Roles of Us8A and its phosphorylation mediated by Us3 in herpes simplex virus 1 pathogenesis

The herpes simplex virus 1 (HSV-1) Us8A gene overlaps the gene that encodes glycoprotein E (gE). Previous studies have investigated the roles of Us8A in HSV-1 infection using null mutations in Us8A and gE; therefore, the role of Us8A remains to be elucidated. In this study, we investigated the function of Us8A and its phosphorylation at serine 61 (Ser-61), which we recently identified as a phosphorylation site by mass spectrometry-based phosphoproteomic analysis of HSV-1-infected cells, in HSV-1 pathogenesis. We observed that (i) the phosphorylation of Us8A Ser-61 in infected cells was dependent on the activity of the virus-encoded Us3 protein kinase; (ii) the Us8A null mutant virus exhibited a 10-fold increase in the 50% lethal dose for virulence in the central nervous system (CNS) of mice following intracranial infection compared with a repaired virus; (iii) replacement of Ser-61 with alanine (S61A) in Us8A had little effect on virulence in the CNS of mice following intracranial infection, whereas it significantly reduced the mortality of mice following ocular infection to levels similar to the Us8A null mutant virus; (iv) the Us8A S61A mutation also significantly reduced viral yields in mice following ocular infection, mainly in the trigeminal ganglia and brains; and (v) a phosphomimetic mutation at Us8A Ser-61 restored wild-type viral yields and virulence. Collectively, these results indicate that Us8A is a novel HSV-1 virulence factor and suggest that the Us3-mediated phosphorylation of Us8A Ser-61 regulates Us8A function for viral invasion into the CNS from peripheral sites. IMPORTANCE The DNA genomes of viruses within the subfamily Alphaherpesvirinae are divided into unique long (UL) and unique short (Us) regions. Us regions contain alphaherpesvirus-specific genes. Recently, high-throughput sequencing of ocular isolates of HSV-1 showed that Us8A was the most highly conserved of 13 herpes simplex virus 1 (HSV-1) genes mapped to the Us region, suggesting Us8A may have an important role in the HSV-1 life cycle. However, the specific role of Us8A in HSV-1 infection remains to be elucidated. Here, we show that Us8A is a virulence factor for HSV-1 infection in mice, and the function of Us8A for viral invasion into the central nervous system from peripheral sites is regulated by Us3-mediated phosphorylation of the protein at Ser-61. This is the first study to report the significance of Us8A and its regulation in HSV-1 infection.