scholarly journals Influenza Virus Usurps an Interferon-Induced Translational Program to Promote Viral Replication

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 134
Author(s):  
Mitchell P. Ledwith ◽  
Vy Tran ◽  
Thiprampai Thamamongood ◽  
Christina A. Higgins ◽  
Shashank Tripathi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Influenza Virus ◽  
Viral Replication ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Translational Efficiency ◽  
A Genome ◽  
Cellular Mrnas ◽  
Stimulation Of

Hosts mount prudently tuned responses to viral infection in an attempt to block nearly every step of the replication cycle. Viruses must adapt to replicate in this hostile antiviral cellular state. Interferon stimulation or pathogen challenge robustly induces expression of IFIT (interferon-induced proteins with tetratricopeptide repeats) proteins. IFITs are a family of proteins that bind RNA and play antiviral roles during infection. Thus, we were surprised to identify the IFIT family as top candidate proviral host factors for influenza A virus (IAV) in a genome-wide CRISPR–Cas9 knockout screen. We validated the proviral activity of IFIT2 by showing that IFIT2-deficient cells support lower levels of IAV replication and exhibit defects in viral gene expression. The molecular functions of IFIT2, let alone how they are used by influenza virus, are unknown. Using CLIP-seq, we showed that IFIT2 binds directly to viral and cellular mRNAs in AU-rich regions largely in the 3’UTR, with a preference for a subset of interferon-stimulated mRNAs. IFIT2 also associates with actively translating ribosomes in infected cells to facilitate the translation of viral messages. IFIT2-responsive elements from an IAV mRNA were sufficient to confer translational enhancement to exogenous transcripts in cis. Conversely, mutation of these elements or the use of an IFIT2 RNA-binding mutant ablated stimulation of viral gene expression. Together, these data link the RNA-binding capability of IFIT2 to changes in translational efficiency of target viral mRNAs and the stimulation of viral replication. They establish a model for the normal function of IFIT2 as an antiviral protein affecting the post-transcriptional fate of cellular mRNAs and explain how influenza virus repurposes IFIT2 to support viral replication. Our work highlights a new node for the regulation of translation during interferon responses and highlights how canonical antiviral responses may be repurposed to support viral replication.

scholarly journals Influenza Virus NS1 Protein-RNA Interactome Reveals Intron Targeting

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Liang Zhang ◽  
Juan Wang ◽  
Raquel Muñoz-Moreno ◽  
Min Kim ◽  
Ramanavelan Sakthivel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Influenza Virus ◽  
Virus Infection ◽  
Influenza A ◽  
Rna Binding ◽  
Influenza Virus Infection ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Ns1 Protein ◽  
Cellular Processes

ABSTRACTThe NS1 protein of influenza A virus is a multifunctional virulence factor that inhibits cellular processes to facilitate viral gene expression. While NS1 is known to interact with RNA and proteins to execute these functions, the cellular RNAs that physically interact with NS1 have not been systematically identified. Here we reveal a NS1 protein-RNA interactome and show that NS1 primarily binds intronic sequences. Among this subset of pre-mRNAs is the RIG-I pre-mRNA, which encodes the main cytoplasmic antiviral sensor of influenza virus infection. This suggested that NS1 interferes with the antiviral response at a posttranscriptional level by virtue of its RNA binding properties. Indeed, we show that NS1 is necessary in the context of viral infection and sufficient upon transfection to decrease the rate of RIG-I intron removal. This NS1 function requires a functional RNA binding domain and is independent of the NS1 interaction with the cleavage and polyadenylation specificity factor CPSF30. NS1 has been previously shown to abrogate RIG-I-mediated antiviral immunity by inhibiting its protein function. Our data further suggest that NS1 also posttranscriptionally alters RIG-I pre-mRNA processing by binding to the RIG-I pre-mRNA.IMPORTANCEA key virulence factor of influenza A virus is the NS1 protein, which inhibits various cellular processes to facilitate viral gene expression. The NS1 protein is localized in the nucleus and in the cytoplasm during infection. In the nucleus, NS1 has functions related to inhibition of gene expression that involve protein-protein and protein-RNA interactions. While several studies have elucidated the protein interactome of NS1, we still lack a clear and systematic understanding of the NS1-RNA interactome. Here we reveal a nuclear NS1-RNA interactome and show that NS1 primarily binds intronic sequences within a subset of pre-mRNAs, including the RIG-I pre-mRNA that encodes the main cytoplasmic antiviral sensor of influenza virus infection. Our data here further suggest that NS1 is necessary and sufficient to impair intron processing of the RIG-I pre-mRNA. These findings support a posttranscriptional role for NS1 in the inhibition of RIG-I expression.

