RNA conformational propensities determine cellular activity.

Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules.

Epigenetic silencing by the SMC5/6 complex mediates HIV-1 latency.

After viral entry and reverse transcription, HIV-1 proviruses that fail to integrate are epigenetically silenced, but the underlying mechanism has remained unclear. Using a genome-wide CRISPR/Cas9 knockout screen, we identified the host SMC5/6 complex as essential for this epigenetic silencing. We show that SMC5/6 binds to and then SUMOylates unintegrated chromatinized HIV-1 DNA.

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique.

Pseudouridine (Ψ) is the most common noncanonical ribonucleoside present on mammalian noncoding RNAs (ncRNAs), including rRNAs, tRNAs, and snRNAs, where it contributes ∼7% of the total uridine level. However, Ψ constitutes only ∼0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear.

Epitranscriptomic addition of mA regulates HIV-1 RNA stability and alternative splicing.

Previous work has demonstrated that the epitranscriptomic addition of mA to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, yet the underlying mechanisms responsible for this effect have remained unclear. It is known that mA function is largely mediated by cellular mA binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that mA addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear mA reader YTHDC1 and the cytoplasmic mA reader YTHDF2.

Mapping RNA Modifications Using Photo-Crosslinking-Assisted Modification Sequencing.

Epitranscriptomic RNA modifications function as an important layer of gene regulation that modulates the function of RNA transcripts. A key step in understanding how RNA modifications regulate biological processes is the mapping of their locations, which is most commonly done by RNA immunoprecipitation (RIP) using modification-specific antibodies. Here, we describe the use of a photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) method, in conjunction with RNA modification-specific antibodies, to map modification sites.

Understanding the characteristics of nonspecific binding of drug-like compounds to canonical stem-loop RNAs and their implications for functional cellular assays.

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities.

Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability.

Epitranscriptomic RNA modifications, including methylation of adenine and cytidine residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression.

Epigenetic and epitranscriptomic regulation of viral replication.

Eukaryotic gene expression is regulated not only by genomic enhancers and promoters, but also by covalent modifications added to both chromatin and RNAs. Whereas cellular gene expression may be either enhanced or inhibited by specific epigenetic modifications deposited on histones (in particular, histone H3), these epigenetic modifications can also repress viral gene expression, potentially functioning as a potent antiviral innate immune response in DNA virus-infected cells. However, viruses have evolved countermeasures that prevent the epigenetic silencing of their genes during lytic replication, and they can also take advantage of epigenetic silencing to establish latent infections.

Reversal of Epigenetic Silencing Allows Robust HIV-1 Replication in the Absence of Integrase Function.

Integration of the proviral DNA intermediate into the host cell genome normally represents an essential step in the retroviral life cycle. While the reason(s) for this requirement remains unclear, it is known that unintegrated proviral DNA is epigenetically silenced. Here, we demonstrate that human immunodeficiency virus 1 (HIV-1) mutants lacking a functional integrase (IN) can mount a robust, spreading infection in cells expressing the Tax transcription factor encoded by human T-cell leukemia virus 1 (HTLV-1).

Probing RNA Conformational Equilibria within the Functional Cellular Context.

Low-abundance short-lived non-native conformations referred to as excited states (ESs) are increasingly observed in vitro and implicated in the folding and biological activities of regulatory RNAs. We developed an approach for assessing the relative abundance of RNA ESs within the functional cellular context. Nuclear magnetic resonance (NMR) spectroscopy was used to estimate the degree to which substitution mutations bias conformational equilibria toward the inactive ES in vitro.

Epitranscriptomic Addition of mC to HIV-1 Transcripts Regulates Viral Gene Expression.

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (mC) and 2'O-methyl modifications being particularly prevalent.

Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression.

While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of -methyladenosine (mA), 5-methylcytosine (mC), and 2'O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of mA and mC residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites.

Targeting HPV16 DNA using CRISPR/Cas inhibits anal cancer growth .

Aim: The goal of this study was to determine if a single AAV vector, encoding Cas9 and guide RNAs specific for the HPV16 E6 and E7 genes, could inhibit the growth of an HPV16-induced tumor .

Materials & Methods: We grew HPV16, patient-derived anal cancer explants in immunodeficient mice and then challenged these by injection of AAV-based vectors encoding Cas9 and control or HPV16-specific guide RNAs.

Results & Conclusion: We observed a significant and selective reduction in tumor growth when the HPV16 E6 and E7 genes were targeted using Cas9.

Influenza A virus-derived siRNAs increase in the absence of NS1 yet fail to inhibit virus replication.

While the issue of whether RNA interference (RNAi) ever forms part of the antiviral innate immune response in mammalian somatic cells remains controversial, there is considerable evidence demonstrating that few, if any, viral small interfering RNAs (siRNAs) are produced in infected cells. Moreover, inhibition of RNAi by mutational inactivation of key RNAi factors, such as Dicer or Argonaute 2, fails to enhance virus replication. One potential explanation for this lack of inhibitory effect is that mammalian viruses encode viral suppressors of RNAi (VSRs) that are so effective that viral siRNAs are not produced in infected cells.

Insights into the mechanisms underlying the inactivation of HIV-1 proviruses by CRISPR/Cas.

DNA editing using CRISPR/Cas has emerged as a potential treatment for diseases caused by pathogenic human DNA viruses. One potential target is HIV-1, which replicates via a chromosomally integrated DNA provirus. While CRISPR/Cas can protect T cells from de novo HIV-1 infection, HIV-1 frequently becomes resistant due to mutations in the chosen single guide RNA (sgRNA) target site.

Addition of m6A to SV40 late mRNAs enhances viral structural gene expression and replication.

Polyomaviruses are a family of small DNA tumor viruses that includes several pathogenic human members, including Merkel cell polyomavirus, BK virus and JC virus. As is characteristic of DNA tumor viruses, gene expression in polyomaviruses is temporally regulated into an early phase, consisting of the viral regulatory proteins, and a late phase, consisting of the viral structural proteins. Previously, the late transcripts expressed by the prototypic polyomavirus simian virus 40 (SV40) were reported to contain several adenosines bearing methyl groups at the N6 position (m6A), although the precise location of these m6A residues, and their phenotypic effects, have not been investigated.

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis.

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant (∆123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3'UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein.

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

Many viral RNAs are modified by methylation of the N position of adenosine (mA). mA is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses mA-modified RNAs, but the effects of mA on this segmented RNA virus remain unclear.

RNA Interference in Mammals: The Virus Strikes Back.

The importance of RNA interference (RNAi) as a mammalian antiviral defense mechanism has been controversial. Qiu et al. (2017) now present data suggesting that the difficulty of detecting RNAi in virus-infected mammalian cells reflects the expression of highly effective viral suppressors of RNAi.