Biology subjects

Glaunsinger, B. A.

Publications and source records attributed to Glaunsinger, B. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Mutational screening reveals a cluster of residues within the SARS-CoV-2 nsp1 N-terminus that confers RNA-targeting selectivity

The SARS-CoV-2 nsp1 protein is a virulence factor that broadly inhibits cellular gene expression. Although cellular mRNAs are translationally inhibited by nsp1 and subsequently degraded, viral transcripts possess a 5' leader sequence (CoV2L) that enables them to escape nsp1-mediated repression. Both transcript targeting selectivity and mRNA decay require coordination by the nsp1 N-terminal domain (NTD) through an unknown mechanism. Here, we generated an alanine-scanning library of mutations encompassing all residues in the nsp1 NTD to gain a deeper understanding of how this domain coordinates nsp1 function. We screened this library for the ability to repress mRNA bearing a host- or CoV2L-derived 5' untranslated region, revealing two predominant clusters of residues required for target selectivity. These largely comprised adjacent surface-exposed beta-sheets on the NTD, whose deletion rendered CoV2L-containing mRNA susceptible to repression and prevented nsp1-induced mRNA decay. This work provides residue-level information on the role of the NTD in distinguishing among mRNA targets and further links nsp1 target selectivity to mRNA decay.

molecular biology↗

bioRxiv2026

Promoter-associated RNA polymerase III shapes RNA polymerase II-dependent inflammatory gene expression during viral infection

Cells must be primed to rapidly induce inflammatory gene expression upon infection while also tuning the level of induction to avoid immunopathology. Here, we identify RNA polymerase III (Pol III), best known for transcribing noncoding RNAs, as a dual-function regulator of RNA polymerase II (Pol II)-dependent inflammatory gene expression. Pol III is selectively enriched at promoters of innate immune, pro-inflammatory, and stress-response genes, where it maintains chromatin accessibility and supports basal transcription. Upon infection with murine gammaher-pesvirus 68 (MHV68), Pol III redistributes from these promoters to retrotransposon loci, coinciding with enhanced expression of inflammatory genes. Depletion of the Pol III transcription factor Brf1 further amplifies inflammatory transcription during infection with MHV68, herpes simplex virus-1, and influenza A virus. Genes restrained by Pol III have TATA-box-enriched promoters and are functionally dependent on TATA-binding protein (TBP), suggesting that Pol III modulates inflammatory gene expression by competing with Pol II for shared transcriptional machinery. Thus, Pol III is a chromatin licensor in uninfected cells and an inflammation rheostat during viral infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/738346v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@14e9026org.highwire.dtl.DTLVardef@d9bccborg.highwire.dtl.DTLVardef@f9d5b6org.highwire.dtl.DTLVardef@3dd384_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

RNA polymerase III transcription-associated polyadenylation promotes the accumulation of noncoding retrotransposons during infection

The accumulation of RNA Polymerase III (Pol III) transcribed short interspersed nuclear element (SINE) retrotransposon RNA is a hallmark of various cellular stressors, including DNA virus infection. However, the molecular mechanisms driving the induction of these normally repressed loci are largely undefined. Here, we reveal that in addition to Pol III transcriptional induction, gammaherpesvirus infection stimulates mRNA-like 3 end processing of SINE RNAs that leads to their stabilization. We developed a convolutional neural network (CNN)-based model that identified a polyadenylation-associated motif as the key hallmark of infection-induced SINEs. Indeed, mRNA polyadenylation machinery is recruited in a Pol III-dependent manner to virus-induced loci, including B2 SINE and tRNA genes. Infection causes enhanced polyadenylation of SINE ncRNA, which is required for its stable accumulation. This virus-host interaction therefore highlights an inducible, coupled relationship between Pol III transcription and mRNA-like polyadenylation. It also reveals that co-option of the polyadenylation machinery by Pol III is a mechanism to increase the abundance of noncoding RNA during pathogenic stress. SIGNIFICANCEShort interspersed nuclear elements (SINEs) are hyperabundant and transcribed by RNA polymerase III (Pol III) to produce noncoding retrotransposons. Although generally not detectable in healthy somatic cells, SINE RNA expression is upregulated during stress, including viral infection and inflammatory diseases. We used gammaherpesvirus infection to uncover pathways leading to increased SINE RNA expression. Using a newly developed deep learning model and genomics analyses, we reveal that infection-induced accumulation of SINEs is driven by increased Pol III transcription and Pol III-dependent recruitment of polyadenylation machinery. This stimulates polyadenylation of SINEs, which is a known stabilizer of these noncoding transcripts. Our findings suggest that inducible alterations to Pol III transcript 3 end processing modulate the abundance of noncoding retrotransposons during pathogenic stress.

