Check out our most current work—we now deposit every research manuscript on the bioRxiv preprint server at the time of peer-reviewed journal submission. We believe in open access science!
Preprints (on bioRxiv, currently undergoing peer review)
Recent Publications (2015-pres)
Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing
Michael Ly, Hannah M. Burgess, Ian Mohr, Britt A. Glaunsinger
PLoS Pathogens, 2022 Feb 24;18(2):e1010099. doi: 10.1371/journal.ppat.1010099
The mRNA decapping activity of the vaccinia virus D10 enzyme is known to play key roles in poxvirus immune evasion and viral mRNA translation, however neither its breadth of targeting nor whether there are RNA features that influence its decapping activity in cells are known. Here, we addressed these questions by providing the first genome-wide evaluation of viral decapping enzyme activity on host and viral RNA abundance. We discovered that while D10 is a promiscuous decapper that targets the majority of host mRNA, a central (and unexpected) determinant of it targeting efficiency is the splicing status of an mRNA. This is notable for two reasons: first, this represents a novel connection between these arms of the gene expression cascade. Second, poxviruses replicate in the cytoplasm and their transcripts lack introns; thus, preferential decapping of transcripts from intron-encoding genes provides a clear mechanism for the virus to disproportionately deplete host over viral transcripts.
A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains
David W Morgens, Divya Nandakumar, Allison L Didychuk, Kevin J Yang, Britt Glaunsinger
PLoS Pathogens, January 2022 https://doi.org/10.1371/journal.ppat.1010236
While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel. Here, we used this approach to study the human oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV). We designed a sgRNA library containing all possible ~22,000 guides targeting the genome of KSHV and used this tool to phenotype all possible Cas9-targeted viruses for transcription of KSHV late genes, which is required to produce structural components of the viral capsid. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we discovered a novel hit, ORF46 – and more specifically its DNA binding domain – is required for viral DNA replication. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.
Manipulation of RNA polymerase III by Herpes Simplex Virus-1.
Dremel SE, Sivrich FL, Tucker JM, Glaunsinger BA, DeLuca NA.
Nature Communications, 2022 Feb 2;13(1):623. doi: 10.1038/s41467-022-28144-8.
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.
The N-terminal domain of CoV-2 nsp1 plays key functional roles in suppression of cellular gene expression and preservation of viral gene expression
Aaron S. Mendez*, Michael Ly*, Angélica M. González-Sánchez, Ella Hartenian, Nicholas T. Ingolia, Jamie H. Cate, Britt A. Glaunsinger (*equal contribution)
Cell Reports 2021; https://doi.org/10.1016/j.celrep.2021.109841
The coronavirus nsp1 protein is a virulence factor that restricts cellular gene expression by inducing translational repression and mRNA cleavage. How it coordinates both activities against cellular mRNAs while sparing viral transcripts is a central open question. Here, we report the most detailed structure-function analysis to date of CoV-2 nsp1, revealing that residues outside of the expected ribosome-binding domain play key roles in stabilizing nsp1-40S subunit interaction. We also identify CoV-2 nsp1 point mutants that gain the ability to repress translation of the normally protected viral leader-containing transcripts. These findings provide insight into the functional contribution of nsp1 regions outside of the 40S ‘docking’ domain and how their alteration could decrease viral pathogenesis.
Cytoplasmic mRNA decay represses RNA polymerase II transcription during early apoptosis
Christopher Duncan-Lewis, Ella Hartenian, Valeria King and Britt A. Glaunsinger
eLife 2021; DOI: 10.7554/eLife.58342
Crosstalk between RNA polymerase II transcription and cytoplasmic mRNA degradation often leads to compensatory changes in gene expression. Here, we reveal that widespread mRNA decay during early apoptosis represses RNAPII transcription, indicative of positive (rather than compensatory) feedback. This repression requires active degradation of cytoplasmic mRNA, which leads to impaired recruitment of components of the transcription preinitiation complex to promoter DNA. Thus, accelerated mRNA decay leads to the repression of mRNA transcription, thereby amplifying the shutdown of gene expression. This highlights a conserved gene regulatory mechanism by which cells respond to threats.
