DESCRIPTION (provided by applicant): HSV-1 is a widespread human pathogen. In addition, it serves as an important model to study how pathogenic human herpesviruses regulate their genes during infections. One protein that is critical for HSV-1 gene expression is the immediate-early protein ICP27. In this application, we propose to study two distinct functions of ICP27 that regulate viral gene expression. In Specific Aim 1, we will study how ICP27 stimulates mRNA accumulation from delayed-early (DE) and late (L) target genes. The current leading model for how ICP27 turns on viral genes is that it functions as a mRNA export factor that transports intronless viral transcripts to the cytoplasm. However, this model cannot readily explain how ICP27 dramatically promotes the accumulation of certain viral mRNAs, including that encoding glycoprotein C (gC). Our recent work on the gC gene suggests a novel model in which ICP27 prevents newly transcribed but inherently unstable viral transcripts from being rapidly degraded in the nucleus by a host cell nuclease. This nuclease targets the gC mRNA in response to a specific cis-acting sequence in the body of the message. We have named this sequence the silencing element (SE). Intriguingly, the SE can be transferred to another gene, where it functions both to silence the gene as well as to confer ICP27-dependent expression. In this Aim, we will study how the SE and ICP27 functionally interact to regulate mRNA stability. We will also study how ICP27 regulates other viral genes DE and L genes, as well as the gene for the viral latency-associated transcript (LAT), which we have recently shown is regulated by ICP27 at the level of mRNA stability. In Specific Aim 2, we will study how ICP27 promotes the retention of introns in viral mRNAs. This Aim is based on our recent discovery that the gC gene contains a functional intron, the retention of which is enhanced by ICP27. The spliced transcript encodes a secreted variant of gC, called gCsec. It is not known how ICP27 promotes the retention of introns in mature mRNA. However, very few cellular or viral regulators of intron-retention have been identified, so ICP27 serves as a valuable model to understand how this important cellular process can be regulated. Three models are proposed to explain how ICP27 regulates intron-retention, and experiments are outlined to test these. The second part of this Aim seeks to identify novel introns in the HSV-1 genome that are regulated by ICP27. Viral genes harboring such introns may encode biologically important protein variants, analogous to gCsec. One such intron has already been identified in the UL24 gene, which encodes a protein important for neuronal replication. Together, our studies will illuminate how an essential but poorly understood viral regulatory protein controls the fate of viral mRNAs, and in so doing, helps determine the outcome of HSV-1 infections.

PUBLIC HEALTH RELEVANCE: HSV-1 and its closely related cousin HSV-2 are comon human viruses that causes several serious disesases including encephalitis, disseminated newborn infections, and stromal keratitis resulting in blindness. This research will help us better understand the replication of these viruses and other related pathogenic herpesviruses. It is also possible that these studies will lead to novel strategies for therapeutic modalities to prevent or treat herpesvirus infections.