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Keeping quiet: microRNAs in HIV-1 latency; commentary by Siliciano and Han
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Nature Medicine 13, 1138 - 1140 (Oct 2007)
News and Views
Yefei Han1 & Robert F Siliciano1
1. Yefei Han and Robert F. Siliciano are in The Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, USA.
MicroRNAs contribute to HIV-1 latency in resting T cells. This finding could potentially be used in the development of therapies targeted to purge the latent reservoir in an effort to clear the body of virus (pages 1241-1247).
Advances in the treatment of HIV-1 infection with highly active antiretroviral therapy (HAART) have greatly reduced mortality and changed this fatal infection into a chronic disease for many1. HAART can reduce viremia to below the clinical limit of detection. HIV-1 infection has not yet been completely eradicated from patients on HAART, however, as the virus can establish latent infection in a small pool of resting memory CD4+ T cells, in which the provirus is stably integrated into the host genome but does not produce viral proteins2. This state of latency allows HIV-1 to evade immune responses and antiretroviral drugs. Upon host T-cell activation, replication-competent viruses can be released quickly from the latent reservoir to rekindle the infection. Because of its extremely slow decay rate3, the latent reservoir is a major barrier to curing HIV-1 infection.
Understanding precisely how HIV-1 latency is maintained is essential for developing strategies to 'purge' the reservoir4, 5. In this issue of Nature Medicine, Huang et al.6 unearth an additional layer of complexity by providing evidence that multiple cellular microRNAs (miRNAs) are involved in maintaining latency. Further, they show that specific antisense inhibitors of these miRNAs can induce virus production in resting T cells from HIV-1-infected individuals on HAART6.
Several factors may contribute to HIV-1 latency, including transcriptional and post-transcriptional mechanisms4 (Fig. 1). First, the initiation of transcription of the HIV-1 genome is inhibited because crucial activation-dependent host transcription factors, such as nuclear factor-kB (NF-kB) and nuclear factor of activated T cells (NFAT), are excluded from the nucleus in resting cells. Even when transcription is initiated, transcriptional elongation fails because the HIV Tat protein, which recruits cyclin-dependent kinase-9 and cyclin T1, is absent, and this complex is responsible for the phosphorylation of the C-terminal domain of RNA polymerase II that allows transcriptional elongation to occur.
As a result of these blocks in transcriptional initiation and elongation, only a small number of full-length HIV transcripts are made in latently infected cells. The primary transcript, unspliced HIV RNA, is retained in the nucleus in the absence of the HIV Rev protein. Unspliced HIV RNA can be processed to yield spliced forms including multiply spliced HIV RNAs, which encode the regulatory proteins Tat and Rev. If these proteins are not made, neither transcript elongation or export can be achieved. The net effect of all of these blocks is a profound silencing of HIV-1 gene expression in resting CD4+ T cells. It now appears that host cell miRNAs expressed in resting T cells impose an additional block.
miRNAs are small (22 nucleotides in length) noncoding RNAs expressed by metazoans and viruses7 that bind to messenger mRNAs and mark them for post-transcriptional regulation. If the complementarity is complete between an miRNA and its target, the mRNA is cleaved and degraded. If the complementarity is imperfect, translation of the target mRNA is inhibited7. Although it has not been fully proven that RNA viruses express miRNAs8, host miRNAs clearly have important roles in the life cycles and tropism of some viruses, such as primate foamy virus type 1 (ref. 9) and hepatitis C virus10. About 500 human miRNAs are registered in the miRNA database (miRBase release 10.0, http://microrna.sanger.ac.uk). A recent study has shown that the miRNA silencing machinery is involved in the control of HIV-1 in vitro: HIV-1 replication can be promoted by lowering expression of Dicer and Drosher, components of the miRNA silencing machinery11. Whether and how cellular miRNAs might affect HIV-1 latency has remained elusive.
In a series of elegant experiments, Huang et al.6 mapped the potential miRNA target sites in the 3' untranslated region (UTR) of HIV-1 RNAs from several different HIV-1 strains with a GFP reporter system in resting T cells. By dissecting the 3' UTR into small fragments, they identified regions that appeared to function as target sites for miRNAs. Among the cellular miRNAs with predicted binding sites in these regions, five are upregulated in resting T cells when compared with activated T cells (miR-28, miR-125b, miR-150, miR-223 and miR-382), consistent with the notion that these miRNAs might contribute to the latency observed in quiescent cells. Together, the results provide intriguing evidence for the involvement of miRNAs in HIV-1 latency.
miRNAs can inhibit translational initiation. However, several lines of evidence suggest that HIV-1 latency is maintained in part through regulation of the initiation and elongation of viral transcripts (Fig. 1). In latently infected cells, only low levels of HIV-1 transcripts are detected4. Huang et al.6 hypothesized that neutralizing miRNAs in latently infected cells would reverse latency6. To address this question, the authors counteracted inhibition by the five miRNAs mentioned above with specific antisense oligonucleotide inhibitors of these miRNAs. Upon transfection of resting T cells with all five antisense inhibitors and a plasmid encoding an infectious HIV-1 genome, virus production was significantly increased. To further test this, Huang et al.6 collected resting T cells from HIV-infected individuals treated with HAART and applied the five miRNA inhibitors to these samples6. Unlike in untreated cells, replication-competent virus was recovered with this coculture method. Notably, global T-cell activation induced several-fold more virus production than the five miRNA inhibitors. This observation is consistent with previous studies indicating that multiple factors are involved in HIV-1 latency.
This study demonstrates that a new silencing mechanism has a role in HIV latency, contributing to an emerging picture of how this system functions. Latency does not result from any single mechanism; rather, it seems to be the accidental consequence of HIV-1 evolution to better achieve infection of and virus production in activated T cells.
Activated T cells undergo a profound change, however, when they revert back to a resting state as memory cells. The intracellular microenvironment of resting T cells is suboptimal for HIV-1 gene expression, and so the viral genome is profoundly silenced4. Curiously, removing any one of the multiple restrictions on HIV-1 gene expression can lead to virus production. This may be a consequence of the strong positive feedback loop that operates through the HIV-1 protein Tat (Fig. 1) as any change that allows the synthesis of a small amount of Tat protein will result in a large increase in HIV gene expression because Tat promotes processive transcription.
In principle, miRNA inhibitors could potentially be used to purge the reservoir of latent virus in vivo, if the miRNA inhibitors could be delivered for therapeutic advantage. Before this can be attempted, however, two immediate questions remain. First, because of the small size of miRNAs and the partial homology needed to inhibit the target mRNAs, there are an estimated 200 mRNA targets per mammalian miRNA, on average12. It will be necessary to study how the host cell is affected by the neutralization of each of the miRNAs identified in this study, both individually and together. More importantly, it will be necessary to ensure that each infected cell is targeted by such an approach. The cure for HIV-1 will remain unreachable without a reversal of HIV-1 gene silencing in every latently infected cell, as virus from a single cell could potentially restart the infection. Nevertheless, the work of Huang et al.6 has certainly set the stage for interesting future developments in this area.
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