for selection of certain tRNAs for high-level replication in PBMC

Effect of Universal Testing and Treatment on HIV Incidence - HPTN 071 (PopART).

BACKGROUND: A universal testing and treatment strategy is a potential approach to reduce the incidence of human immunodeficiency virus (HIV) infection, yet previous trial results are inconsistent. METHODS: In the HPTN 071 (PopART) community-randomized trial conducted from 2013 through 2018, we randomly assigned 21 communities in Zambia and South Africa (total population, approximately 1 million) to group A (combination prevention intervention with universal antiretroviral therapy [ART]), group B (the prevention intervention with ART provided according to local guidelines [universal since 2016]), or group C (standard care). The prevention intervention included home-based HIV testing delivered by community workers, who also supported linkage to HIV care and ART adherence. The primary outcome, HIV incidence between months 12 and 36, was measured in a population cohort of approximately 2000 randomly sampled adults (18 to 44 years of age) per community. Viral suppression (<400 copies of HIV RNA per milliliter) was assessed in all HIV-positive participants at 24 months. RESULTS: The population cohort included 48,301 participants. Baseline HIV prevalence was 21% or 22% in each group. Between months 12 and 36, a total of 553 new HIV infections were observed during 39,702 person-years (1.4 per 100 person-years; women, 1.7; men, 0.8). The adjusted rate ratio for group A as compared with group C was 0.93 (95% confidence interval [CI], 0.74 to 1.18; P = 0.51) and for group B as compared with group C was 0.70 (95% CI, 0.55 to 0.88; P = 0.006). The percentage of HIV-positive participants with viral suppression at 24 months was 71.9% in group A, 67.5% in group B, and 60.2% in group C. The estimated percentage of HIV-positive adults in the community who were receiving ART at 36 months was 81% in group A and 80% in group B. CONCLUSIONS: A combination prevention intervention with ART provided according to local guidelines resulted in a 30% lower incidence of HIV infection than standard care. The lack of effect with universal ART was unanticipated and not consistent with the data on viral suppression. In this trial setting, universal testing and treatment reduced the population-level incidence of HIV infection. (Funded by the National Institute of Allergy and Infectious Diseases and others; HPTN 071 [PopArt] ClinicalTrials.gov number, NCT01900977.)

Female Genital Schistosomiasis and HIV-1 Incidence in Zambian Women: A Retrospective Cohort Study.

BACKGROUND: Female genital schistosomiasis (FGS) has been associated with prevalent HIV-1. We estimated the incidence of HIV-1 infection in Zambian women with and without FGS. METHODS: Women (aged 18-31, nonpregnant, sexually active) were invited to participate in this study in January-August 2018 at the final follow-up of the HPTN 071 (PopART) Population Cohort. HIV-1-negative participants at enrollment (n = 492) were included in this analysis, with testing to confirm incident HIV-1 performed in HPTN 071 (PopART). The association of incident HIV-1 infection with FGS (Schistosoma DNA detected by polymerase chain reaction [PCR] in any genital specimen) was assessed with exact Poisson regression. RESULTS: Incident HIV-1 infections were observed in 4.1% (20/492) of participants. Women with FGS were twice as likely to seroconvert as women without FGS but with no statistical evidence for a difference (adjusted rate ratio, 2.16; 95% CI, 0.21-12.30; P = .33). Exploratory analysis suggested an association with HIV-1 acquisition among women with ≥2 positive genital PCR specimens (rate ratio, 6.02; 95% CI, 0.58-34.96; P = .13). CONCLUSIONS: Despite higher HIV seroconversion rates in women with FGS, there was no statistical evidence of association, possibly due to low power. Further longitudinal studies should investigate this association in a setting with higher schistosomiasis endemicity

Forced selection of tRNA(Glu) reveals the importance of two adenosine-rich RNA loops within the U5-PBS for SIV(smmPBj) replication.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV-1) preferentially select and use tRNA(Lys,3) as the primer for initiation of reverse transcription. Previous studies have shown that HIV-1 can be forced to use tRNA(Glu) if mutations are made within the primer-binding site (PBS) and a region upstream, A-loop, to be complementary to the 3'-terminal 18 nucleotides and anticodon loop of tRNA(Glu). To examine the primer preference of SIV, mutations were made within the PBS of SIV(smmPBj) to be complementary to tRNA(Glu). Analysis of the production of infectious virus revealed that SIV(smmPBj) with the PBS complementary to tRNA(Glu) retained approximately 80% infectivity of the wild type. However, modification of the U5 of SIV(smmPBj) to alter nucleotides to be complementary to the anticodon of tRNA(Glu), in combination with the PBS complementary to tRNA(Glu), drastically reduced the production of infectious SIV(smmPBj) to less than 1% that of wild type. The replication of SIV(smmPBj) with the PBS complementary to tRNA(Glu) was similar to that of the wild type virus, while the replication of SIV(smmPBj) with PBS and A-loop complementary to tRNA(Glu) was delayed compared to that of wild type virus. Analysis of the PBS regions revealed that the virus with the PBS complementary to tRNA(Glu) reverted quickly, within 4 days, to be complementary to tRNA(Lys,3), while the virus with PBS and A-loop complementary to tRNA(Glu) retained the PBS for a longer time during in vitro culture although following extended replication both the A-loop and PBS of SIV(smmPBj) reverted to be complementary to tRNA(Lys,3). RNA modeling of SIV(smmPBj) U5-PBS by m-fold revealed two potential A-loop regions. Mutations in either A-loop drastically effected replication in human PBMC. Analysis of the A-loops following in vitro replication revealed that both reverted to the wild type sequence. The results of these studies demonstrate that SIV(smmPBj), like HIV-1, preferentially utilizes tRNA(Lys,3) as a primer for reverse transcription for high level replication, but unlike HIV-1 selection may involve the use of two adenosine-rich loops

tRNA Isoacceptor Preference prior to Retrovirus Gag-Pol Junction Links Primer Selection and Viral Translation▿