molecular biology↗

bioRxiv2023

Coding and non-coding elements comprise a regulatory network controlling transcription in Kaposi's sarcoma-associated herpesvirus

Complex transcriptional control is a conserved feature of both eukaryotes and the viruses that infect them. Here, we illustrate this by combining high-density functional genomics, expression profiling, and viral-specific chromosome conformation capture to define with unprecedented detail the transcriptional regulation of a single gene, ORF68, from Kaposis sarcoma-associated herpesvirus (KSHV). We first identified seven cis-regulatory regions by densely tiling the [~]154 kb KSHV genome with CRISPRi. A parallel Cas9 nuclease screen indicated that three of these regions act as promoters of genes that regulate ORF68. RNA expression profiling demonstrated that three more of these regions act by either repressing or enhancing other distal viral genes involved in ORF68 transcriptional regulation. Finally, we tracked how the 3D structure of the viral genome changes during its lifecycle, revealing that these enhancing regulatory elements are physically closer to their targets when active, and that disrupting some elements caused large- scale changes to the 3D genome. These data enable us to construct a complete model revealing that the mechanistic diversity of this essential regulatory circuit matches that of human genes.

microbiology↗

bioRxiv2023

The viral packaging motor potentiates late gene expression in Kaposi's sarcoma-associated herpesvirus

{beta}- and {gamma}-herpesviruses transcribe their late genes in a manner distinct from host transcription. This process is directed by a complex of viral transcriptional activator proteins that hijack cellular RNA polymerase II and an unknown set of additional factors. We employed proximity labeling coupled with mass spectrometry, followed by CRISPR and siRNA screening to identify proteins functionally associated with the Kaposis sarcoma-associated herpesvirus (KSHV) late gene transcriptional complex. These data revealed that the catalytic subunit of the viral DNA packaging motor, ORF29, is both dynamically associated with the viral transcriptional activator complex and potentiates late gene expression. Through genetic mutation and deletion of ORF29, we establish that its catalytic activity potentiates viral transcription and is required for robust accumulation of essential late proteins during infection. Thus, we propose an expanded role for ORF29 that encompasses its established function in viral packaging and its newly discovered contributions to viral transcription and late gene expression in KSHV. Author summary{beta}- and {gamma}-herpesviruses express a class of genes essential for completion of the viral life cycle late during infection. A specialized complex of viral transcriptional activator proteins drives expression of these late genes in a manner dependent on viral genome replication, although the mechanisms and regulation of this process are largely unknown. Here, we identified factors that physically and functionally associate with the late gene transcription complex and unexpectedly found that the viral DNA packaging motor in Kaposis sarcoma-associated herpesvirus (KSHV) contributes to late gene expression. We show that the catalytic activity of this protein is not only required for genomic packaging but also for the robust expression of late genes to ensure the successful production of progeny virions. Thus, late gene transcription is mechanistically linked to the conserved processes of viral genome replication and packaging.

molecular biology↗

bioRxiv2022

High-Throughput Discovery and Characterization of Viral Transcriptional Effectors in Human Cells

Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression. Given their multifunctional nature and high sequence divergence, it is unclear which viral proteins can affect transcription and which specific sequences contribute to this function. Using a high-throughput assay, we measured the transcriptional regulatory potential of over 60,000 protein tiles across [~]1,500 proteins from 11 coronaviruses and all nine human herpesviruses. We discovered hundreds of new transcriptional effector domains, including a conserved repression domain in all coronavirus Spike homologs, dual activation-repression domains in VIRFs, and an activation domain in six herpesvirus homologs of the single-stranded DNA-binding protein that we show is important for viral replication and late gene expression in KSHV. For the effector domains we identified, we investigated their mechanisms via high-throughput sequence and chemical perturbations, pinpointing sequence motifs essential for function. This work massively expands viral protein annotations, serving as a springboard for studying their biological and health implications and providing new candidates for compact gene regulation tools.

systems biology↗

bioRxiv2022

DNA processing by the Kaposi's sarcoma-associated herpesvirus alkaline exonuclease SOX contributes to viral gene expression and infectious virion production

