Hidden regulation of herpes simplex virus 1 pre-mRNA splicing and polyadenylation by virally encoded immediate early gene ICP27

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion. Author summary Little is known regarding to how HSV, a large DNA virus and known to contain very few spliced genes, escapes host pre-mRNA splicing machinery. Here, by establishing a high throughput splice junction identification platform and quantitative analysis method to assess splicing efficiency based on high throughput data, we find that HSV-1 encodes hundreds of previously unknown alternative splice junctions; however, splicing of these novel spliced genes is largely silenced in wild-type HSV-1 infected cells, explaining why only very few spliced genes have been previously identified in HSV-1. Moreover, ICP27 is required for splicing inhibition and 3' end formation of ICP34.5, the major viral neurovirulence factor and also the major target of latently expressed viral miRNAs. These findings not only fundamentally change the view of HSV gene structure, but also reveal a mechanism by which HSV employs host splicing and polyadenylation machineries to achieve optimal gene expression during acute infection and may also contribute to immune evasion during latency when ICP27 is not expressed.