Abstract
Viral genetic studies typically focus on large open reading frames (ORFs) identified during genome annotation (ORF-based annotation). Here we describe tools for examining viral gene expression nucleotide by nucleotide across the genome. Using these tools on the 119,450 base pair (bp) genome of murine gammaherpesvirus 68 (gammaHV68) allowed us to establish that gammaHV68 RNA expression was significantly more complex than predicted from ORF-based annotation, including over 73,000 nucleotides of unexpected transcription within 30 expressed genomic regions (EGRs). Approximately 90% of this RNA expression was antisense to genomic regions containing known large ORFs. We verified the existence of previously undefined transcripts in three EGRs and determined which parts of the transcriptome depend on protein or viral DNA synthesis. This study redefines the genetic map of gammaHV68, indicating that herpesviruses contain significantly more genetic complexity than predicted from ORF-based genome annotations, and provides alternative tools and approaches for viral genetic studies.