Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection.

To globally profile circRNAs, we employ RNA-Sequencing paired with chimeric junction analysis for alpha-, beta-, and gamma-herpesvirus infection. We find circRNAs are, as a population, resistant to host shutoff. We validate this observation using ectopic expression assays of human and murine herpesvirus endoribonucleases. During lytic infection, four circRNAs are commonly induced across all subfamilies of human herpesviruses, suggesting a shared mechanism of regulation. We test one such mechanism, namely how interferon-stimulation influences circRNA expression. 67 circRNAs are upregulated by either interferon-ß or -γ treatment, with half of these also upregulated during lytic infection. Using gain and loss of function studies we find an interferon-stimulated circRNA, circRELL1, inhibits lytic Herpes Simplex Virus-1 infection. We previously reported circRELL1 inhibits lytic Kaposi sarcoma-associated herpesvirus infection, suggesting a pan-herpesvirus antiviral activity. We propose a two-pronged model in which interferon-stimulated genes may encode both mRNA and circRNA with antiviral activity. This is critical in cases of host shutoff, such as alpha- and gamma-herpesvirus infection, where the mRNA products are degraded but circRNAs escape.

Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection.

A first line of defense during infection is expression of interferon (IFN)-stimulated gene products which suppress viral lytic infection. To combat this, herpesviruses express endoribonucleases to deplete host RNAs. Here we demonstrate that IFN-induced circular RNAs (circRNAs) can escape viral-mediated degradation. We performed comparative circRNA expression profiling for representative alpha- (Herpes simplex virus-1, HSV-1), beta- (human cytomegalovirus, HCMV), and gamma-herpesviruses (Kaposi sarcoma herpesvirus, KSHV; murine gamma-herpesvirus 68, MHV68). Strikingly, we found that circRNAs are, as a population, resistant to host shutoff. This observation was confirmed by ectopic expression assays of human and murine herpesvirus endoribonucleases. During primary lytic infection, ten circRNAs were commonly regulated across all subfamilies of human herpesviruses, suggesting a common mechanism of regulation. We tested one such mechanism, namely how interferon-stimulation influences circRNA expression. 67 circRNAs were upregulated by either IFN-ß or -γ treatment, with half of these also upregulated during lytic infection. Using gain and loss of function studies we found an interferon-stimulated circRNA, circRELL1, inhibited lytic HSV-1 infection. We have previously reported circRELL1 inhibits lytic KSHV infection, suggesting a pan-herpesvirus antiviral activity. We propose a two-pronged model in which interferon-stimulated genes may encode both mRNA and circRNA with antiviral activity. This is critical in cases of host shutoff, such as alpha- and gamma-herpesvirus infection, where the mRNA products are degraded but circRNAs escape.

"Transfer" of power: The intersection of DNA virus infection and tRNA biology.

Transfer RNAs (tRNAs) are at the heart of the molecular biology central dogma, functioning to decode messenger RNAs into proteins. As obligate intracellular parasites, viruses depend on the host translation machinery, including host tRNAs. Thus, the ability of a virus to fine-tune tRNA expression elicits the power to impact the outcome of infection. DNA viruses commonly upregulate the output of RNA polymerase III (Pol III)-dependent transcripts, including tRNAs. Decades after these initial discoveries we know very little about how mature tRNA pools change during viral infection, as tRNA sequencing methodology has only recently reached proficiency. Here, we review perturbation of tRNA biogenesis by DNA virus infection, including an emerging player called tRNA-derived fragments (tRFs). We discuss how tRNA dysregulation shifts the power landscape between the host and virus, highlighting the potential for tRNA-based antivirals as a future therapeutic.

Better late than never: A unique strategy for late gene transcription in the beta- and gammaherpesviruses.

During lytic replication, herpesviruses express their genes in a temporal cascade culminating in expression of "late" genes. Two subfamilies of herpesviruses, the beta- and gammaherpesviruses (including human herpesviruses cytomegalovirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus), use a unique strategy to facilitate transcription of late genes. They encode six essential viral transcriptional activators (vTAs) that form a complex at a subset of late gene promoters. One of these vTAs is a viral mimic of host TATA-binding protein (vTBP) that recognizes a strikingly minimal cis-acting element consisting of a modified TATA box with a TATTWAA consensus sequence. vTBP is also responsible for recruitment of cellular RNA polymerase II (Pol II). Despite extensive work in the beta/gammaherpesviruses, the function of the other five vTAs remains largely unknown. The vTA complex and Pol II assemble on the promoter into a viral preinitiation complex (vPIC) to facilitate late gene transcription. Here, we review the properties of the vTAs and the promoters on which they act.

