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

AbstractCovalent modifications added to individual nucleotides on mRNAs, called epitranscriptomic modifications, have recently emerged as key regulators of both cellular and viral mRNA function1,2 and RNA methylation has now been shown to enhance the replication of human immunodeficiency virus 1 (HIV-1) and several other viruses3–11. Recently, acetylation of the N4 position of cytidine (ac4C) was reported to boost cellular mRNA function by increasing mRNA translation and stability12. We therefore hypothesized that ac4C and N-acetyltransferase 10 (NAT10), the cellular enzyme that adds ac4C to RNAs, might also have been subverted by HIV-1 to increase viral gene expression. We now confirm that HIV-1 transcripts are indeed modified by addition of ac4C at multiple discreet sites and demonstrate that silent mutagenesis of a subset of these ac4C addition sites inhibits HIV-1 gene expression in cis. Moreover, reduced expression of NAT10, and the concomitant decrease in the level of ac4C on viral RNAs, inhibits HIV-1 replication by reducing HIV-1 RNA stability. Interestingly Remodelin, a previously reported inhibitor of NAT10 function13,14, also inhibits HIV-1 replication without affecting cell viability, thus raising the possibility that the addition of ac4C to viral mRNAs might emerge as a novel cellular target for antiviral drug development.

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Identifying HIV-1 RNA splice variant protein interactomes using HyPR-MSSV

10.1101/2020.09.15.298190 ◽

2020 ◽

Author(s):

Rachel A. Knoener ◽

Edward L. Evans ◽

Jordan T. Becker ◽

Mark Scalf ◽

Bayleigh E. Benner ◽

...

Keyword(s):

Gene Expression ◽

Splice Variant ◽

Rna Stability ◽

Mass Spectrometric ◽

Host Protein ◽

Single Population ◽

Mass Spectrometric Identification ◽

Variant Protein ◽

Hiv 1

ABSTRACTHIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs; each playing distinct roles in viral replication. Elucidating their host protein “interactomes” is crucial to understanding virus-host interplay. Here, we present HyPR-MSSV for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes; including, preferential association of regulators of RNA stability with US- and PS-transcripts and, unexpectedly, mitochondria-linked proteins with US-transcripts. Remarkably, >80 of these factors screened by siRNA knock-down impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MSSV for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.

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Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing

10.1101/2021.02.23.432449 ◽

2021 ◽

Author(s):

Kevin Tsai ◽

Hal P. Bogerd ◽

Edward M. Kennedy ◽

Ann Emery ◽

Ronald Swanstrom ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Rna Binding ◽

Binding Protein ◽

Rna Stability ◽

Rna Binding Protein ◽

Viral Gene ◽

Viral Mrnas ◽

Wide Range ◽

Hiv 1

AbstractPrevious work in several laboratories has demonstrated that the epitranscriptomic addition of m6A to viral transcripts promotes the replication and pathogenicity of a wide range of DNA and RNA viruses, yet the underlying mechanisms responsible for this positive effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how m6A readers regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is in large part mediated by the the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs while YTHDF2 binding correlates with increased HIV-1 transcript stability.Author SummaryThis manuscript reports that the expression of mRNAs encoded by the pathogenic human retrovirus HIV-1 is regulated by the methylation of a small number of specific adenosine residues. These in turn recruit a nuclear RNA binding protein, called YTHDC1, which modulates the alternative splicing of HIV-1 transcripts, as well as a cytoplasmic RNA binding protein, called YTHDF2, which stabilizes viral mRNAs. The regulation of HIV-1 gene expression by adenosine methylation is therefore critical for the effective and ordered expression of HIV-1 mRNAs and could represent a novel target for antiviral development.