scholarly journals Bacteriophage self-counting in the presence of viral replication

2021 ◽  
Vol 118 (51) ◽  
pp. e2104163118
Author(s):  
Tianyou Yao ◽  
Seth Coleman ◽  
Thu Vu Phuc Nguyen ◽  
Ido Golding ◽  
Oleg A. Igoshin
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Viral Gene Expression ◽  
Host Cells ◽  
Optimal Decision ◽  
Viral Gene ◽  
Viral Genomes ◽  
Viral Copy Number ◽  
Viral Copy ◽  
Type Phage

When host cells are in low abundance, temperate bacteriophages opt for dormant (lysogenic) infection. Phage lambda implements this strategy by increasing the frequency of lysogeny at higher multiplicity of infection (MOI). However, it remains unclear how the phage reliably counts infecting viral genomes even as their intracellular number increases because of replication. By combining theoretical modeling with single-cell measurements of viral copy number and gene expression, we find that instead of hindering lambda’s decision, replication facilitates it. In a nonreplicating mutant, viral gene expression simply scales with MOI rather than diverging into lytic (virulent) and lysogenic trajectories. A similar pattern is followed during early infection by wild-type phage. However, later in the infection, the modulation of viral replication by the decision genes amplifies the initially modest gene expression differences into divergent trajectories. Replication thus ensures the optimal decision—lysis upon single-phage infection and lysogeny at higher MOI.

B-Catenin Signaling Regulates the In Vivo Distribution of Hepatitis B Virus Biosynthesis across the Liver Lobule

2021 ◽  
Author(s):  
Grant Tarnow ◽  
Alan McLachlan
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Viral Replication ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Central Vein ◽  
Liver Lobule ◽  
Liver Gene ◽  
Mice Liver

β-catenin (Ctnnb1) supports high levels of liver gene expression in hepatocytes in proximity to the central vein functionally defining zone 3 of the liver lobule. This region of the liver lobule supports the highest levels of viral biosynthesis in wildtype HBV transgenic mice. Liver-specific β-catenin-null HBV transgenic mice exhibit a stark loss of high levels of pericentral viral biosynthesis. Additionally, viral replication that does not depend directly on β-catenin activity appears to expand to include hepatocytes of zone 1 of the liver lobule in proximity to the portal vein, a region of the liver that typically lacks significant HBV biosynthesis in wildtype HBV transgenic mice. While the average amount of viral RNA transcripts does not change, viral DNA replication is reduced approximately three-fold. Together, these observations demonstrate that β-catenin signaling represents a major determinant of HBV biosynthesis governing the magnitude and distribution of viral replication across the liver lobule in vivo. Additionally, these findings reveal a novel mechanism for the regulation of HBV biosynthesis that is potentially relevant to the expression of additional liver-specific genes. IMPORTANCE Viral biosynthesis is highest around the central vein in the HBV transgenic mouse model of chronic infection. The associated HBV biosynthetic gradient across the liver lobule is primarily dependent upon β-catenin. In the absence of β-catenin, the gradient of viral gene expression spanning the liver lobule is absent and HBV replication is reduced. Therefore, therapeutically manipulating β-catenin activity in the liver of chronic HBV carriers may reduce circulating infectious virions without greatly modulating viral protein production. Together, these change in viral biosynthesis might limit infection of additional hepatocytes while permitting immunological clearance of previously infected cells, potentially limiting disease persistence.

