The microRNA miR-29a is associated with human immunodeficiency virus latency
© Patel et al.; licensee BioMed Central Ltd. 2014
Received: 9 August 2014
Accepted: 11 November 2014
Published: 9 December 2014
Latent reservoirs of HIV-1 provide a major challenge to its cure. There are increasing reports of interplay between HIV-1 replication and host miRNAs. Several host miRNAs, which potentially target the nef-3′LTR region of HIV-1 RNA, including miR-29a, are proposed to promote latency.
We used two established cellular models of HIV-1 latency – the U1 monocytic and J1.1 CD4+ T cell lines to show an inverse relationship between HIV-1 replication and miR-29a levels, which was mediated by the HIV-1 Nef protein. Using a miR-29a responsive luciferase reporter plasmid, an expression plasmid and an anti-miR29a LNA, we further demonstrate increased miR-29a levels during latency and reduced levels following active HIV replication. Finally, we show that miR-29a levels in the PBMCs and plasma of HIV infected persons also correlate inversely with latency and active viral replication.
The levels of miR-29a correlate inversely with active HIV-1 replication in cell culture models and in HIV infected persons. This links miR-29a to viral latency and suggests another approach to activate and destroy latent HIV-1 reservoirs.
MicroRNAs (miRNAs) are 18–23 nucleotides non-coding, regulatory RNA molecules, which are involved in post-transcriptional gene regulation and have recently been shown to be important for regulating host responses in vertebrates . During viral infections, miRNAs can either directly affect viral replication or modulate the expression of host genes and pathways essential for it . For example, miR-32 and miR-181 restrict the replication of primate foamy virus 1 replication , and porcine reproductive and respiratory syndrome virus and Mink enteritis virus ,, respectively. Alternatively, miR-122 is highly expressed in the liver and facilitates hepatitis C virus replication ,. The human immunodeficiency virus (HIV) may also be targeted by several host miRNAs . The HIV-1 Tat and Nef proteins inhibit key components of the host miRNA pathway ,, knockdown of the miRNA biogenesis proteins Drosha and Dicer in latently infected cells increases HIV-1 replication, and host miRNAs such as the miR-17/92 cluster indirectly modulate HIV-1 replication through the p300/CBP-associated factor . Thus, there is significant functional interplay between HIV-1 and miRNA-mediated silencing in host cells.
Following highly active antiretroviral therapy (HAART), the HIV-1 load is reduced dramatically to undetectable levels. However, replication competent viruses survive in latent reservoirs whose size is determined by the initial viral loads. On therapy interruption or failure, the virus replicates and repopulates peripheral sites . The ability of HIV-1 to cause latent and persistent infection is a major impediment to its cure, making it important to understand the mechanisms of latency . Profiling studies using various HIV-1 infection models have revealed different miRNAs associated with viral replication -. Although their contribution to HIV-1 latency is unclear, comparative miRNA expression in resting versus activated primary CD4+ T lymphocytes identified several host miRNAs, which potentially target the nef-3′UTR region to promote latency . Several recent studies have shown that miR-29a, which targets the nef-3′UTR, is a potent inhibitor of HIV-1 replication ,,. Another report also showed miR-29a, −29b, −9 and -146a to target the SIV/HIV 3′UTR . We hypothesize that miR-29a levels are high during latency and are reduced during active viral replication, and have explored this with cellular models of latent HIV-1 infection as well as PBMC and plasma from HIV infected individuals. All cell lines, other materials and methods used are detailed in Additional file 1: Materials and Methods.
Previous studies on the role of miRNAs in HIV infection and pathogenesis have largely focused on acute infection models that involve actively replicating virus. Microarray analysis of acutely infected PBMCs showed downregulation of miR-29a and similar results were observed in PBMCs isolated from HIV infected individuals with high viral load ,. These results directly support our hypothesis. In the U1 and J1.1 latency models we observed miR-29a levels to reduce following PMA activation and HIV-1 replication, and this correlated with expression of the Nef protein. Higher levels of miR-29a in the PBMCs and plasma of HIV-infected asymptomatic individuals compared to those with symptomatic disease, also support a role for miR-29a in HIV latency. Whether the size of the latent reservoir is determined early in infection or is related to peak viral loads or CD4/CD8 ratio is currently debatable. Our data support its relation to viral load, which is lower in asymptomatic individuals.
A functional miR-29a site is located in a highly conserved region within the HIV-1 nef-3′UTR. Previous studies have shown that co-transfection of HeLa cells with the HIV-1 pNL4-3 infectious clone and a miR-29a mimic leads to reduced p24 levels ,. In physiologically relevant cell line models, we observed that overexpression of miR-29a further reduced HIV-1 replication, and a knockdown of miR-29a induced HIV-1 replication in latently infected cells without the need for PMA. Previous studies have shown that HIV-1 latency is controlled at the transcriptional level, and histone deacetylase (HDAC) inhibitors have been studied to break HIV-1 latency. Different HDAC inhibitors can induce HIV-1 replication from 2–20 folds in U1 cells . Though the effects of blocking miR-29a on HIV-1 replication are small compared to those of HDAC inhibitors, these are still significant. Our findings suggest that HIV-1 latency may also be controlled at the post-transcriptional levels. This offers the opportunity for synergism with HDAC inhibitors in purging viral reservoirs.
The interaction of miR-29a and 3′UTR of HIV-1 RNA is sufficient for targeting the latter to the P bodies; the disruption of P bodies results in enhanced viral replication . Host or environmental cues might release viral mRNAs from P bodies and thus release the suppression on viral replication . Along these lines, increased miR-29a might target more viral mRNAs to P bodies and promote latency. The present study shows that HIV-1 Nef and miR-29a levels are inversely correlated, suggesting another mechanism for its activation of HIV-1 replication. Our data also suggest a role for miR-29a in maintaining HIV-1 latency and provides a new approach to activate (and destroy) latent HIV-1 reservoirs by inhibiting miR-29a. Further investigations are required to understand the tripartite regulatory axis of Nef, miR-29a and latency.
We gratefully acknowledge receiving the HIV-1 infectious clone pNL4-3 from Dr. Malcolm Martin through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, and plasmids pMIR-Report-Nef3′UTR, pMIR-Report and pEGFP-miR-29a from Dr. Beena Pillai at the Institute of Genomics and Integrative Biology, New Delhi, India. This work was supported by funds from Department of Biotechnology, Government of India.
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