Latent gammaherpesvirus 68 infection induces distinct transcriptional changes in different organs
Ontology highlight
ABSTRACT: Previous studies identified a role for latent herpesvirus infection in cross-protection to infection and exacerbation of chronic inflammatory diseases. Here, we compared the gene expression signature from livers, spleens and brains of mice infected with wild-type gammaherpesvirus 68 (MHV68), a mutant virus defective in the establishment of latency (ORF73.stop) or mockulum. We identified over 600 genes differentially expressed in organs of mice latently infected with MHV68 and found distinct sets of genes linked to different pathways were altered in spleen compared to liver. Several of the most differentially expressed latency-specific genes (e.g. IFNγ, Cxcl9, Ccl5) are associated with known latency-specific phenotypes.
Project description:Previous studies identified a role for latent herpesvirus infection in cross-protection to infection and exacerbation of chronic inflammatory diseases. Here, we compared the gene expression signature from livers, spleens and brains of mice infected with wild-type gammaherpesvirus 68 (MHV68), a mutant virus defective in the establishment of latency (ORF73.stop) or mockulum. We identified over 600 genes differentially expressed in organs of mice latently infected with MHV68 and found distinct sets of genes linked to different pathways were altered in spleen compared to liver. Several of the most differentially expressed latency-specific genes (e.g. IFNγ, Cxcl9, Ccl5) are associated with known latency-specific phenotypes. RNA was extracted from livers, spleens and brains of 7-9 week old male C57Bl/6 mice infected with gammaherpesvirus 68 (MHV68), a virus defective in establishment of latency (ORF73.stop) or mockulum. RNA from 3-4 mice per treatment was pooled and analyzed by M430 2.0 Affymetrix Gene Chip. Three biologic replicates were analyzed for all conditions, except mock livers, for which four biologic replicates were analyzed.
Project description:We applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a timecourse of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently-infected B cell line. During de novo infection, all ORFs were transcribed and clustered into four major temporal groups that were overlapping, yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation timecourse. High-density transcript analysis at two-hour intervals during de novo infection mapped gene boundaries with a 20-nt resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of KSHV vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5' RACE. The ~1.3 kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation is dynamic and distinct, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection. This SuperSeries is composed of the following subset Series: GSE35863: Tiled Array Experiment of Murine Gammaherpesvirus 68 Transcripts In Newly Infected Fibroblasts GSE35865: Tiled Array Experiment of Murine Gammaherpesvirus 68 Transcripts Upon TPA-Stimulated Reactivation From Latency Refer to individual Series
Project description:Murine NIH3T3 fibroblasts were infected with MHV68 and RNA expression was examined over an 18 hour timecourse experiment. Study includes 8 samples isolated at 0, 2, 4, 6, 8, 10, 12, and 18 hours after infection of NIH3T3 fibroblasts with murine gammaherpesvirus 68 at a multiplicity of infection of 10.0.
Project description:We applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a timecourse of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently-infected B cell line. During de novo infection, all ORFs were transcribed and clustered into four major temporal groups that were overlapping, yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation timecourse. High-density transcript analysis at two-hour intervals during de novo infection mapped gene boundaries with a 20-nt resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of KSHV vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5' RACE. The ~1.3 kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation is dynamic and distinct, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection. This SuperSeries is composed of the SubSeries listed below.
Project description:Gammaherpesviruses, including the human pathogens Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus, establish lifelong latent infection in B cells and are associated with a variety of tumors. In addition to protein coding genes, these viruses encode numerous microRNAs (miRNAs) within their genomes. While putative host targets of EBV and KSHV miRNAs have been previously identified, the specific functions of these miRNAs during in vivo infection are largely unknown. Murine gammaherpesvirus 68 is a natural pathogen of rodents that is genetically related to both EBV and KSHV, and thus serves as an excellent model for the study of EBV and KSHV elements such as miRNAs in the context of infection and disease. However, MHV68 the specific targets of miRNAs remain unknown. Using a technique known as quick CLASH (crosslinking, ligation, and sequencing of hybrids), we have now identified specific, Ago-associated mRNA targets of MHV68 miRNAs during lytic infection, latent infection and reactivation from latency.
Project description:RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. LPS/TLR4 engagement enhances rhadinovirus reactivation. We developed the HE-RIT cell line, a latent murine A20 B cell inducible for Flag-RTA expression and murine gammaherpesvirus 68 reactivation. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of FLAG-RTA. We applied RNAseq to examine for genome-wide changes in viral gene expression in response to doxycycline-induced RTA-FLAG, alone or in combination with LPS.
Project description:Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. Moreover, mounting evidence supports a noncanonical role for tRNA cleavage products in the control of gene expression during diverse conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to altered tRNA transcription, suggesting that tRNA regulation may play an important role in mediating viral replication or the host response. To better understand how viral infection alters tRNA expression, we combined Ordered Two Template Relay (OTTR) with tRNA-specific bioinformatic software called tRAX to profile full-length tRNAs and fragmented tRNA-derived RNAs (tDRs) during infection with the model gammaherpesvirus, MHV68. We find that OTTR-tRAX is a powerful sequencing strategy for combined tRNA/tDR profiling, and reveal that MHV68 infection triggers pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tDRs. Fragments of virally-encoded tRNAs (virtRNAs), as well as virtRNA base modification signatures are also detectable during infection. We further dissected the biogenesis pathway of an MHV68-induced cleavage product from a pre-tRNA. Our data shows that pre-tDR-Tyr expression is dependent on the tRNA splicing factor, TSEN2, and that pre-tDR-Tyr expression is inhibited by the kinase, CLP1, which regulates tRNA splicing. Significantly, our findings suggest that CLP1 kinase is required for infectious gammaherpesvirus production, offering new insight into the importance of tRNA processing during viral infection.
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).
Project description:Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and KaposiM-bM-^@M-^Ys sarcoma-associated herpesvirus (KSHV) and provides a small animal model to study the pathogenesis of gammaherpesvirus (M-NM-3HV) infections. To completely explore the potential of the MHV-68 system for the investigation of gHV miRNAs, it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By using small RNA deep sequencing, we systematically investigated the MHV-68 miRNA expression profiles in both lytically and persistently infected cells. In addition to the known nine MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68 infected versus non-infected NIH3T3 fibroblasts and in TPA-treated versus non-treated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH3T3 cells, indicating a potential role of cellular miRNAs during MHV-68 infection. Our data will aid to fully explore the functions of gHV miRNAs. A mouse fibroblast cell line infected with/without MHV-68 and a MHV-68 infected mouse B lymphoma cell line treated with/without TPA (4 samples in total) were examined.