A pentameric protein ring with novel architecture is required for herpesviral packaging
Allison L. Didychuk, Stephanie N. Gates, Matthew R. Gardern, Lisa M. Strong, Andreas Martin and Britt Glaunsinger
eLife 2021;10:e62261 DOI: 10.7554/eLife.62261
Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids. In addition to this motor complex, packaging of herpesviral DNA requires an accessory factor whose function has remained unknown. Here, we present structures of this essential packaging protein from both KSHV (ORF68) and EBV (BFLF1), revealing that these proteins adopt a novel fold and assemble into a highly similar homopentameric ring. The central channel of this ring is positively charged and binds double-stranded DNA, suggesting a model whereby ORF68 facilitates the transfer of newly replicated viral genomes to the packaging motor.
Alteration of the premature tRNA landscape by gammaherpesvirus infection
Jessica M Tucker, Aaron M. Schaller, Ian Willis, Britt A. Glaunsinger
mBio, Dec 2020 DOI: 10.1128/mBio.02664-20
Transfer RNAs (tRNAs) are transcribed by RNA polymerase III (RNAPIII) and play a central role in decoding our genome. Many DNA tumor viruses enhance the activity of RNAPIII, yet whether infection alters tRNA expression is largely unknown. Here, we present the first genome wide analysis of how viral infection alters the tRNAome, revealing that tRNA transcription and processing are perturbed by the gammaherpesvirus MHV68.
The gammaherpesviral TATA box binding protein directly interacts with RNA Polymerase II to direct late gene transcription
Angelica F Castañeda*, Allison L Didychuk*, Robert K Louder, Chloe O McCollum, Zoe H Davis, Eva Nogales and Britt Glaunsinger (*equal contribution) PLoS Pathogens, Sept 2020 https://doi.org/10.1371/journal.ppat.1008843
Near the end of their replication cycle, β- and γ-herpesviruses transcribe their late genes in a manner distinct from host transcription. Late gene transcription requires six virally-encoded proteins, one of which is a functional mimic of host TATA-box-binding protein (TBP). Using a combination of electron microscopy and biochemical protein interaction assays, we found that this protein, encoded by ORF24 in Kaposi’s sarcoma associated herpesvirus, makes a direct protein-protein contact with the C-terminal domain of host RNA polymerase II. Thus, these herpesviruses employ an unprecedented strategy in eukaryotic transcription, wherein promoter recognition and polymerase recruitment are facilitated by a single transcriptional activator with functionally distinct domains.
The Molecular Virology of Coronaviruses
Ella Hartenian*, Divya Nandakumar*, Azra Lari, Michael Ly, Jessica M Tucker, Britt A Glaunsinger. (*equal contribution) Journal of Biological Chemistry, July 13, 2020 doi: 10.1074/jbc.REV120.013930
We present the molecular virology of coronavirus infection, including its entry into cells, its remarkably sophisticated gene expression and replication mechanisms, its extensive remodeling of the intracellular environment and its multifaceted immune evasion strategies. We highlight aspects of the viral lifecycle that may be amenable to antiviral targeting as well as key features of its biology that await discovery.
The conserved herpesviral kinase ORF36 activates B2 retrotransposons during murine gammaherpesvirus infection
Aaron M. Schaller, Jessica Tucker, Ian Willis, Britt A. Glaunsinger. J. Virol. 2020 Jul 1;94(14):e00262-20. bioRxiv link.
Selected as JVI spotlight article: ‘Unique Action of a Herpesvirus Kinase: Retrotransposon Induction’
Viral 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 explored the mechanisms underlying activation of the B2 class of SINE retrotransposons by the murine gammaherpesvirus MHV68. 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 noncoding RNA 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.
Herpesvirus infection reduces Pol II occupancy of host promoters but spares viral promoters
Ella Hartenian and Britt Glaunsinger. PLoS Pathogens. 2020 Feb 7;16(2):e1008269.
