Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression

Oko, Lauren; Kimball, Abigail K.; Kaspar, Rachael E.; Knox, Ashley N.; Chang, Tim; Alderete, Benjamin; Dyk, Linda F. van; Clambey, Eric T.

doi:10.1101/482042

Cited by 2 publications

(2 citation statements)

References 39 publications

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“…No.1708880) with the following conditions: (i) 95°C for 3 min, (ii) 40 cycles of 95°C for 15 sec, 60°C for 1 min, and (iii) 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. Amplification of viral genes were normalized to murine 18S expression to calculate the relative difference of target gene expression using the Pfaffl method, as previously described (49,50) : Target primer efficiency^Target𝛥𝐶𝑡/18S primer efficiency^18𝑆𝛥𝐶𝑡. A single product for each target was confirmed by melt curve analysis.…”

Section: Rt-qpcrmentioning

confidence: 99%

Murine Gammaherpesvirus 68 Efficiently Infects Myeloid Cells Resulting In An Atypical, Restricted Form Of Infection

Vragel,

Gomez,

Kostelecky

et al. 2023

Preprint

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The gammaherpesviruses (γHVs) establish a lifelong infection in their hosts, with the cellular outcome of infection intimately regulated by target cell type. Murine gammaherpesvirus 68 (MHV68), a small animal model of γHV infection, infects macrophages in vivo, resulting in a range of outcomes, from lytic replication to latent infection. Here, we have further investigated the nature of MHV68 macrophage infection using reductionist and primary in vivo infection studies. While MHV68 readily infected the J774 macrophage cell line, viral gene expression and replication were significantly impaired relative to a fully permissive fibroblast cell line. Lytic replication only occurred in a small subset of MHV68-infected J774 cells, despite the fact that these cells were fully competent to support lytic replication following pre-treatment with interleukin-4, a known potentiator of replication in macrophages. In parallel, we harvested virally-infected macrophages at 16 hours after MHV68 infectionin vivoand analyzed gene expression by single cell RNA-sequencing. Among virally infected macrophages, only rare (0.25%) cells had lytic cycle gene expression, characterized by detection of multiple lytic cycle RNAs. In contrast, ∼50% of virally-infected macrophages were characterized by expression of ORF75A, ORF75B and/or ORF75C, in the absence of other detectable viral RNAs. Selective transcription of the ORF75 locus also occurred in MHV68-infected J774 cells. In total, these studies indicate that MHV68 efficiently infects macrophages, with the majority of cells characterized by an atypical state of restricted viral transcription, and only rare cells undergoing lytic replication.ImportanceThe human gammaherpesviruses Epstein-Barr virus and Kaposi’s sarcoma associated herpesvirus are DNA viruses that cause lifelong infection and are associated with multiple diseases, especially in immunocompromised individuals. Murine gammaherpesvirus 68 (MHV68) is a powerful mouse model that permits close examination of these viruses. Previous studies of MHV68 identified that macrophages are an important in vivo target of infection; how infection within these cells is regulated remains incompletely understood. Here, we demonstrate that MHV68 infection of macrophages is characterized by two divergent outcomes across a population of infected cells: while a small subset of cells undergo lytic replication, to make new virus progeny, the majority of cells are characterized by an atypical, restricted form of infection characterized by a distinct viral gene transcription program not previously reported. These studies highlight important cell-type specific outcomes of gammaherpesvirus infection and identify a potential alternate program by which these viruses usurp macrophages.

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Section: Rt-qpcrmentioning

confidence: 99%

Murine Gammaherpesvirus 68 Efficiently Infects Myeloid Cells Resulting In An Atypical, Restricted Form Of Infection

Vragel,

Gomez,

Kostelecky

et al. 2023

Preprint

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“…Interestingly, some γHV ncRNAs are transcribed by RNA polymerase III (pol III), lending them unique transcriptional regulation and characteristics. The γHV68 tRNA-miRNA-encoded RNAs (TMERs; TMER1-TMER8) are expressed during lytic and latent infection (6). The TMERs are dispensable for lytic replication and the establishment of latency, but required for pathogenesis (7,8).…”

Section: Introductionmentioning

confidence: 99%

Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

Knox

Medina

Oldenburg

et al. 2022

Preprint

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Gammaherpesvirus (γHV) non-coding RNAs (ncRNAs) are integral modulators of viral infection. The γHVs engage RNA polymerase III (pol III)-dependent transcription of both host and viral ncRNAs, which contribute to viral establishment, gene expression, and pathogenesis. Viral ncRNAs, such as the EBV-encoded RNAs (EBERs), reportedly interact with multiple host RNA-binding proteins (RBPs) and contribute to inflammatory responses implicated in the development of malignancies. Here, we examined RBP interactions of the pol III-transcribed tRNA-miRNA encoded non-coding RNAs (TMERs) of murine γHV68, and the potential contributions of these and the related EBERs to in vivo pathogenesis. Using sequential enzymatic treatments, we found that several TMER1 forms retain a 5’-triphosphate, lending the possibility of recognition by the innate immune sensor RIG-I. We further examined the interactions of TMERs and EBERs with host RBPs, and found that multiple TMERs and EBERs interact with the La protein, though minimal interaction was detected with RIG-I during primary virus infection. Finally, we investigated the contributions of the TMERs and EBERs to disease in an immune-compromised mouse model with a series of viral recombinants. We found that expression of multiple single TMERs, or the EBERs expressed in place of the TMERs, was capable of restoring virulence to a viral recombinant lacking expression of all TMERs. Ultimately, these studies demonstrate that divergent pol III-transcribed γHV ncRNAs share interaction characteristics with two host RBPs and conserved contributions to disease, despite little to no significant sequence conservation. These findings support a model of convergent functions of the sequence-variable pol III-transcribed γHV ncRNAs.IMPORTANCEViruses manipulate the infected cell and host inflammatory responses through expression of coding and non-coding RNAs. The gammaherpesviruses are a subfamily of herpesviruses associated with chronic inflammatory diseases and malignancies, especially in immune-compromised individuals. Among these, the human Epstein-Barr virus and murine gammaherpesvirus 68 (γHV68) express highly abundant, RNA polymerase III-dependent, short non-coding RNAs. Whether these sequence-divergent ncRNAs have conserved functional properties is unknown. By using viral recombinants to allow direct comparison of these ncRNAs during primary infection, we find that the sequence-divergent ncRNAs of γHV68 and EBV share a conserved property to bind to the host RNA binding protein, La, and function interchangeably to facilitate in vivo pathogenesis. These studies demonstrate that abundant, RNA polymerase III-dependent viral ncRNAs can potently function to alter the host cell landscape and promote disease in a sequence-independent manner.

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Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression

Cited by 2 publications

References 39 publications

Murine Gammaherpesvirus 68 Efficiently Infects Myeloid Cells Resulting In An Atypical, Restricted Form Of Infection

Murine Gammaherpesvirus 68 Efficiently Infects Myeloid Cells Resulting In An Atypical, Restricted Form Of Infection

Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

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