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Host and viral gene expression in murine gammaherpesvirus 68 infected primary peritoneal mouse macrophages harvested 16 hours post-infection in vivo

ABSTRACT: The primary goal of this project was to identify host and viral RNA expression from virally infected cells, analyzing primary, peritoneal mouse macrophages after acute infection with a mouse model of gammaherpesvirus infection, murine gammaherpesvirus 68 (MHV68). The gammaherpesviruses (gammaHVs) establish a lifelong infection in their hosts, with the outcome of infection at the cellular level intimately regulated by target cell type. Murine gammaherpesvirus 68 (MHV68), a small animal model of gammaHV 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 focused on analysis of primary in vivo infection. We harvested virally-infected macrophages at 16 hours after MHV68 infection in vivo using a wild-type MHV68 reporter virus to directly identify virally infected cells, followed by polyA-based 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 is a unique and unanticipated viral transcription signature that is not predicted based on current models of either lytic replication or latent infection. These studies, in combination with related experimental approaches, indicate that MHV68 efficiently infects macrophages, with the majority of cells characterized by an atypical state of restricted viral transcription, with only rare cells undergoing lytic replication at early times post-infection.

ORGANISM(S): Mus musculus

PROVIDER: GSE233202 | GEO | 2026/09/01

REPOSITORIES: GEO

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Project description:MicroRNA (miRNA) and endogenous siRNA (endo-siRNA) are two essential classes of small noncoding RNAs (sncRNAs) in eukaryotic organisms. The class of miRNA is diverse and there exist noncanonical miRNAs that bypass the canonical miRNA biogenesis pathway. In order to identify noncanonical miRNAs and endo-siRNAs responding to virus infection and study their potential function, we sequenced small-RNA species from cells lytically infected with murine gammaherpesvirus 68. In addition to 3 novel canonical miRNAs in mouse, two antisense miRNAs in virus and 25 novel noncanonical miRNAs, including miRNAs derived from tRNAs, snoRNAs and introns, in the host were identified. These noncanonical miRNAs exhibited features distinct from canonical miRNAs in the lengths and structures of miRNA hairpins as well as base pairings and first nucleotide preference. Many of the novel miRNAs are conserved in mammals. In addition to several known murine endo-siRNAs detected by the sequencing profiling, a novel locus in the mouse genome was identified to give rise to endo-siRNAs. This novel endo-siRNA locus is comprised of two tandem inverted B4 short interspersed nuclear elements (SINEs). Unexpectedly, the SINE-derived endo-siRNAs were found in a variety of sequencing data as well as virus-infected cells. Moreover, a murine miRNA was up-regulated more than 35 fold in infected than in mock-treated cells. The putative target genes of the viral and the up-regulated murine miRNAs were potentially involved in processes of gene transcription and protein phosphorylation and localized to membranes, suggesting their role in manipulating the host basal immune system during lytic infection. Our results extended the number of noncanonical miRNAs in mammals and shed new lights on their potential functions of lytic infection of MHV68. Mouse NIH 3T12 cells infectd with MHV68 (3 samples) and mock-treated (2 samples) were examined. Noncanonical microRNAs and endogenous siRNAs discovery in lytic infection of murine gammaherpesvirus MHV68 (NC_001826.2).

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Host and viral gene expression in murine gammaherpesvirus 68 infected primary peritoneal mouse macrophages harvested 16 hours post-infection in vivo

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