Protein-RNA interactome analysis reveals wide association of KSHV ORF57 with host non-coding RNAs and polysomes

2021 ◽  
Author(s):  
Beatriz Alvarado-Hernandez ◽  
Yanping Ma ◽  
Nishi R. Sharma ◽  
Vladimir Majerciak ◽  
Alexei Lobanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Splicing ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
28S Rrna ◽  
Protein Coding ◽  
Infected Cells ◽  
Non Coding Rnas ◽  
Rna Interaction

Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding post-transcriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57-CLIP (Cross-linking Immunoprecipitation) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIPed RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host non-coding and protein-coding mRNAs. We found ORF57 binds and regulates expression of a subset of host lncRNAs, including LINC00324, LINC00355, and LINC00839 which are involved in cell growth. ORF57 binds snoRNAs responsible for 18S and 28S rRNA modifications, but does not interact with fibrillarin and NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57-RNA immunoprecipitation (RIP)-snoRNA-array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap with the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57-RIP and Northern blot analyses using a 32 P-labeled oligo probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligos from the rRNA regions that ORF57 binds for oligo pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and its association with polysomes increases PABPC1 binding to, but prevent Ago2 from polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. Significance As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus infected cells. In this report, ORF57 was found to interact many host non-coding RNAs, including lncRNAs, snoRNAs and ribosomal RNAs to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the identified RNA motifs by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins, and with ribosomal RNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4, but not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC-1 but prevents Ago2 association with polysomes. Data provide a compelling evidence on how ORF57 in KSHV infected cells might regulate protein synthesis by blocking Ago2’s hostile activities on translation.

scholarly journals Interaction between STAT-3 and HNF-3 Leads to the Activation of Liver-Specific Hepatitis B Virus Enhancer 1 Function

2002 ◽  
Vol 76 (6) ◽  
pp. 2721-2729 ◽  
Author(s):  
Gulam Waris ◽  
Aleem Siddiqui
Keyword(s):  
Gene Expression ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Transcriptional Control ◽  
Viral Gene Expression ◽  
Cooperative Interaction ◽  
Viral Gene ◽  
Mobility Shift ◽  
B Virus ◽  
Stimulation Of

ABSTRACT The signal transducer and activator of transcription 3 (STAT-3), a member of the STAT family of proteins, binds to a large number of transcriptional control elements and regulates gene expression in response to cytokines. While it binds to its cognate nucleotide sequences, it has been recently shown to directly interact with other transcriptional factors in the absence of DNA. We report here one such novel interaction between STAT-3 and hepatocyte nuclear factor 3 (HNF-3) in the absence of DNA. We have identified a STAT-3 binding site within the core domain of hepatitis B virus (HBV) enhancer 1. The HBV enhancer 1 DNA-STAT-3 protein interaction is shown to be stimulated by interleukin-6 (IL-6) and epidermal growth factor, which leads to an overall stimulation of HBV enhancer 1 function and viral gene expression. Using mobility shift assays and transient transfection schemes, we demonstrate a cooperative interaction between HNF-3 and STAT-3 in mediating the cytokine-mediated HBV enhancer function. Cytokine stimulation of HBV gene expression represents an important regulatory scheme of direct relevance to liver disease pathogenesis associated with HBV infection.

scholarly journals Extensive epitranscriptomic methylation of A and C residues on murine leukemia virus transcripts enhances viral gene expression

2019 ◽  
Author(s):  
David G. Courtney ◽  
Andrea Chalem ◽  
Hal P. Bogerd ◽  
Brittany A. Law ◽  
Edward M. Kennedy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Positive Role ◽  
Viral Rnas ◽  
Order Of Magnitude ◽  
Murine Leukemia

