scholarly journals Widespread remodeling of the m6A RNA-modification landscape by a viral regulator of RNA processing and export

2021 ◽  
Vol 118 (30) ◽  
pp. e2104805118
Author(s):  
Kalanghad Puthankalam Srinivas ◽  
Daniel P. Depledge ◽  
Jonathan S. Abebe ◽  
Stephen A. Rice ◽  
Ian Mohr ◽  
...  
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Rna Modification ◽  
Viral Gene ◽  
Viral Mrnas ◽  
Simplex Virus ◽  
Processing Stability ◽  
Hsv 1

N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification, contributing to the processing, stability, and function of methylated RNAs. Methylation occurs in the nucleus during pre-mRNA synthesis and requires a core methyltransferase complex consisting of METTL3, METTL14, and WTAP. During herpes simplex virus (HSV-1) infection, cellular gene expression is profoundly suppressed, allowing the virus to monopolize the host transcription and translation apparatus and antagonize antiviral responses. The extent to which HSV-1 uses or manipulates the m6A pathway is not known. Here, we show that, in primary fibroblasts, HSV-1 orchestrates a striking redistribution of the nuclear m6A machinery that progresses through the infection cycle. METTL3 and METTL14 are dispersed into the cytoplasm, whereas WTAP remains nuclear. Other regulatory subunits of the methyltransferase complex, along with the nuclear m6A-modified RNA binding protein YTHDC1 and nuclear demethylase ALKBH5, are similarly redistributed. These changes require ICP27, a viral regulator of host mRNA processing that mediates the nucleocytoplasmic export of viral late mRNAs. Viral gene expression is initially reduced by small interfering RNA (siRNA)-mediated inactivation of the m6A methyltransferase but becomes less impacted as the infection advances. Redistribution of the nuclear m6A machinery is accompanied by a wide-scale reduction in the installation of m6A and other RNA modifications on both host and viral mRNAs. These results reveal a far-reaching mechanism by which HSV-1 subverts host gene expression to favor viral replication.

scholarly journals Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Benjamin A. Diner ◽  
Krystal K. Lum ◽  
Jared E. Toettcher ◽  
Ileana M. Cristea
Keyword(s):  
Gene Expression ◽  
Live Cell Imaging ◽  
Nuclear Periphery ◽  
Viral Gene Expression ◽  
Live Cell ◽  
Viral Gene ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTThe human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses.IMPORTANCEHow mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16 rapidly oligomerizes at incoming herpesvirus genomes at the nuclear periphery to transcriptionally repress viral gene expression and limit viral replicative capacity. We further demonstrate that IFI16 does not initiate upstream activation of the canonical STING/TBK-1/IRF3 signaling pathway but is required for downstream antiviral cytokine expression. In contrast, we find that, upon DNA sensing during herpesvirus infection, cGAS triggers apoptosis in a STING-dependent manner. Our live-cell imaging, mass spectrometry-based proteomics, CRISPR-based cellular assays, and optogenetics underscore the value of integrative approaches to uncover complex cellular responses against pathogens.

scholarly journals Regulation of viral gene expression by the herpes simplex virus 1 UL24 protein (HSV-1 UL24 inhibits accumulation of viral transcripts)

Virology ◽  
2016 ◽  
Vol 495 ◽  
pp. 148-160 ◽  
Author(s):  
Carolina Sanabria-Solano ◽  
Carmen Elena Gonzalez ◽  
Nicolas Richerioux ◽  
Luc Bertrand ◽  
Slimane Dridi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

scholarly journals Herpes Simplex Virus Is Equipped with RNA- and Protein-Based Mechanisms To Repress Expression of ATRX, an Effector of Intrinsic Immunity

2012 ◽  
Vol 86 (18) ◽  
pp. 10093-10102 ◽  
Author(s):  
Igor Jurak ◽  
Leah B. Silverstein ◽  
Mayuri Sharma ◽  
Donald M. Coen
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Intrinsic Immunity ◽  
Gene Encoding ◽  
Simplex Virus ◽  
In Cells ◽  
Hsv 1

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3′ untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection.

scholarly journals Mapping of Functional Regions in the Amino-Terminal Portion of the Herpes Simplex Virus ICP27 Regulatory Protein: Importance of the Leucine-Rich Nuclear Export Signal and RGG Box RNA-Binding Domain

2002 ◽  
Vol 76 (23) ◽  
pp. 11866-11879 ◽  
Author(s):  
Joy Lengyel ◽  
Chandra Guy ◽  
Vivian Leong ◽  
Sarah Borge ◽  
Stephen A. Rice
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Nuclear Export Signal ◽  
Viral Gene Expression ◽  
Vero Cells ◽  
Viral Gene ◽  
Functional Regions ◽  
Hsv 1

