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Tiled Microarray Identification of Novel Viral Transcript Structures and Distinct Transcriptional Profiles During Two Modes of Productive Murine Gammaherpesvirus 68 Infection.

ABSTRACT: 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.

ORGANISM(S): Mus musculus Murid gammaherpesvirus 4

PROVIDER: GSE35866 | GEO | 2012/02/16

SECONDARY ACCESSION(S): PRJNA151915

REPOSITORIES: GEO

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Project description:Interaction of the Kaposi’s sarcoma-associated herpesvirus (KSHV) Rta protein with the cellular Notch signaling effector, Recombination Signaling Protein (RBP)-Jk (aka CSL and CBF-1), is essential for viral reactivation from latency. We previously showed that Rta binds to a DNA motif repeated in the viral Mta promoter (called “CANT” or Rta-c) to stimulate RBP-Jk DNA binding, and distinguished Rta from the activated Notch-1 protein. To determine whether Rta’s mechanism would apply generally to other viral promoters, we employed chromatin immunoprecipitation/deep sequencing (ChIP/Seq) to identify Rta and RBP-Jk binding sites across the KSHV genome. We show that RBP-Jk binds nearly exclusively to unique genome sites during latency and reactivation. Many, but not all, reactivation-specific RBP-Jk peaks were associated with Rta bound to single Rta-c motifs. Other motifs that were over-represented with stimulated RBP-Jk DNA binding including those for the cellular DNA binding proteins BCL11A, MNT, MAF B, and TCF12. Four of the top seven motifs that were most over-represented with inhibition of RBP-Jk DNA binding were putative binding sites for the Pit/Oct/Unc (POU) family of proteins, including POU3F3 (Oct-8) and POU5F1 (Oct-4). Interestingly, two other POU motifs, including one for the POU2F1 (Oct-1) protein, are associated with inhibition of RBP-Jk DNA binding unless Rta bound to a nearby Rta-c motif. The relative distances between the Rta-c, POU, and RBP-Jk motifs were conserved at reactivation-specific RBP-Jk peaks in three promoters that Rta transactivated, and the Rta-c and Oct-1 motifs overlapped in two of those. The proximity of the Rta-c/Oct-1 motif to an RBP-Jk motif is critical for Rta transactivation of the ORF50AS/KbZIP promoter, and knockdown of Oct-1 protein debilitated reactivation and production of infectious virus. Our data suggest a broad role for POU proteins in regulating DNA binding of RBP-Jk and its associated transactivators.

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Tiled Microarray Identification of Novel Viral Transcript Structures and Distinct Transcriptional Profiles During Two Modes of Productive Murine Gammaherpesvirus 68 Infection.

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