Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape

One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection

Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a DNA virus that is linked to several human malignancies. The cGMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) pathway is able to detect KSHV during primary infection and regulates the reactivation of KSHV from latency. We screened KSHV proteins for their ability to inhibit this pathway and block IFN-β activation. One KSHV protein, vIRF1, inhibited this pathway by preventing STING from interacting with TBK1 and inhibiting STING’s phosphorylation and concomitant activation. Moreover, depletion of vIRF1 in the context of KSHV infection prevented efficient viral reactivation and replication, and increased the host IFN response to KSHV. Collectively, our results demonstrate that the modulation of this pathway is important for viral transmission and the lifelong persistence of gammaherpesviruses in the human population

Aberrant Herpesvirus-Induced Polyadenylation Correlates With Cellular Messenger RNA Destruction

Inhibition of host cell gene expression by the human herpesvirus KSHV occurs via a novel mechanism involving polyadenylation-linked RNA turnover

Coordinated Destruction of Cellular Messages in Translation Complexes by the Gammaherpesvirus Host Shutoff Factor and the Mammalian Exonuclease Xrn1

Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells

The Anti-interferon Activity of Conserved Viral dUTPase ORF54 is Essential for an Effective MHV-68 Infection

Gammaherpesviruses such as KSHV and EBV establish lifelong persistent infections through latency in lymphocytes. These viruses have evolved several strategies to counteract the various components of the innate and adaptive immune systems. We conducted an unbiased screen using the genetically and biologically related virus, MHV-68, to find viral ORFs involved in the inhibition of type I interferon signaling and identified a conserved viral dUTPase, ORF54. Here we define the contribution of ORF54 in type I interferon inhibition by ectopic expression and through the use of genetically modified MHV-68. ORF54 and an ORF54 lacking dUTPase enzymatic activity efficiently inhibit type I interferon signaling by inducing the degradation of the type I interferon receptor protein IFNAR1. Subsequently, we show in vitro that the lack of ORF54 causes a reduction in lytic replication in the presence of type I interferon signaling. Investigation of the physiological consequence of IFNAR1 degradation and importance of ORF54 during MHV-68 in vivo infection demonstrates that ORF54 has an even greater impact on persistent infection than on lytic replication. MHV-68 lacking ORF54 expression is unable to efficiently establish latent infection in lymphocytes, although it replicates relatively normally in lung tissues. However, infection of IFNAR−/− mice alleviates this phenotype, emphasizing the specific role of ORF54 in type I interferon inhibition. Infection of mice and cells by a recombinant MHV-68 virus harboring a site specific mutation in ORF54 rendering the dUTPase inactive demonstrates that dUTPase enzymatic activity is not required for anti-interferon function of ORF54. Moreover, we find that dUTPase activity is dispensable at all stages of MHV-68 infection analyzed. Overall, our data suggest that ORF54 has evolved anti-interferon activity in addition to its dUTPase enzymatic activity, and that it is actually the anti-interferon role that renders ORF54 critical for establishing an effective persistent infection of MHV-68

Modulation of the Translational Landscape During Herpesvirus Infection.

Herpesviral mRNAs are produced and translated by cellular machinery, rendering them susceptible to the network of regulatory events that impact translation. In response, these viruses have evolved to infiltrate and hijack translational control pathways as well as to integrate specialized host translation strategies into their own repertoire. They are robust systems to dissect mechanisms of mammalian translational regulation and continue to offer insight into cis-acting mRNA features that impact assembly and activity of the translation apparatus. Here, I discuss recent advances revealing the extent to which the three herpesvirus subfamilies regulate both host and viral translation, thereby dramatically impacting the landscape of protein synthesis in infected cells

Feedback to the central dogma: cytoplasmic mRNA decay and transcription are interdependent processes

Transcription and RNA decay are key determinants of gene expression; these processes are typically considered as the uncoupled beginning and end of the messenger RNA (mRNA) lifecycle. Here we describe the growing number of studies demonstrating interplay between these spatially disparate processes in eukaryotes. Specifically, cells can maintain mRNA levels by buffering against changes in mRNA stability or transcription, and can also respond to virally induced accelerated decay by reducing RNA polymerase II gene expression. In addition to these global responses, there is also evidence that mRNAs containing a premature stop codon can cause transcriptional upregulation of homologous genes in a targeted fashion. In each of these systems, RNA binding proteins (RBPs), particularly those involved in mRNA degradation, are critical for cytoplasmic to nuclear communication. Although their specific mechanistic contributions are yet to be fully elucidated, differential trafficking of RBPs between subcellular compartments are likely to play a central role in regulating this gene expression feedback pathway

RNA polymerase II subunit modulation during viral infection and cellular stress

Control of gene expression, including transcription, is central in dictating the outcome of viral infection. One of the profound alterations induced by viruses is modification to the integrity and function of eukaryotic RNA polymerase II (Pol II). Here, we discuss how infection perturbs the Pol II complex by altering subunit phosphorylation and turnover, as well as how cellular genotoxic stress (e.g. DNA damage) elicits similar outcomes. By highlighting emerging parallels and differences in Pol II control during viral infection and abiotic stress, we hope to bolster identification of pathways that target Pol II and regulate the transcriptome

Deep sequencing reveals direct targets of gammaherpesvirus-induced mRNA decay and suggests that multiple mechanisms govern cellular transcript escape.

One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection

Murine Gammaherpesvirus 68 ORF45 Stimulates B2 Retrotransposon and Pre-tRNA Activation in a Manner Dependent on Mitogen-Activated Protein Kinase (MAPK) Signaling.

RNA polymerase III (RNAPIII) transcribes a variety of noncoding RNAs, including tRNA (tRNA) and the B2 family of short interspersed nuclear elements (SINEs). B2 SINEs are noncoding retrotransposons that possess tRNA-like promoters and are normally silenced in healthy somatic tissue. Infection with the murine gammaherpesvirus MHV68 induces transcription of both SINEs and tRNAs, in part through the activity of the viral protein kinase ORF36. Here, we identify the conserved MHV68 tegument protein ORF45 as an additional activator of these RNAPIII loci. MHV68 ORF45 and ORF36 form a complex, resulting in an additive induction RNAPIII and increased ORF45 expression. ORF45-induced RNAPIII transcription is dependent on its activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway, which in turn increases the abundance of the RNAPIII transcription factor Brf1. Other viral and nonviral activators of MAPK/ERK signaling also increase the levels of Brf1 protein, B2 SINE RNA, and tRNA, suggesting that this is a common strategy to increase RNAPIII activity. IMPORTANCE Gammaherpesviral infection alters the gene expression landscape of a host cell, including through the induction of noncoding RNAs transcribed by RNA polymerase III (RNAPIII). Among these are a class of repetitive genes known as retrotransposons, which are normally silenced elements and can copy and spread throughout the genome, and transfer RNAs (tRNAs), which are fundamental components of protein translation machinery. How these loci are activated during infection is not well understood. Here, we identify ORF45 from the model murine gammaherpesvirus MHV68 as a novel activator of RNAPIII transcription. To do so, it engages the MAPK/ERK signaling pathway, which is a central regulator of cellular response to environmental stimuli. Activation of this pathway leads to the upregulation of a key factor required for RNAPIII activity, Brf1. These findings expand our understanding of the regulation and dysregulation of RNAPIII transcription and highlight how viral cooption of key signaling pathways can impact host gene expression