Alkaline exonucleases (AE) are present in several large DNA viruses including bacteriophage {lambda} and herpesviruses, where they play roles in viral DNA processing during genome replication. Given the genetic conservation of AEs across viruses infecting different kingdoms of life, these enzymes likely assume central roles in the lifecycles of viruses where they have yet to be well characterized. Here, we applied a structure-guided functional analysis of the bifunctional AE in the oncogenic human gammaherpesvirus Kaposis sarcoma-associated herpesvirus (KSHV), called SOX. In addition to identifying a preferred DNA substrate preference for SOX, we define key residues important for DNA binding and DNA processing, and how SOX activity on DNA partially overlaps with its functionally separable cleavage of mRNA. By engineering these SOX mutants into KSHV, we reveal roles for its DNase activity in viral gene expression and infectious virion production. Our results provide mechanistic insight into gammaherpesviral AE activity as well as areas of functional conservation between this mammalian virus AE and its distant relative in phage {lambda}.

microbiology↗

bioRxiv2022

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 transfer RNA (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 encoded by 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 non-viral 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. ImportanceGammaherpesviral infection alters the gene expression landscape of a host cell, including through induction of non-coding RNAs transcribed by RNA polymerase III (RNAPIII). Among these are a class of repetitive genes known as retrotransposons, which are normally silenced elements that 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 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 co-option of key signaling pathways can impact host gene expression.

microbiology↗

bioRxiv2021

Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing

The mRNA 5 cap structure serves both to protect transcripts from degradation and promote their translation. Cap removal is thus an integral component of mRNA turnover that is carried out by cellular decapping enzymes, whose activity is tightly regulated and coupled to other stages of the mRNA decay pathway. The poxvirus vaccinia virus (VACV) encodes its own decapping enzymes, D9 and D10, that act on cellular and viral mRNA, but may be regulated differently than their cellular counterparts. Here, we evaluated the targeting potential of these viral enzymes using RNA sequencing from cells infected with wild-type and decapping mutant versions of VACV as well as in uninfected cells expressing D10. We found that D9 and D10 target an overlapping subset of viral transcripts but that D10 plays a dominant role in depleting the vast majority of human transcripts, although not in an indiscriminate manner. Unexpectedly, the splicing architecture of a gene influences how robustly its corresponding transcript is targeted by D10, as transcripts derived from intronless genes are less susceptible to enzymatic decapping by D10. As all VACV genes are intronless, preferential decapping of transcripts from intron-encoding genes provides an unanticipated mechanism for the virus to disproportionately deplete host transcripts and remodel the infected cell transcriptome.

microbiology↗

bioRxiv2021

Manipulation of RNA Polymerase III by Herpes Simplex Virus-1

RNA Polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and acts as a pathogen sensor during the innate immune response. To promote enhanced proliferation, the Pol III machinery is commonly targeted during cancer and viral infection. Herein we employ DM-RNA-Seq, 4SU-Seq, ChIP-Seq, and ATAC-Seq to characterize how Herpes Simplex Virus-1 (HSV-1) perturbs the Pol III landscape. We find that HSV-1 stimulates tRNA expression 10-fold, with mature tRNAs exhibiting a 2-fold increase within 12 hours of infection. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts, nascent viral genomes, or viral progeny. Host 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 is known to mediate host transcriptional shut off and lead to a 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 HSV genome, which suggests a previously unrecognized role in HSV-1 gene expression. These data provide insight into novel mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.

molecular biology↗

bioRxiv2020

A pentameric protein ring with novel architecture is required for herpesviral packaging

Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids. This process appears mechanistically similar in two evolutionarily distant viruses, the herpesviruses and the tailed bacteriophages, which infect different kingdoms of life. While the motor and mechanism as a whole are thought to be conserved, accessory factors that influence packaging are divergent and poorly understood, despite their essential roles. An accessory factor required for herpesviral packaging is encoded by ORF68 in the oncogenic virus Kaposis sarcoma-associated herpesvirus (KSHV), whose homolog in Epstein Barr Virus (EBV) is BFLF1. Here, we present structures of both KSHV ORF68 and EBV BFLF1, revealing that these proteins form a highly similar homopentameric ring. The central channel of this ring is positively charged, and we demonstrate that this region of KSHV ORF68 binds double-stranded DNA. Mutation of individual positively charged residues within but not outside the channel ablates DNA binding, and in the context of KSHV infection these mutants fail to package the viral genome or produce progeny virions. Thus, we propose a model in which ORF68 facilitates the transfer of newly replicated viral genomes to the packaging motor.

microbiology↗

bioRxiv2020

The conserved herpesviral kinase ORF36 activates B2 retrotransposons during murine gammaherpesvirus infection