An essential step in the replication of all retroviruses is the capture of a cellular tRNA that is used as the primer for reverse transcription. The 3′-terminal 18 nucleotides of the tRNA are complementary to the primer binding site (PBS). Moloney murine leukemia virus (MuLV) preferentially captures tRNAPro. To investigate the specificity of primer selection, the PBS of MuLV was altered to be complementary to different tRNAs. Analysis of the infectivity of the virus and stability of the PBS following in vitro replication revealed that MuLV prefers to select tRNAPro, tRNAGly, or tRNAArg. Previous studies from our laboratory have suggested that tRNA primer capture is coordinated with translation. Coincidentally, a cluster of proline, arginine, and glycine precedes the Gag-Pol junction of MuLV. Human immunodeficiency virus type 1 (HIV-1), which prefers \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} as the primer, can be forced to utilize tRNAMet, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{1,2}^{{\mathrm{Lys}}}\end{equation*}\end{document}, tRNAHis, or tRNAGlu, although these viruses replicate poorly. Codons for methionine, lysine, histidine, or glutamic acid are found prior to the Gag-Pol frameshift site. HIV-1 was mutated so that the 5 lysine codons prior to the Gag-Pol frameshift region were specific for \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{1,2}^{{\mathrm{Lys}}}\end{equation*}\end{document}. HIV-1 forced to use \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{1,2}^{{\mathrm{Lys}}}\end{equation*}\end{document} as the primer, with the mutation of codons specific for \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{1,2}^{{\mathrm{Lys}}}\end{equation*}\end{document} prior to the Gag-Pol junction, had enhanced infectivity and replicated similarly to the wild-type virus. The results demonstrate that codon preference prior to the Gag-Pol junction influences primer selection and suggest a coordination of Gag-Pol synthesis and acquisition of the tRNA primer required for retrovirus replication

Bis-Anthracycline Antibiotics Inhibit Human Immunodeficiency Virus Type 1 Transcription

The increasing numbers of human immunodeficiency virus type 1 (HIV-1) strains that exhibit resistance to antiretroviral agents used at present require the development of new effective antiretroviral compounds. Tat transactivation was recognized early on as an attractive target for drug interference. To screen for and analyze the effects of compounds that interfere with Tat transactivation, we developed several cell-based reporter systems in which enhanced green fluorescence protein is a direct and quantitative marker of HIV-1 expression or Tat-dependent long terminal repeat activity. Using these reporter cell lines, we found that the bis-anthracycline WP631, a recently developed DNA intercalator, efficiently inhibits HIV-1 expression at subcytotoxic concentrations. WP631 also abrogated acute HIV-1 replication in peripheral blood mononuclear cells infected with various primary virus isolates. We demonstrate that WP631-mediated HIV-1 inhibition is caused by the inhibition of Tat transactivation. The data presented suggest that WP631 could serve as a lead compound for a new type of HIV-1 inhibitor

Field comparison of OraQuick ADVANCE Rapid HIV-1/2 antibody test and two blood-based rapid HIV antibody tests in Zambia.

BACKGROUND: Zambia's national HIV testing algorithm specifies use of two rapid blood based antibody assays, DetermineHIV-1/2 (Inverness Medical) and if positive then Uni-Gold Recombigen HIV-1/2 (Trinity Biotech). Little is known about the performance of oral fluid based HIV testing in Zambia. The aims of this study are two-fold: 1) to compare the diagnostic accuracy (sensitivity and specificity) under field conditions of the OraQuick ADVANCE Rapid HIV-1/2 (OraSure Technologies, Inc.) to two blood-based rapid antibody tests currently in use in the Zambia National Algorithm, and 2) to perform a cost analysis of large-scale field testing employing the OraQuick. METHODS: This was a operational retrospective research of HIV testing and questionnaire data collected in 2010 as part of the ZAMSTAR (Zambia South Africa TB and AIDS reduction) study. Randomly sampled individuals in twelve communities were tested consecutively with OraQuick test using oral fluid versus two blood-based rapid HIV tests, Determine and Uni-Gold. A cost analysis of four algorithms from health systems perspective were performed: 1) Determine and if positive, then Uni-Gold (Determine/Uni-Gold); based on current algorithm, 2) Determine and if positive, then OraQuick (Determine/OraQuick), 3) OraQuick and if positive, then Determine (OraQuick/Determine), 4) OraQuick and if positive, then Uni-Gold (OraQuick/Uni-Gold). This information was then used to construct a model using a hypothetical population of 5,000 persons with varying prevalence of HIV infection from 1-30%. RESULTS: 4,458 participants received both a Determine and OraQuick test. The sensitivity and specificity of the OraQuick test were 98.7 (95%CI, 97.5-99.4) and 99.8 (95%CI, 99.6-99.9), respectively when compared to HIV positive serostatus. The average unit costs per algorithm were US$3.76, US$4.03, US$7.35, and US$7.67 for Determine/Uni-Gold, Determine/OraQuick, OraQuick/Determine, and OraQuick/Uni-Gold, respectively, for an HIV prevalence of 15%. CONCLUSIONS: An alternative HIV testing algorithm could include OraQuick test which had a high sensitivity and specificity. The current Determine/Uni-Gold testing algorithm is the least expensive when compared to Determine/OraQuick, OraQuick/Determine, and OraQuick/Uni-Gold in the Zambian setting. From our field experience, oral fluid based testing offers many advantages over blood-based testing, especially with self testing on the horizon