While cytoplasmic mRNA degradation and nuclear mRNA synthesis are generally considered to be independent events in gene expression, recent evidence suggests that these processes are intimately linked in eukaryotic cells. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. Viral genes, despite the fact that they require Pol II for transcription, escape this transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We hypothesize that the ability to escape from transcriptional repression is linked to the localization of viral DNA in replication compartments, providing a means for these viruses to counteract decay-induced viral transcript loss.
Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34
Didychuk AL, Castañeda AF, Kushnir LO, Huang CJ, Glaunsinger BA. J Virol. 2020 Jan 6;94(2).
In gammaherpesviruses, virion production requires synthesis of viral late genes, which are transcriptionally controlled by a complex of virally encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function the majority of 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 depends upon its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examined the assembly of the viral preinitiation complex at late gene promoters and found that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral preinitiation complex.
Feedback to the central dogma: cytoplasmic mRNA decay and transcription are interdependent processes
Hartenian E, Glaunsinger BA. Crit Rev Biochem Mol Biol. 2019 Aug;54(4):385-398. doi: 10.1080/10409238.2019.1679083.
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.
An integrative approach identifies direct targets of the late viral transcription complex and an expanded promoter recognition motif in Kaposi’s sarcoma-associated herpesvirus
Divya Nandakumar and Britt Glaunsinger. PLoS Pathog. 2019 May 16;15(5):e1007774
Strategies underlying expression of DNA virus late genes are often unique and distinct from gene regulation of other temporal classes. In the beta- and gamma-herpesviruses, this process involves an essential six-component viral complex that directs late gene transcription, largely by unknown mechanisms. Here, we have used complementary gene expression and promoter binding experiments to reveal which KHSV genes are directly controlled by the late gene transcription complex. Using these data, we uncovered a new regulatory element in KSHV late promotershelping to explain how the late gene transcription complex is able to bind seemingly minimal promoters with high specificity, ensuring robust expression of viral factors necessary for assembly of progeny virions.
Not immune to modification
Charles R Hesser and Britt A Glaunsinger. Nat Immunol. 2019 Feb;20(2):116-118.
The interaction between ORF18 and ORF30 is required for late gene transcription in Kaposi’s sarcoma-associated herpesvirus
Angelica F Castañeda and Britt A Glaunsinger. Journal of Virology, Oct 2018, doi: 10.1128/JVI.01488-18
All DNA viruses express a class of essential ‘late’ genes whose transcription occurs only after the viral genome is replicated. In the beta- and gammaherpesviruses, a group of six viral proteins forms a complex that is required for late gene activation. Here, we find that disrupting a single protein-protein interaction within this complex blocks late gene transcription and destabilizes the complex as a whole, underscoring how the precise organization of this protein complex is critical for its function–and for completion of the viral lifecycle.
Site specific target binding controls RNA cleavage efficiency by the Kaposi’s sarcoma-associated herpesvirus endonuclease SOX
Aaron Stephen Mendez, Carolin Vogt, Jens Bohne, Britt A Glaunsinger. Nucleic Acids Research, Oct 13 2018, https://doi.org/10.1093/nar/gky932
During lytic KHSV infection, the host cell’s gene expression landscape is dramatically remodeled by the viral endonuclease SOX, which cleaves the majority of cytoplasmic messenger RNA. We have now developed an in vitro assay that recapitulates target site specificity observed for SOX in cells, and use it to reveal key RNA sequence and structural features that dictate the strength of SOX binding and subsequent cleavage efficiency.
Global changes in mRNA abundance drive differential shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription
Sarah Gilbertson, Joel Federspiel, Ella Hartenian, Ileana Cristea, Britt Glaunsinger. eLife, 2018, 7:e37663 doi: 10.7554/eLife.37663
Alterations in the rates of mRNA decay can broadly impact both upstream and downstream components of the gene expression pathway, yet signals that govern such changes are often poorly understood. Here, we demonstrate that cells can convey mRNA abundance information between subcellular compartments through translocation of select RNA binding proteins. A notable example is poly(A) binding protein, whose nuclear translocation in response to accelerated cytoplasmic mRNA degradation restricts RNAPII transcription.