ABSTRACT Infected-cell protein 27 (ICP27) is an essential herpes simplex virus type 1 (HSV-1) regulatory protein that activates a subset of viral delayed-early and late genes, at least in part through posttranscriptional mechanisms. Previous studies have shown that the amino (N)-terminal half of the protein contains important functional regions, including a leucine-rich nuclear export signal (NES). However, to date, the phenotype of an HSV-1 ICP27 NES mutant has not been reported. In this study, we engineered and characterized dLeu, an HSV-1 deletion mutant that specifically lacks ICP27's NES (amino acids 6 to 19). The phenotype of dLeu was analyzed alongside those of eight other ICP27 N-terminal deletion mutants. We found that in Vero cells, dLeu displays modest defects in viral gene expression and an approximately 100-fold reduction in the production of viral progeny. Unlike wild-type (WT) ICP27, which exhibits a cytoplasmic distribution in addition to its predominant nuclear localization, dLeu ICP27 is highly restricted to the cell nucleus. This strongly suggests that the N-terminal leucine-rich sequence functions as an NES during viral infection. Our analysis of dLeu and the other mutants has enabled us to genetically define the regions in the N-terminal 200 residues of ICP27 which are required for efficient viral growth in Vero cells. Only two regions appear to be important: (i) the leucine-rich NES and (ii) the RGG box RNA-binding domain, encoded by residues 139 to 153. A virus lacking the RGG box-encoding sequence, d4-5, has a phenotype similar to that of dLeu in that it displays modest defects in viral gene expression and grows poorly. Interestingly, deletion of both the NES and RGG box, as well as the sequences in between, is lethal. The resulting virus, d1-5, displays severe defects in viral gene expression and DNA synthesis and is unable to produce significant amounts of infectious progeny. Therefore, the N-terminal portion of ICP27 contains at least two functional domains which collectively are absolutely essential for viral infection.

scholarly journals Cellular Transcriptional Coactivator RanBP10 and Herpes Simplex Virus 1 ICP0 Interact and Synergistically Promote Viral Gene Expression and Replication

2016 ◽  
Vol 90 (6) ◽  
pp. 3173-3186 ◽  
Author(s):  
Yuka Sato ◽  
Akihisa Kato ◽  
Yuhei Maruzuru ◽  
Masaaki Oyama ◽  
Hiroko Kozuka-Hata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Remodeling ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Null Mutation ◽  
Null Mutant ◽  
Transcriptional Coactivator ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTTo investigate the molecular mechanism(s) by which herpes simplex virus 1 (HSV-1) regulatory protein ICP0 promotes viral gene expression and replication, we screened cells overexpressing ICP0 for ICP0-binding host cell proteins. Tandem affinity purification of transiently expressed ICP0 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP0 interacted with cell protein RanBP10, a known transcriptional coactivator, in HSV-1-infected cells. Knockdown of RanBP10 in infected HEp-2 cells resulted in a phenotype similar to that observed with the ICP0-null mutation, including reduction in viral replication and in the accumulation of viral immediate early (ICP27), early (ICP8), and late (VP16) mRNAs and proteins. In addition, RanBP10 knockdown or the ICP0-null mutation increased the level of histone H3 association with the promoters of these viral genes, which is known to repress transcription. These effects observed in wild-type HSV-1-infected HEp-2 RanBP10 knockdown cells or those observed in ICP0-null mutant virus-infected control HEp-2 cells were remarkably increased in ICP0-null mutant virus-infected HEp-2 RanBP10 knockdown cells. Our results suggested that ICP0 and RanBP10 redundantly and synergistically promoted viral gene expression by regulating chromatin remodeling of the HSV-1 genome for efficient viral replication.IMPORTANCEUpon entry of herpesviruses into a cell, viral gene expression is restricted by heterochromatinization of the viral genome. Therefore, HSV-1 has evolved multiple mechanisms to counteract this epigenetic silencing for efficient viral gene expression and replication. HSV-1 ICP0 is one of the viral proteins involved in counteracting epigenetic silencing. Here, we identified RanBP10 as a novel cellular ICP0-binding protein and showed that RanBP10 and ICP0 appeared to act synergistically to promote viral gene expression and replication by modulating viral chromatin remodeling. Our results provide insight into the mechanisms by which HSV-1 regulates viral chromatin remodeling for efficient viral gene expression and replication.

scholarly journals General and Specific Alterations in Programming of Global Viral Gene Expression during Infection by VP16 Activation-Deficient Mutants of Herpes Simplex Virus Type 1

