Genome-wide transcript structure resolution in murine gammaherpesvirus 68: HE 2.1 BGI RNA-Seq
ABSTRACT: Strand-specific RNA-Seq was used in conjunction with Pacific Biosciences Iso-Seq and deepCAGE to globally resolve transcript structures in lytic murine gammaherpesvirus 68. Overall design: Strand-specific BGI RNA-Seq of MHV68-infected fibroblasts
Project description:Strand-specific RNA-Seq was used in conjunction with Pacific Biosciences Iso-Seq and deepCAGE to globally resolve transcript structures in lytic murine gammaherpesvirus 68. Overall design: Strand-specific BGI RNA-Seq of MHV68-infected fibroblasts
Project description:Strand-specific Illumina RNA-Seq was used in conjunction with Pacific Biosciences Iso-Seq and deepCAGE to globally resolve transcript structures in lytic murine gammaherpesvirus 68. Overall design: Strand-specific Illumina RNA-Seq of MHV68-infected fibroblasts
Project description:Pacific Biosciences Iso-Seq was used in conjunction with Illumina RNA-Seq and deepCAGE to globally resolve transcript structures in lytic murine gammaherpesvirus 68. Overall design: Pacific Biosciences Iso-Seq of MHV68-infected fibroblasts
Project description:deepCAGE was used in conjunction with Pacific Biosciences Iso-Seq and Illumina RNA-Seq to globally resolve transcript structures in lytic murine gammaherpesvirus 68. Overall design: deepCAGE of MHV68-infected fibroblasts
Project description:deepCAGE was used in conjunction with Pacific Biosciences Iso-Seq and Illumina RNA-Seq to globally resolve transcript structures in lytic murine gammaherpesvirus 68. Overall design: deepCAGE of B cells with latent or reactivated MHV68
Project description:Herpesviruses are a large group of DNA viruses infecting mainly vertebrates. Murine gammaherpesvirus 68 (MHV68) is often used as a model in studies of the pathogenesis of clinically important human gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This rodent virus appears to be geographically widespread; however, its natural transmission cycle is unknown. Following detection of MHV68 in field-collected ticks, including isolation of the virus from tick salivary glands and ovaries, we investigated whether MHV68 is a tick-borne virus. Uninfected Ixodes ricinus ticks were shown to acquire the virus by feeding on experimentally infected laboratory mice. The virus survived tick molting, and the molted ticks transmitted the virus to uninfected laboratory mice on which they subsequently fed. MHV68 was isolated from the tick salivary glands, consistent with transmission via tick saliva. The virus survived in ticks without loss of infectivity for at least 120 days, and subsequently was transmitted vertically from one tick generation to the next, surviving more than 500 days. Furthermore, the F1 generation (derived from F0 infected females) transmitted MHV68 to uninfected mice on which they fed, with MHV68 M3 gene transcripts detected in blood, lung, and spleen tissue of mice on which F1 nymphs and F1 adults engorged. These experimental data fulfill the transmission criteria that define an arthropod-borne virus (arbovirus), the largest biological group of viruses. Currently, African swine fever virus (ASFV) is the only DNA virus recognized as an arbovirus. Like ASFV, MHV68 showed evidence of pathogenesis in ticks. Previous studies have reported MHV68 in free-living ticks and in mammals commonly infested with I. ricinus, and neutralizing antibodies to MHV68 have been detected in large mammals (e.g., deer) including humans. Further studies are needed to determine if these reports are the result of tick-borne transmission of MHV68 in nature, and whether humans are at risk of infection.
Project description:The ORF75c tegument protein of murine gammaherpesvirus 68 (MHV68) promotes the degradation of the antiviral promyelocytic leukemia (PML) protein. Surprisingly, MHV68 expressing a degradation-deficient ORF75c replicated in cell culture and in mice similar to the wild-type virus. However, in cells infected with this mutant virus, PML formed novel track-like structures that are induced by ORF61, the viral ribonucleotide reductase large subunit. These findings may explain why ORF75c mutant viruses unable to degrade PML had no demonstrable phenotype after infection.
Project description:The gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, MuHV-4, ?HV68), are etiologic agents of a wide range of lymphomas and non-hematological malignancies. These viruses possess large and highly dense dsDNA genomes that feature >80 bidirectionally positioned open reading frames (ORFs). The abundance of overlapping transcripts and extensive splicing throughout these genomes have until now prohibited high throughput-based resolution of transcript structures. Here, we integrate the capabilities of long-read sequencing with the accuracy of short-read platforms to globally resolve MHV68 transcript structures using the transcript resolution through integration of multi-platform data (TRIMD) pipeline. This approach reveals highly complex features, including: (1) pervasive overlapping transcript structures; (2) transcripts containing intra-gene or trans-gene splices that yield chimeric ORFs; (3) antisense and intergenic transcripts containing ORFs; and (4) noncoding transcripts. This work sheds light on the underappreciated complexity of gammaherpesvirus transcription and provides an extensively revised annotation of the MHV68 transcriptome.
Project description:Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), 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 (MHV68) 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 genetic elements such as miRNAs in the context of infection and disease. However, the specific targets of MHV68 miRNAs remain completely unknown. Using a technique known as qCLASH (quick crosslinking, ligation, and sequencing of hybrids), we have now identified thousands of Ago-associated, direct miRNA-mRNA interactions during lytic infection, latent infection and reactivation from latency. Validating this approach, detailed molecular analyses of specific interactions demonstrated repression of numerous host mRNA targets of MHV68 miRNAs, including Arid1a, Ctsl, Ifitm3 and Phc3. Notably, of the 1,505 MHV68 miRNA-host mRNA targets identified in B cells, 86% were shared with either EBV or KSHV, and 64% were shared among all three viruses, demonstrating significant conservation of gammaherpesvirus miRNA targeting. Pathway analysis of MHV68 miRNA targets further revealed enrichment of cellular pathways involved in protein synthesis and protein modification, including eIF2 Signaling, mTOR signaling and protein ubiquitination, pathways also enriched for targets of EBV and KSHV miRNAs. These findings provide substantial new information about specific targets of MHV68 miRNAs and shed important light on likely conserved functions of gammaherpesvirus miRNAs.
Project description:Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine gammaherpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by interferon-? (IFN-?). Using a lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16l1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5 deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-?, and an IFN-?-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-?. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency.