Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68

O’Grady, Tina; Feswick, April; Hoffman, Brett; Wang, Yiping; Medina, Eva M.; Kara, Mehmet; Dyk, Linda F. van; Flemington, Erik K.; Tibbetts, Scott A.

doi:10.1016/j.celrep.2019.05.086

Cited by 32 publications

(27 citation statements)

References 59 publications

(67 reference statements)

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“…The effect of loxP insertion on adjacent gene expression has not been assessed. The coding capacity of the MHV68 genome is densely packed (40, 41), and it is possible that loxP insertion or deletion of the locus inadvertently disrupts the function of overlapping or adjacent transcripts. To better characterize the potential off target effects that could occur following M2 deletion, further analysis such as single-cell RNA sequencing is required.…”

Section: Discussionmentioning

confidence: 99%

Deletion of Murine Gammaherpesvirus GeneM2in AID-Expressing B Cells Impairs Host Colonization and Viral Reactivation

Owens

Oldenburg

White

et al. 2020

Preprint

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1 Gammaherpesviruses (GHVs) are DNA tumor viruses that establish life-long, chronic 2 infections in lymphocytes of humans and other mammals. GHV infections are associated with 3 numerous cancers, especially in immune compromised hosts. While it is known that GHVs utilize 4 host germinal center (GC) B cell responses during latency establishment, an understanding of 5 how viral gene products function in specific B cell subsets to regulate this process is incomplete. 6 Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of 7 GHV pathogenesis in vivo, we generated a virus in which the M2 gene was flanked by loxP sites 8 (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in 9 infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that 10 promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-11 expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency 12 in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency 13 establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of 14 mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for 15 M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, 16 neither colonization of draining lymph nodes after IN infection nor the spleen after intraperitoneal 17(IP) infection required M2, although the reactivation defect was retained. Together, these data 18 confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-19 expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and 20 reactivation from latency. Our study reveals that a viral latency gene functions within a distinct 21 subset of cells to facilitate host colonization. 22 131 M2 is required in CD19 + B cells for latency establishment and reactivation 132RT-PCR analyses suggest that a properly spliced M2 transcript capable of yielding the 133 functional M2 protein is only present in B cells (4, 23, 31). From this observation it is hypothesized 134 that M2 primarily functions in B cells to promote MHV68 latency and reactivation. To more 135 conclusively test this hypothesis, we infected mice that express Cre recombinase under control 136

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Section: Discussionmentioning

confidence: 99%

Deletion of Murine Gammaherpesvirus GeneM2in AID-Expressing B Cells Impairs Host Colonization and Viral Reactivation

Owens

Oldenburg

White

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…These results suggest that while ORF36 contributes to B2 induction during infection, one or more other viral activities may be involved. Our ORF screen encompassed a significant percentage of the annotated MHV68 genome [76], however it should be noted that recent work from O’Grady et al [77] shows pervasive alternate isoform usage overlapping ORF isoforms, suggesting that MHV68 encodes a more diverse proteome than previously anticipated. One or more of these untested proteins may also contribute to B2 induction, either via independent mechanisms or in cooperation with ORF36.…”

Section: Discussionmentioning

confidence: 99%

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

Schaller

Tucker

Willis

et al. 2020

Preprint

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ABSTRACTShort 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.

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“…The closely related human gammaherpesvirus, KSHV, also has 4 different Orf50 promoters that induce expression of 6 different RTA transcripts [36]. There are also other herpesvirus open reading frames that are controlled by multiple promoters that produce alternative transcripts [37–39]. One possibility is that these different promoters respond to different acute, latent, and reactivation signals.…”

Section: Discussionmentioning

confidence: 99%

Th2 Cytokine Modulates Herpesvirus Reactivation in a Cell Type Specific Manner

Zarek

Chang

Tao

et al. 2020

Preprint

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Gammaherpesviruses, such as Epstein-Barr virus (EBV), Kaposi's sarcoma associated virus (KSHV), and murine g-herpesvirus 68 (MHV68), establish latent infection in B cells, macrophages, and non-lymphoid cells, and can induce both lymphoid and non-lymphoid cancers. Research on these viruses has relied heavily on immortalized B cell and endothelial cell lines. Therefore, we know very little about the cell type specific regulation of virus infection. We have previously shown that treatment of MHV68-infected macrophages with the cytokine interleukin-4 (IL-4) or challenge of MHV68-infected mice with an IL-4-inducing parasite leads to virus reactivation. However, we do not know if all latent reservoirs of the virus, including B cells, reactivate the virus in response to IL-4. Here we used an in vivo approach to address the question of whether all latently infected cell types reactivate MHV68 in response to a particular stimulus. We found that IL-4 receptor expression on macrophages was required for IL-4 to induce virus reactivation, but that it was dispensable on B cells. We further demonstrated that the transcription factor, STAT6, which is downstream of the IL-4 receptor and binds a viral promoter in macrophages, did not bind to the viral promoter in B cells. These data suggest that stimuli that promote herpesvirus reactivation may only affect latent virus in particular cell types, but not in others.

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Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68

Cited by 32 publications

References 59 publications

Deletion of Murine Gammaherpesvirus GeneM2in AID-Expressing B Cells Impairs Host Colonization and Viral Reactivation

Deletion of Murine Gammaherpesvirus GeneM2in AID-Expressing B Cells Impairs Host Colonization and Viral Reactivation

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

Th2 Cytokine Modulates Herpesvirus Reactivation in a Cell Type Specific Manner

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