Short interspersed nuclear elements (SINEs) are RNA polymerase III (RNAPIII) transcribed, retrotransposable noncoding RNA (ncRNA) elements ubiquitously spread throughout mammalian genomes. While normally silenced in healthy somatic tissue, SINEs can be induced during infection with DNA viruses, including the model murine gammaherpesvirus MHV68. Here, we explored the mechanisms underlying MHV68 activation of SINE ncRNAs. We demonstrate that lytic MHV68 infection of B cells, macrophages and fibroblasts leads to robust activation of the B2 family of SINEs in a cell autonomous manner. B2 ncRNA induction requires neither host innate immune signaling factors nor involvement of the RNAPIII master regulator Maf1. However, we identify MHV68 ORF36, the conserved herpesviral kinase, as playing a key role in B2 induction during lytic infection. SINE activation is linked to ORF36 kinase activity and can also be induced by HDAC1/2 inhibition, which is one of the known ORF36 functions. Collectively, our data suggest that ORF36-mediated changes in chromatin modification contribute to B2 activation during MHV68 infection, and that this activity is conserved in other herpesviral protein kinase homologs. AUTHOR SUMMARYViral infection dramatically changes the levels of many types of RNA in a cell. In particular, certain oncogenic viruses activate expression of repetitive genes called retrotransposons, which are normally silenced due to their ability to copy and spread throughout the genome. Here, we established that infection with the gammaherpesvirus MHV68 leads to a dramatic induction of a class of noncoding retrotransposons called B2 SINEs in multiple cell types. We then explored how MHV68 activates B2 SINEs, revealing a role for the conserved herpesviral protein kinase ORF36. Both ORF36 kinase-dependent and kinase-independent functions contribute to B2 induction, perhaps through ORF36 targeting of proteins involved in controlling the accessibility of chromatin surrounding SINE loci. Understanding features underlying induction of these elements following MHV68 infection should provide insight into core elements of SINE regulation, as well as dis-regulation of SINE elements associated with disease.

molecular biology↗

bioRxiv2019

Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally-encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposis sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is mediated by a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF66 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we find that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV vPIC and suggests that it functions as a complex to recognize late gene promoters.\n\nIMPORTANCEKaposis sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. Release of infectious virions requires production of capsid proteins and other late genes, whose production are transcriptionally controlled by a complex of six virally-encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function five of its six components play in transcription. Here, we demonstrate that ORF66 is an essential component of this complex in KSHV and that its inclusion in the complex is mediated through its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examine assembly of the viral pre-initiation complex at late gene promoters and find that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral pre-initation complex.

molecular biology↗

bioRxiv2018

The interaction between ORF18 and ORF30 is required for late gene transcription in Kaposi’s sarcoma-associated herpesvirus

In the beta- and gammaherpesviruses, a specialized complex of viral transcriptional activators (vTAs) coordinate to direct expression of virus-encoded late genes, which are critical for viral assembly and whose transcription initiates only after the onset of viral DNA replication. The vTAs in Kaposis sarcoma-associated herpesvirus (KSHV) are ORF18, ORF24, ORF30, ORF31, ORF34, and ORF66. While the general organization of the vTA complex has been mapped, the individual roles of these proteins, and how they coordinate to activate late gene promoters, remains largely unknown. Here, we performed a comprehensive mutational analysis of the conserved residues in ORF18, which is a highly interconnected vTA component. Surprisingly, the mutants were largely selective for disrupting the interaction with ORF30 but not the other three ORF18 binding partners. Furthermore, disrupting the ORF18-ORF30 interaction weakened the vTA complex as a whole, and an ORF18 point mutant that failed to bind ORF30 was unable to complement an ORF18 null virus. Thus, contacts between individual vTAs are critical, as even small disruptions in this complex result in profound defects in KSHV late gene expression.

molecular biology↗

bioRxiv2018

Kaposi’s sarcoma-associated herpesvirus ORF68 is a DNA binding protein required for viral genome cleavage and packaging

Herpesviral DNA packaging into nascent capsids requires multiple conserved viral proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 protein of Kaposis sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA encapsidation whose function remains largely unresolved across the herpesviridae. We found that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication compartments, although it is dispensable for viral DNA replication and gene expression. However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B-capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside from a series of putative zinc finger motifs. However, in vitro biochemical analyses of purified ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. These activities provide new insights into the role of KSHV ORF68 in viral genome encapsidation.\n\nImportanceKaposis sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposis sarcoma and several B-cell cancers, causing significant morbidity and mortality in immunocompromised individuals. A critical step in the production of infectious viral progeny is the packaging of the newly replicated viral DNA genome into the capsid, which involves coordination between at least seven herpesviral proteins. While the majority of these packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, we show that the purified ORF68 protein directly binds DNA and is associated with a metal-dependent cleavage activity on double stranded DNA in vitro. These activities suggest a novel role for ORF68 in herpesviral genome processing and encapsidation.