Kaposi’s sarcoma-associated herpesvirus ORF68 is a DNA binding protein required for viral genome cleavage and packaging
Matthew R. Gardner, Britt A. Glaunsinger. Journal of Virology, 2018, doi: 10.1128/JVI.00840-18
Once herpesviral DNA is amplified in the nucleus of an infected cell, numerous viral proteins coordinate to thread the DNA through a portal into the tight space of each new isocahedral capsid. Here, we investigated the role of one of the least well understood components of this ‘encapsidation’ machinery, and found that it is a DNA binding protein that is essential for viral genome packaging.
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.
Hesser CR, Karijolich J, Dominissini D, He C, Glaunsinger BA. PLoS Pathog. 2018 Apr 16;14(4):e1006995.
Host noncoding retrotransposons induced by DNA viruses: a SINE of infection?
Tucker JM, Glaunsinger BA. J Virol. 2017 Nov 14;91(23).
Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases.
Muller M, Glaunsinger B. PLoS Pathog. 2017 Aug 25;13(8):e1006593.
Genome-wide mapping of infection-induced SINE RNAs reveals a role in selective mRNA export.
Karijolich J, Yang Y, Glaunsinger B. Nucleic Acids Res. 2017 Jun 2;45(10):6194-6208.
Pseudouridylation of 7SK snRNA promotes 7SK snRNP formation to suppress HIV-1 transcription and escape from latency.
Zhao Y, Karijolich J, Glaunsinger B, Zhou Q. EMBO Reports. 2016 Oct;17(10):1441-1451.
Transcriptome-wide cleavage site mapping on cellular mRNAs reveals features underlying sequence-specific cleavage by the viral ribonuclease SOX.
Gaglia M, Rycroft C, and Glaunsinger B. PLoS Pathogens; 2015 Dec 8;11(12):e1005305
Infection-Induced Retrotransposon-Derived Noncoding RNAs Enhance Herpesviral Gene Expression via the NF-kB Pathway.
Karijolich J, Abernathy E, and Glaunsinger B. PLoS Pathogens; 2015 11(11): e1005260. *PLoS Pathogens ‘Featured Article’
Interaction Between ORF24 and ORF34 in the Kaposi’s Sarcoma-Associated Herpesvirus Late Gene Transcription Factor Complex is Essential For Viral Late Gene Expression.
Davis ZH, Hesser C, Park J, Glaunsinger BA. Journal of Virology, 2015 Oct 14;90(1):599-604.
Viral nucleases reveal an mRNA degradation-transcription feedback loop in mammalian cells.
Abernathy E, Gilberson S, Alla R, and Glaunsinger B. Cell Host & Microbe, 2015 Aug 12;18(2):243-53.*Featured in Cell Host & Microbe commentary: doi:10.1016/j.chom.2015.07.011
Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses.
Zhe, M, Jacobs, SR, West, JA, Stopford, C, Zhang, Z, Davis, Z, Barber, GN, Glaunsinger, B, Dittmer, DP and B. Damania. PNAS, 2015 Aug 4;112(31):E4306-15.
Modulation of the translational landscape during herpesvirus infection.
Glaunsinger B. Annual Review of Virology; 2015, 2:9.1–9.23.
A Ribonucleoprotein Complex Protects the Interleukin-6 mRNA from Degradation by Distinct Herpesviral Endonucleases.
Muller M, Hutin S, Marigold O, Lee KH, Burlingame A, and Glaunsinger BA. PLoS Pathogens; 2015, May 12;11(5):e1004899.
Emerging roles for RNA degradation in viral replication and antiviral defense.
Abernathy E, Glaunsinger B. Virology 60th Anniversary Edition; 2015, May;479-480C:600-608.
Global mapping of herpesvirus-host protein complexes reveals a novel transcription strategy for late genes.
Davis ZH, Verschueren E, Jang GM, Kleffman K, Johnson JR, Park J, Von Dollen J, Maher MC, Johnson T, Newton W, Jäger S, Shales M, Horner J, Hernandez RD, Krogan NJ, Glaunsinger BA. Molecular Cell, 2015, Jan 22;57(2):349‐360
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