Pervasive Transcription of a Herpesvirus Genome Generates Functionally Important RNAs

T.A.R. and L.S.J. contributed equally to this work. ABSTRACT Pervasive transcription is observed in a wide range of organisms, including humans, mice, and viruses, but the func-tional significance of the resulting transcripts remains uncertain. Current genetic approaches are often limited by their emphasis on protein-coding open reading frames (ORFs). We previously identified extensive pervasive transcription from the murine gammaherpesvirus 68 (MHV68) genome outside knownORFs and antisense to known genes (termed expressed genomic regions [EGRs]). Similar antisense transcripts have been identified in many other herpesviruses, including Kaposi’s sarcoma-associated herpesvirus and human andmurine cytomegalovirus. Despite their prevalence, whether these RNAs have any functional impor-tance in the viral life cycle is unknown, and one interpretation is that these are merely “noise ” generated by functionally unim-portant transcriptional events. To determine whether pervasive transcription of a herpesvirus genome generates RNAmolecules that are functionally important, we used a strand-specific functional approach to target transcripts from thirteen EGRs in MHV68.We found that targeting transcripts from six EGRs reduced viral protein expression, proving that pervasive transcrip-tion can generate functionally important RNAs.We characterized transcripts emanating from EGRs 26 and 27 in detail using several methods, including RNA sequencing, and identified several novel polyadenylated transcripts that were enriched in the nuclei of infected cells. These data provide the first evidence of the functional importance of regions of pervasive transcription emanating fromMHV68 EGRs. Therefore, studies utilizing mutation of a herpesvirus genomemust account for possible effect