molecular biology↗

bioRxiv2018

Site specific target binding controls RNA cleavage efficiency by the Kaposi’s sarcoma-associated herpesvirus endonuclease SOX

During lytic replication of Kaposis sarcoma-associated herpesvirus (KSHV), the gene expression landscape of a cell is remodeled to evade the immune response and create an environment favorable to viral replication. A major driver of these gene expression changes is a virally encoded, messenger RNA (mRNA)-specific endonuclease termed SOX. SOX cleaves the majority of cytoplasmic mRNAs, but does so at specific internal sites loosely defined by a degenerate sequence motif. If and how RNA sequence directs SOX targeting remained unknown. To address these questions, we used recombinant, highly purified SOX endonuclease in a series of biochemical assays to reconstitute the cleavage reaction in vitro and gain significant insight into the biochemical mechanism of both SOX target recognition and endonucleolytic cleavage. Using this system, we determined that cut site specificity is preserved with purified SOX and a validated target RNA and thus does not require additional cellular cofactors. Furthermore, we showed that SOX displays robust, sequence-specific RNA binding to residues proximal to the cleavage site, which must be presented in a particular structural context. The strength of SOX binding dictates cleavage efficiency, providing an explanation for the breadth of target RNA susceptibility observed in cells.\n\nSignificance StatementKaposis sarcoma-associated herpesvirus (KSHV) is an oncogenic human virus that causes Kaposis sarcoma, primary effusion lymphoma, and multicentric Castleman disease. During viral replication, KSHV expresses an enzyme called SOX that cuts and inactivates the majority of cellular messenger RNAs, preventing their translation into proteins. Some mRNAs are efficiently cleaved by SOX, while others are poorly cleaved, but the mechanistic basis underlying this selectivity has remained largely unknown. Here, we reveal that the efficiency of RNA cleavage is heavily impacted by RNA sequences proximal to the cleavage site, which serve as a SOX binding platform. This helps explain both the range of RNA cleavage efficiency observed in SOX-expressing cells as well as the sequence specificity underlying SOX targeting.

microbiology↗

bioRxiv2018

An Integrin-Independent Kaposi’s Sarcoma-Associated Herpesvirus Infection Mechanism

Host receptor usage by KSHV has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding, then interacts with a complex of EphA2, xct, and integrins 3{beta}1, V{beta}3, V{beta}5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of V- and {beta}1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.\n\nIMPORTANCEOur data reveals an integrin-independent route of KSHV infection and suggests that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.

microbiology↗

bioRxiv2017

N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi’s sarcoma-associated herpesvirus infection

Methylation at the N6 position of adenosine (m6A) is a highly prevalent and reversible modification within eukaryotic mRNAs that has been linked to many stages of RNA processing and fate. Recent studies suggest that m6A deposition and proteins involved in the m6A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m6A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposis sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m6A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m6A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m6A levels across much of the host transcriptome. However, we found that depletion of the m6A machinery had differential pro- and anti-viral impacts on viral gene expression depending on the cell-type analyzed. In iSLK.219 and iSLK.BAC16 cells the pathway functioned in a pro-viral manner, as depletion of the m6A writer METTL3 and the reader YTHDF2 significantly impaired virion production. In iSLK.219 cells the defect was linked to their roles in the post-transcriptional accumulation of the major viral lytic transactivator ORF50, which is m6A modified. In contrast, although the ORF50 mRNA was also m6A modified in KSHV infected B cells, ORF50 protein expression was instead increased upon depletion of METTL3, or, to a lesser extent, YTHDF2. These results highlight that the m6A pathway is centrally involved in regulating KSHV gene expression, and underscore how the outcome of this dynamically regulated modification can vary significantly between cell types.\n\nAuthor SummaryIn addition to its roles in regulating cellular RNA fate, methylation at the N6 position of adenosine (m6A) of mRNA has recently emerged as a mechanism for regulating viral infection. While it has been known for over 40 years that the mRNA of nuclear replicating DNA viruses contain m6A, only recently have studies began to examine the distribution of this modification across viral transcripts, as well as characterize its functional impact upon viral lifecycles. Here, we apply m6A-sequencing to map the location of m6A modifications throughout the transcriptome of the oncogenic human DNA virus Kaposis sarcoma-associated herpesvirus (KSHV). We show that the m6A machinery functions in a cell type specific manner to either promote or inhibit KSHV gene expression. Thus, the KSHV lifecycle is impacted by the m6A pathway, but the functional outcome may depend on cell lineage specific differences in m6A-based regulation.

microbiology↗