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Role of small nuclear RNAs in eukaryotic gene expression

Essays in Biochemistry ◽

10.1042/bse0540079 ◽

2013 ◽

Vol 54 ◽

pp. 79-90 ◽

Cited By ~ 34

Author(s):

Saba Valadkhan ◽

Lalith S. Gunawardane

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Rna Stability ◽

Splice Sites ◽

Branch Site ◽

Small Nuclear Rnas ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene ◽

Rna Biogenesis

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the first and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

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Faculty Opinions recommendation of Gene expression profiles of HIV-1-infected glia and brain: toward better understanding of the role of astrocytes in HIV-1-associated neurocognitive disorders.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1166271.627210 ◽

2009 ◽

Author(s):

Bruce Brew

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Neurocognitive Disorders ◽

Hiv 1

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Faculty Opinions recommendation of Distinct gene-expression profiles associated with the susceptibility of pathogen-specific CD4 T cells to HIV-1 infection.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717980795.793471790 ◽

2013 ◽

Author(s):

Paul Goepfert ◽

Anju Bansal

Keyword(s):

Gene Expression ◽

T Cells ◽

Cd4 T Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Distinct Gene ◽

Hiv 1

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STING and cGAS gene expressions were downregulated among HIV-1-infected persons after antiretroviral therapy

Virology Journal ◽

10.1186/s12985-021-01548-6 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Lorena Leticia Peixoto de Lima ◽

Allysson Quintino Tenório de Oliveira ◽

Tuane Carolina Ferreira Moura ◽

Ednelza da Silva Graça Amoras ◽

Sandra Souza Lima ◽

...

Keyword(s):

Gene Expression ◽

Viral Load ◽

T Cell ◽

Antiretroviral Therapy ◽

Cell Count ◽

Enzyme Linked Immunosorbent Assay ◽

Type I ◽

Α Level ◽

The Impact ◽

Hiv 1

Abstract Background The HIV-1 epidemic is still considered a global public health problem, but great advances have been made in fighting it by antiretroviral therapy (ART). ART has a considerable impact on viral replication and host immunity. The production of type I interferon (IFN) is key to the innate immune response to viral infections. The STING and cGAS proteins have proven roles in the antiviral cascade. The present study aimed to evaluate the impact of ART on innate immunity, which was represented by STING and cGAS gene expression and plasma IFN-α level. Methods This cohort study evaluated a group of 33 individuals who were initially naïve to therapy and who were treated at a reference center and reassessed 12 months after starting ART. Gene expression levels and viral load were evaluated by real-time PCR, CD4+ and CD8+ T lymphocyte counts by flow cytometry, and IFN-α level by enzyme-linked immunosorbent assay. Results From before to after ART, the CD4+ T cell count and the CD4+/CD8+ ratio significantly increased (p < 0.0001), the CD8+ T cell count slightly decreased, and viral load decreased to undetectable levels in most of the group (84.85%). The expression of STING and cGAS significantly decreased (p = 0.0034 and p = 0.0001, respectively) after the use of ART, but IFN-α did not (p = 0.1558). Among the markers evaluated, the only markers that showed a correlation with each other were STING and CD4+ T at the time of the first collection. Conclusions ART provided immune recovery and viral suppression to the studied group and indirectly downregulated the STING and cGAS genes. In contrast, ART did not influence IFN-α. The expression of STING and cGAS was not correlated with the plasma level of IFN-α, which suggests that there is another pathway regulating this cytokine in addition to the STING–cGAS pathway.

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The [KIL-d] Element Specifically Regulates Viral Gene Expression in Yeast

Genetics ◽

10.1093/genetics/155.2.601 ◽

2000 ◽

Vol 155 (2) ◽

pp. 601-609 ◽

Cited By ~ 1

Author(s):

Zsolt Tallóczy ◽

Rebecca Mazar ◽

Denise E Georgopoulos ◽

Fausto Ramos ◽

Michael J Leibowitz

Keyword(s):

Gene Expression ◽

Phenotypic Expression ◽

Viral Gene Expression ◽

Virus Genome ◽

Viral Rna ◽

High Rate ◽

Viral Gene ◽

Double Stranded Rna ◽

Yeast Prions ◽

Killer Virus

Abstract The cytoplasmically inherited [KIL-d] element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae. [KIL-d] results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are “healed” in diploids. We report that the [KIL-d] element is spontaneously lost with a frequency of 10−4–10−5 and reappears with variegated phenotypic expression with a frequency of ≥10−3. This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions. However, [KIL-d] is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance. Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the [KIL-d] trait. [KIL-d] epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA. This specificity remained even after a cycle of mating and meiosis. Due to its unique genetic properties and viral RNA specificity, [KIL-d] represents a new type of genetic element that interacts with a viral RNA genome.

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