Dataset Information

Deep sequencing of HIV-infected cells: insights into nascent transcription and host-directed therapy.

ABSTRACT: Polyadenylated mature mRNAs are the focus of standard transcriptome analyses. However, the profiling of nascent transcripts, which often include nonpolyadenylated RNAs, can unveil novel insights into transcriptional regulation. Here, we separately sequenced total RNAs (Total RNAseq) and mRNAs (mRNAseq) from the same HIV-1-infected human CD4(+) T cells. We found that many nonpolyadenylated RNAs were differentially expressed upon HIV-1 infection, and we identified 8 times more differentially expressed genes at 12 h postinfection by Total RNAseq than by mRNAseq. These expression changes were also evident by concurrent changes in introns and were recapitulated by later mRNA changes, revealing an unexpectedly significant delay between transcriptional initiation and mature mRNA production early after HIV-1 infection. We computationally derived and validated the underlying regulatory programs, and we predicted drugs capable of reversing these HIV-1-induced expression changes followed by experimental confirmation. Our results show that combined total and mRNA transcriptome analysis is essential for fully capturing the early host response to virus infection and provide a framework for identifying candidate drugs for host-directed therapy against HIV/AIDS.In this study, we used mass sequencing to identify genes differentially expressed in CD4(+) T cells during HIV-1 infection. To our surprise, we found many differentially expressed genes early after infection by analyzing both newly transcribed unprocessed pre-mRNAs and fully processed mRNAs, but not by analyzing mRNAs alone, indicating a significant delay between transcription initiation and mRNA production early after HIV-1 infection. These results also show that important findings could be missed by the standard practice of analyzing mRNAs alone. We then derived the regulatory mechanisms driving the observed expression changes using integrative computational analyses. Further, we predicted drugs that could reverse the observed expression changes induced by HIV-1 infection and showed that one of the predicted drugs indeed potently inhibited HIV-1 infection. This shows that it is possible to identify candidate drugs for host-directed therapy against HIV/AIDS using our genomics-based approach.

SUBMITTER: Peng X

PROVIDER: S-EPMC4136300 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Deep sequencing of HIV-infected cells: insights into nascent transcription and host-directed therapy.

Peng Xinxia X Sova Pavel P Green Richard R RR Thomas Matthew J MJ Korth Marcus J MJ Proll Sean S Xu Jiabao J Cheng Yanbing Y Yi Kang K Chen Li L Peng Zhiyu Z Wang Jun J Wang Jun J Palermo Robert E RE Katze Michael G MG

Journal of virology 20140521 16

<h4>Unlabelled</h4>Polyadenylated mature mRNAs are the focus of standard transcriptome analyses. However, the profiling of nascent transcripts, which often include nonpolyadenylated RNAs, can unveil novel insights into transcriptional regulation. Here, we separately sequenced total RNAs (Total RNAseq) and mRNAs (mRNAseq) from the same HIV-1-infected human CD4(+) T cells. We found that many nonpolyadenylated RNAs were differentially expressed upon HIV-1 infection, and we identified 8 times more d ...[more]

PMID: 24850744

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Project description:Factors that contribute to the transmission of human immunodeficiency virus type 1 (HIV-1), especially drug-resistant HIV-1 variants remain a significant public health concern. In-depth phylogenetic analyses of viral sequences obtained in the screening phase from antiretroviral-naïve HIV-infected patients seeking enrollment in EPZ108859, a large open-label study in the USA, Canada and Puerto Rico (ClinicalTrials.gov NCT00440947) were examined for insights into the roles of drug resistance and epidemiological factors that could impact disease dissemination. Viral transmission clusters (VTCs) were initially predicted from a phylogenetic analysis of population level HIV-1 pol sequences obtained from 690 antiretroviral-naïve subjects in 2007. Subsequently, the predicted VTCs were tested for robustness by ultra deep sequencing (UDS) using pyrosequencing technology and further phylogenetic analyses. The demographic characteristics of clustered and non-clustered subjects were then compared. From 690 subjects, 69 were assigned to 1 of 30 VTCs, each containing 2 to 5 subjects. Race composition of VTCs were significantly more likely to be white (72% vs. 60%; p = 0.04). VTCs had fewer reverse transcriptase and major PI resistance mutations (9% vs. 24%; p = 0.002) than non-clustered sequences. Both men-who-have-sex-with-men (MSM) (68% vs. 48%; p = 0.001) and Canadians (29% vs. 14%; p = 0.03) were significantly more frequent in VTCs than non-clustered sequences. Of the 515 subjects who initiated antiretroviral therapy, 33 experienced confirmed virologic failure through 144 weeks while only 3/33 were from VTCs. Fewer VTCs subjects (as compared to those with non-clustering virus) had HIV-1 with resistance-associated mutations or experienced virologic failure during the course of the study. Our analysis shows specific geographical and drug resistance trends that correlate well with transmission clusters defined by HIV sequences of similarity. Furthermore, our study demonstrates the utility of molecular and epidemiological analysis of VTCs for identifying population-specific risks associated with HIV-1 transmission and developing effective local healthcare strategies.

Project description:ObjectiveTo explore the potential of deep HIV-1 sequencing for adding clinically relevant information relative to viral population sequencing in heavily pre-treated HIV-1-infected subjects.MethodsIn a proof-of-concept study, deep sequencing was compared to population sequencing in HIV-1-infected individuals with previous triple-class virological failure who also developed virologic failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Viral susceptibility was inferred before salvage therapy initiation and at virological failure using deep and population sequencing genotypes interpreted with the HIVdb, Rega and ANRS algorithms. The threshold level for mutant detection with deep sequencing was 1%.Results7 subjects with previous exposure to a median of 15 antiretrovirals during a median of 13 years were included. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively. Self-reported treatment adherence was adequate in 4 and partial in 2; one individual underwent treatment interruption during follow-up. Deep sequencing detected all mutations found by population sequencing and identified additional resistance mutations in all but one individual, predominantly after virological failure to deep salvage therapy. Additional genotypic information led to consistent decreases in predicted susceptibility to etravirine, efavirenz, nucleoside reverse transcriptase inhibitors and indinavir in 2, 1, 2 and 1 subject, respectively. Deep sequencing data did not consistently modify the susceptibility predictions achieved with population sequencing for darunavir, tipranavir or raltegravir.ConclusionsIn this subset of heavily pre-treated individuals, deep sequencing improved the assessment of genotypic resistance to etravirine, but did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility. These data may inform the design of future studies addressing the clinical value of minority drug-resistant variants in treatment-experienced subjects.

Dataset Information

Deep sequencing of HIV-infected cells: insights into nascent transcription and host-directed therapy.

Publications

Deep sequencing of HIV-infected cells: insights into nascent transcription and host-directed therapy.

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