Manipulation of RNA polymerase III by Herpes Simplex Virus-1.

RNA polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and is commonly targeted during cancer and viral infection. We find that Herpes Simplex Virus-1 (HSV-1) stimulates tRNA expression 10-fold. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts. tRNA with a specific codon bias were not targeted-rather increased transcription was observed from euchromatic, actively transcribed loci. tRNA upregulation is linked to unique crosstalk between the Pol II and III transcriptional machinery. While viral infection results in depletion of Pol II on host mRNA promoters, we find that Pol II binding to tRNA loci increases. Finally, we report Pol III and associated factors bind the viral genome, which suggests a previously unrecognized role in HSV-1 gene expression. These findings provide insight into mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.

Herpes simplex viral nucleoprotein creates a competitive transcriptional environment facilitating robust viral transcription and host shut off.

Herpes simplex virus-1 (HSV-1) replicates within the nucleus coopting the host's RNA Polymerase II (Pol II) machinery for production of viral mRNAs culminating in host transcriptional shut off. The mechanism behind this rapid reprogramming of the host transcriptional environment is largely unknown. We identified ICP4 as responsible for preferential recruitment of the Pol II machinery to the viral genome. ICP4 is a viral nucleoprotein which binds double-stranded DNA. We determined ICP4 discriminately binds the viral genome due to the absence of cellular nucleosomes and high density of cognate binding sites. We posit that ICP4's ability to recruit not just Pol II, but also more limiting essential components, such as TBP and Mediator, create a competitive transcriptional environment. These distinguishing characteristics ultimately result in a rapid and efficient reprogramming of the host's transcriptional machinery, which does not occur in the absence of ICP4.

Genome replication affects transcription factor binding mediating the cascade of herpes simplex virus transcription.

In herpes simplex virus type 1 (HSV-1) infection, the coupling of genome replication and transcription regulation has been known for many years; however, the underlying mechanism has not been elucidated. We performed a comprehensive transcriptomic assessment and factor-binding analysis for Pol II, TBP, TAF1, and Sp1 to assess the effect genome replication has on viral transcription initiation and elongation. The onset of genome replication resulted in the binding of TBP, TAF1, and Pol II to previously silent late promoters. The viral transcription factor, ICP4, was continuously needed in addition to DNA replication for activation of late gene transcription initiation. Furthermore, late promoters contain a motif that closely matches the consensus initiator element (Inr), which robustly bound TAF1 postreplication. Continued DNA replication resulted in reduced binding of Sp1, TBP, and Pol II to early promoters. Therefore, the initiation of early gene transcription is attenuated following DNA replication. Herein, we propose a model for how viral DNA replication results in the differential utilization of cellular factors that function in transcription initiation, leading to the delineation of kinetic class in HSV-productive infection.

Replication-Coupled Recruitment of Viral and Cellular Factors to Herpes Simplex Virus Type 1 Replication Forks for the Maintenance and Expression of Viral Genomes.

Herpes simplex virus type 1 (HSV-1) infects over half the human population. Much of the infectious cycle occurs in the nucleus of cells where the virus has evolved mechanisms to manipulate host processes for the production of virus. The genome of HSV-1 is coordinately expressed, maintained, and replicated such that progeny virions are produced within 4-6 hours post infection. In this study, we selectively purify HSV-1 replication forks and associated proteins from virus-infected cells and identify select viral and cellular replication, repair, and transcription factors that associate with viral replication forks. Pulse chase analyses and imaging studies reveal temporal and spatial dynamics between viral replication forks and associated proteins and demonstrate that several DNA repair complexes and key transcription factors are recruited to or near replication forks. Consistent with these observations we show that the initiation of viral DNA replication is sufficient to license late gene transcription. These data provide insight into mechanisms that couple HSV-1 DNA replication with transcription and repair for the coordinated expression and maintenance of the viral genome.