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.

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

2021 ◽  
Author(s):  
Seth Coleman ◽  
Tianyou Yao ◽  
Thu Vu Phuc Nguyen ◽  
Ido Golding ◽  
Oleg Igoshin
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Copy Number ◽  
Viral Gene Expression ◽  
Host Cells ◽  
Optimal Decision ◽  
Viral Gene ◽  
Viral Genomes ◽  
Viral Copy Number ◽  
Viral Copy

SUMMARYWhen 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 due to 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 wildtype 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, 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.

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 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 Cell-to-cell and genome-to-genome variability of adenovirus transcription tuned by the cell cycle

2020 ◽  
Vol 134 (5) ◽  
pp. jcs252544
Author(s):  
Maarit Suomalainen ◽  
Vibhu Prasad ◽  
Abhilash Kannan ◽  
Urs F. Greber
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Single Molecule ◽  
Click Reaction ◽  
Viral Gene Expression ◽  
Early Gene ◽  
Viral Gene ◽  
Viral Genomes ◽  
E1a Gene ◽  
G1 Cells

ABSTRACTIn clonal cultures, not all cells are equally susceptible to virus infection, and the mechanisms underlying this are poorly understood. Here, we developed image-based single-cell measurements to scrutinize the heterogeneity of adenovirus (AdV) infection. AdV delivers, transcribes and replicates a linear double-stranded DNA genome in the nucleus. We measured the abundance of viral transcripts using single-molecule RNA fluorescence in situ hybridization (FISH) and the incoming 5-ethynyl-2′-deoxycytidine (EdC)-tagged viral genomes using a copper(I)-catalyzed azide–alkyne cycloaddition (click) reaction. Surprisingly, expression of the immediate early gene E1A only moderately correlated with the number of viral genomes in the cell nucleus. Intranuclear genome-to-genome heterogeneity was found at the level of viral transcription and, in accordance, individual genomes exhibited heterogeneous replication activity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A gene expression and displayed increased correlation between E1A gene expression and viral genome copy numbers. The combined image-based single-molecule procedures described here are ideally suited to explore the cell-to-cell variability in viral gene expression in a range of different settings, including the innate immune response.

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.

scholarly journals Epigenetic Repression of Herpes Simplex Virus Infection by the Nucleosome Remodeler CHD3

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Jesse H. Arbuckle ◽  
Thomas M. Kristie
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Infection ◽  
Host Cell ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Histone Marks ◽  
Viral Genomes ◽  
Simplex Virus

ABSTRACTUpon infection, the genome of herpes simplex virus is rapidly incorporated into nucleosomes displaying histone modifications characteristic of heterochromatic structures. The initiation of infection requires complex viral-cellular interactions that ultimately circumvent this repression by utilizing host cell enzymes to remove repressive histone marks and install those that promote viral gene expression. The reversion of repression and activation of viral gene expression is mediated by the cellular coactivator HCF-1 in association with histone demethylases and methyltransferases. However, the mechanisms and the components that are involved in the initial repression remain unclear. In this study, the chromatin remodeler chromodomain helicase DNA binding (CHD3) protein is identified as an important component of the initial repression of the herpesvirus genome. CHD3 localizes to early viral foci and suppresses viral gene expression. Depletion of CHD3 results in enhanced viral immediate early gene expression and an increase in the number of transcriptionally active viral genomes in the cell. Importantly, CHD3 can recognize the repressive histone marks that have been detected in the chromatin associated with the viral genome and this remodeler is important for ultimately reducing the levels of accessible viral genomes. A model is presented in which CHD3 represses viral infection in opposition to the actions of the HCF-1 coactivator complex. This dynamic, at least in part, determines the initiation of viral infection.IMPORTANCEChromatin modulation of herpesvirus infection is a dynamic process involving regulatory components that mediate suppression and those that promote viral gene expression and the progression of infection. The mechanisms by which the host cell employs the assembly and modulation of chromatin as an antiviral defense strategy against an invading herpesvirus remain unclear. This study defines a critical cellular component that mediates the initial repression of infecting HSV genomes and contributes to understanding the dynamics of this complex interplay between host cell and viral pathogen.

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

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
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 the present study demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m6A, m5C, and Nm, 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 incis, and 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.

Sign in / Sign up

Export Citation Format

Share Document