title: The UL12 protein of herpes simplex virus 1 is regulated by tyrosine phosphorylation.
abstract: The herpes simplex virus 1 (HSV-1) UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL, and A549 cells, (ii) reduced viral replication, cell-cell spread, and pUL12 expression in infected cells in a cell type-dependent manner, (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner, and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication severalfold more than the nuclease-dead double mutation in a cell type- and multiplicity-of-infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice mainly by upregulating pUL12 nuclease activity and, in part, by regulating the subcellular localization and expression of pUL12 in HSV-1-infected cells.
  
  
  OBJECTIVE
  Herpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been clarified, information on how most of these enzymes are regulated in infected cells is lacking. In the present study, we report that the enzymatic activity of the herpes simplex virus 1 alkaline nuclease pUL12 was regulated by phosphorylation of pUL12 Tyr-371 in infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice, mainly by upregulating pUL12 nuclease activity. Interestingly, pUL12 and tyrosine at pUL12 residue 371 appeared to be conserved in all herpesviruses in the family Herpesviridae, raising the possibility that the herpesvirus pUL12 homologs may also be regulated by phosphorylation of the conserved tyrosine residue.
authors.affiliation: Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Fujii
authors.affiliation: Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Akihisa
authors.initials: A
authors.lastname: Kato
authors.affiliation: Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Michio
authors.initials: M
authors.lastname: Mugitani
authors.affiliation: Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Kashima
authors.affiliation: Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oyama
authors.affiliation: Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kozuka-Hata
authors.affiliation: Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Arii
authors.affiliation: Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan ykawagu@ims.u-tokyo.ac.jp.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawaguchi
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006561:Herpes Simplex / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D006801:Humans; D051379:Mice; D008813:Mice, Inbred ICR; D010766:Phosphorylation; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D014764:Viral Proteins; D014443:Tyrosine; D003851:Deoxyribonucleases; C539238:UL12 protein, herpes simplex virus 1