2002 ◽  
Vol 76 (24) ◽  
pp. 12758-12774 ◽  
Author(s):  
William C. Yang ◽  
G. V. Devi-Rao ◽  
Peter Ghazal ◽  
Edward K. Wagner ◽  
Steven J. Triezenberg
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Gene Expression ◽  
Productive Infection ◽  
Viral Gene ◽  
Virion Protein ◽  
Activation Domain ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT During productive infection by herpes simplex virus 1 (HSV-1), viral gene expression occurs in a temporally regulated cascade in which transcription of the viral immediate-early (IE) genes is strongly stimulated by the virion protein VP16. We have employed an oligonucleotide microarray to examine the effect of VP16 mutations on the overall pattern of viral gene expression following infection of HeLa cells. This microarray detects essentially all HSV-1 transcripts with relative and absolute levels correlating well with known kinetics of expression. This analysis revealed that deletion of the VP16 activation domain sharply reduced overall viral gene expression; moreover, the pattern of this reduced expression varied greatly from the pattern of a wild-type (wt) infection. However, when this mutant virus was delivered at a high multiplicity of infection or in the presence of the cellular stress inducer hexamethylene bisacetamide, expression was largely restored to the wt levels and pattern. Infection with virions that deliver wt VP16 protein at the start of infection but synthesize only truncated VP16 resulted in a normal kinetic cascade. This suggests that newly synthesized VP16 does not play a significant role in the expression of later classes of transcripts. The VP16 activation domain comprises two subregions. Deletion of the C-terminal subregion resulted in minimal changes in the level and profile of gene expression compared to a normal (wt) cascade. In contrast, deletion of the N-terminal subregion reduced the overall expression levels and skewed the relative levels of IE transcripts but did not significantly alter the kinetic pattern of early and late transcript expression. We conclude that the general activation of IE gene transcription by VP16, but not the specific ratios of IE transcripts, is necessary for the subsequent ordered expression of viral genes. Moreover, this report establishes the feasibility of microarray analysis for globally assessing viral gene expression programs as a function of the conditions of infection.

scholarly journals Herpes Simplex Virus Type 1 Immediate-Early Protein ICP27 Is Required for Efficient Incorporation of ICP0 and ICP4 into Virions

2007 ◽  
Vol 82 (1) ◽  
pp. 268-277 ◽  
Author(s):  
Lenka Sedlackova ◽  
Stephen A. Rice
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Cytoplasmic Localization ◽  
Viral Particles ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Early in infection, herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins ICP0 and ICP4 localize to the nucleus, where they stimulate viral transcription. Later in infection, ICP0 and to a lesser extent ICP4 accumulate in the cytoplasm, but their biological role there is unknown. Previously, it was shown that the cytoplasmic localization of ICP0/4 requires the multifunctional IE protein ICP27, which is itself an activator of viral gene expression. Here, we identify a viral ICP27 mutant, d3-4, which is unable to efficiently localize ICP0 and ICP4 to the cytoplasm but which otherwise resembles wild-type HSV-1 in its growth and viral gene expression phenotypes. These results genetically separate the function of ICP27 that affects ICP0/4 localization from its other functions, which affect viral growth and gene expression. As both ICP0 and ICP4 are known to be minor virion components, we used d3-4 to test the hypothesis that the cytoplasmic localization of these proteins is required for their incorporation into viral particles. Consistent with this conjecture, d3-4 virions were found to lack ICP0 in their tegument and to have greatly reduced levels of ICP4. Thus, the cytoplasmic localization of ICP0 and ICP4 appears to be a prerequisite for the assembly of these important transcriptional regulatory proteins into viral particles. Furthermore, our results show that ICP27 plays a previously unrecognized role in determining the composition of HSV-1 virions.

Herpesvirus-mediated stabilization of ICP0 expression neutralizes restriction by TRIM23

2021 ◽  
Vol 118 (51) ◽  
pp. e2113060118
Author(s):  
Xing Liu ◽  
Dhiraj Acharya ◽  
Eric Krawczyk ◽  
Chase Kangas ◽  
Michaela U. Gack ◽  
...  
Keyword(s):  
Viral Replication ◽  
Viral Gene Expression ◽  
Proteasomal Degradation ◽  
Viral Gene ◽  
Posttranslational Regulation ◽  
Early Proteins ◽  
Immediate Early Proteins ◽  
Simplex Virus ◽  
Functional Analyses ◽  
Hsv 1

Herpes simplex virus (HSV) infection relies on immediate early proteins that initiate viral replication. Among them, ICP0 is known, for many years, to facilitate the onset of viral gene expression and reactivation from latency. However, how ICP0 itself is regulated remains elusive. Through genetic analyses, we identify that the viral γ134.5 protein, an HSV virulence factor, interacts with and prevents ICP0 from proteasomal degradation. Furthermore, we show that the host E3 ligase TRIM23, recently shown to restrict the replication of HSV-1 (and certain other viruses) by inducing autophagy, triggers the proteasomal degradation of ICP0 via K11- and K48-linked ubiquitination. Functional analyses reveal that the γ134.5 protein binds to and inactivates TRIM23 through blockade of K27-linked TRIM23 autoubiquitination. Deletion of γ134.5 or ICP0 in a recombinant HSV-1 impairs viral replication, whereas ablation of TRIM23 markedly rescues viral growth. Herein, we show that TRIM23, apart from its role in autophagy-mediated HSV-1 restriction, down-regulates ICP0, whereas viral γ134.5 functions to disable TRIM23. Together, these results demonstrate that posttranslational regulation of ICP0 by virus and host factors determines the outcome of HSV-1 infection.

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