AbstractWhile 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 ofN6-methyladenosine (m6A), 5-methylcytosine (m5C) and 2’O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m6A and m5C 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. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m6A and m5C in viral replication, we also demonstrate that overexpression of the key m6A reader protein YTHDF2 enhances MLV replication, while downregulation of the m5C writer NSUN2 inhibits MLV replication.ImportanceThe data presented in this manuscript demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m6A, m5C and Nm, thus suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these m6A or m5C modifications from MLV transcripts inhibits MLV replication incisand a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors intrans. Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.

scholarly journals Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Ryan T. Behrens ◽  
Mounavya Aligeti ◽  
Ginger M. Pocock ◽  
Christina A. Higgins ◽  
Nathan M. Sherer
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Rna Binding ◽  
Nuclear Export Signal ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Virion Production ◽  
Export Signal ◽  
Hiv 1 ◽  
Rev Protein

ABSTRACT HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. IMPORTANCE HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.

scholarly journals Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication

2002 ◽  
Vol 76 (1) ◽  
pp. 313-326 ◽  
Author(s):  
Jeffery L. Meier ◽  
Michael J. Keller ◽  
James J. McCoy
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Gene Transcription ◽  
Human Cytomegalovirus ◽  
Viral Gene Expression ◽  
Immediate Early ◽  
Viral Gene ◽  
Distal Enhancer ◽  
Cis Acting ◽  
Hcmv Replication

ABSTRACT We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer’s orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at −300 or −345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

scholarly journals The E3 Ubiquitin-Protein Ligase Cullin 3 Regulates HIV-1 Transcription

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2010 ◽  
Author(s):  
Simon Langer ◽  
Xin Yin ◽  
Arturo Diaz ◽  
Alex J. Portillo ◽  
David E. Gordon ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Life Cycle ◽  
Viral Replication ◽  
Viral Gene Expression ◽  
Viral Life Cycle ◽  
Cellular Proteins ◽  
Viral Gene ◽  
Cullin 3 ◽  
Hiv 1

The infectious life cycle of the human immunodeficiency virus type 1 (HIV-1) is characterized by an ongoing battle between a compendium of cellular proteins that either promote or oppose viral replication. On the one hand, HIV-1 utilizes dependency factors to support and sustain infection and complete the viral life cycle. On the other hand, both inducible and constitutively expressed host factors mediate efficient and functionally diverse antiviral processes that counteract an infection. To shed light into the complex interplay between HIV-1 and cellular proteins, we previously performed a targeted siRNA screen to identify and characterize novel regulators of viral replication and identified Cullin 3 (Cul3) as a previously undescribed factor that negatively regulates HIV-1 replication. Cul3 is a component of E3-ubiquitin ligase complexes that target substrates for ubiquitin-dependent proteasomal degradation. In the present study, we show that Cul3 is expressed in HIV-1 target cells, such as CD4+ T cells, monocytes, and macrophages and depletion of Cul3 using siRNA or CRISPR/Cas9 increases HIV-1 infection in immortalized cells and primary CD4+ T cells. Conversely, overexpression of Cul3 reduces HIV-1 infection in single replication cycle assays. Importantly, the antiviral effect of Cul3 was mapped to the transcriptional stage of the viral life cycle, an effect which is independent of its role in regulating the G1/S cell cycle transition. Using isogenic viruses that only differ in their promotor region, we find that the NF-κB/NFAT transcription factor binding sites in the LTR are essential for Cul3-dependent regulation of viral gene expression. Although Cul3 effectively suppresses viral gene expression, HIV-1 does not appear to antagonize the antiviral function of Cul3 by targeting it for degradation. Taken together, these results indicate that Cul3 is a negative regulator of HIV-1 transcription which governs productive viral replication in infected cells.

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