Impact of the changes in substrate specificity of herpes simplex virus 1 protein kinase Us3 on viral infection <i>in vitro</i> and <i>in vivo</i>

A serine-threonine protein kinase (PK), Us3, encoded by herpes simplex virus 1 (HSV-1), shares substrate specificity with host cellular PKs, protein kinase A (PKA), and AKT. Many Us3 substrates have been identified, and it is thought that during HSV-1 infection, Us3 fine-tunes the phosphorylation levels of individual substrates within their repertoire. However, the significance of this regulatory fine-tuning by Us3 during HSV-1 infection is poorly understood. Here, we found alanine at position 326 (Ala-326) in Us3 was required for the proper fine-tuning of Us3-mediated phosphorylation across the target repertoire in HSV-1-infected cells. Using recombinant viruses in which Us3 Ala-326 was replaced with valine (A326V) or isoleucine (A326I), we showed these mutations selectively altered the phosphorylation of only a subset of at least 14 Us3 target proteins tested in HSV-1-infected cells, with each mutation generally affecting different targets. Of note, (i) both mutations significantly reduced plaque sizes without affecting viral replication in cell cultures; (ii) the Us3 A326I mutation impaired viral replication in the brains of mice and improved survival following intracranial infection, whereas the Us3 A326V mutation had little effect; and (iii) the Us3 A326V mutation reduced ocular pathogenic manifestations and viral replication in the trigeminal ganglia and brains of mice, thereby improving survival following ocular infection. Taken together, these results suggest that the proper fine-tuning of Us3-mediated phosphorylation across its target repertoire is required for efficient cell-to-cell spread of HSV-1 <i>in vitro</i>, and its replication and pathogenicity <i>in vivo</i>.IMPORTANCEThe activation loop (A-loop) is a conformationally flexible loop that critically regulates cellular protein kinases (PKs), but its role in viral PKs during infection remains unclear. We demonstrated alanine at position 326 (Ala-326) in the A-loop of herpes simplex virus 1 (HSV-1) PK Us3 was important for the proper fine-tuning of Us3-mediated phosphorylation across the target repertoire in HSV-1-infected cells. This fine-tuning was necessary for efficient HSV-1 cell-cell spread in cell cultures, and replication and pathogenicity in mice. Taken together, fine-tuning phosphorylation levels of individual Us3 targets within its repertoire is important for HSV-1 infection <i>in vitro</i> and <i>in vivo</i>. Different amino acid substitutions at Us3 Ala-326 selectively affected the phosphorylation of most distinct Us3 targets, leading to varied phenotypic outcomes in viral replication and pathogenicity in mice. These results provide important clues to elucidate the mechanisms by which Us3 regulates HSV-1 infection <i>in vivo</i>.