Herpes Simplex Virus 1 Protein Kinase Us3 and Major Tegument Protein UL47 Reciprocally Regulate Their Subcellular Localization in Infected Cells

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We have identified UL47, a major virion protein, as a novel physiological substrate of Us3. In vitro kinase assays and systematic analysis of mutations at putative Us3 phosphorylation sites near the nuclear localization signal of UL47 showed that serine at residue 77 (Ser-77) was required for Us3 phosphorylation of UL47. Replacement of UL47 Ser-77 by alanine produced aberrant accumulation of UL47 at the nuclear rim and impaired the nuclear localization of UL47 in a significant fraction of infected cells. The same defect in UL47 localization was produced by an amino acid substitution in Us3 that inactivated its protein kinase activity. In contrast, a phosphomimetic mutation at UL47 Ser-77 restored wild-type nuclear localization. The UL47 S77A mutation also reduced viral replication in the mouse cornea and the development of herpes stromal keratitis in mice. In addition, UL47 formed a stable complex with Us3 in infected cells, and nuclear localization of Us3 was significantly impaired in the absence of UL47. These results suggested that Us3 phosphorylation of UL47 Ser-77 promoted the nuclear localization of UL47 in cell cultures and played a critical role in viral replication and pathogenesis in vivo. Furthermore, UL47 appeared to be required for efficient nuclear localization of Us3 in infected cells. Therefore, Us3 protein kinase and its substrate UL47 demonstrated a unique regulatory feature in that they reciprocally regulated their subcellular localization in infected cells. Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1) with an amino acid sequence that is conserved in the subfamily Alphaherpesvirinae (11, 40, 56). R nX(S/T)YY is the consensus target sequence of an HSV-1 Us3 homologue encoded by pseudorabies virus (PRV), where ni s2; X can be Arg, Ala, Val, Pro, or Ser; and Y can be any amino acid except an acidic residue (33, 34, 55). This sequence also appears to be the target sequence of HSV-1 Us3, based on reports that the phosphorylation sites of HSV-1 Us3 identified to date match the consensus target sequence (24–26, 47, 62). The phosphorylation target site specificity of HSV-1 Us3 has also been reported to be similar to that of protein kinase A (PKA), a cellular cyclic AMP-dependent protein kinase (2), and Akt (4). Some antibodies to the phosphorylated substrate sequences of PKA can also react with Us3 phosphorylation sites (2, 24, 25). The Us3 protein and its catalytic activity have been suggested to play a critical role in HSV-1 replication and pathogenicity, based on studies showing that recombinant Us3null mutant viruses and recombinant viruses encoding catalytically inactive Us3 have impaired growth properties in cell