Ras-GAP binding and phosphorylation by herpes simplex virus type 2 RR1 PK (ICP10) and activation of the Ras/MEK/MAPK mitogenic pathway are required for timely onset of virus growth.
We used a herpes simplex virus type 2 (HSV-2) mutant with a deletion in the RR1 (ICP10) PK domain (ICP10DeltaPK) and an MEK inhibitor (PD98059) to examine the role of ICP10 PK in virus growth. In HSV-2-infected cells, ICP10 PK binds and phosphorylates the GTPase activating protein Ras-GAP. In vitro binding and peptide competition assays indicated that Ras-GAP N-SH2 and PH domains, respectively, bind ICP10 at phosphothreonines 117 and 141 and a WD40-like motif at positions 160 to 173. Binding and phosphorylation did not occur in cells infected with ICP10DeltaPK. GTPase activity was significantly lower in HSV-2- than in ICP10DeltaPK-infected cells. Conversely, the levels of activated Ras and mitogen-activated protein kinase (MAPK), and the expression and stabilization of the transcription factor c-Fos were significantly increased in cells infected with HSV-2 or a revertant virus [HSV-2(R)] but not with ICP10DeltaPK. PD98059 inhibited MAPK activation and induction-stabilization of c-Fos. Expression from the ICP10 promoter was increased in cells infected with HSV-2 but not with ICP10DeltaPK, and increased expression was ablated by PD98059. ICP10 DNA formed a complex with nuclear extracts from HSV-2-infected cells which was supershifted by c-Fos antibody and was not seen with extracts from ICP10DeltaPK-infected cells. Complex formation was abrogated by PD98059. Onset of HSV-2 replication was significantly delayed by PD98059 (14 h versus 2 h in untreated cells), a delay similar to that seen for ICP10DeltaPK. The data indicate that Ras-GAP phosphorylation by ICP10 PK is involved in the activation of the Ras/MEK/MAPK mitogenic pathway and c-Fos induction and stabilization. This results in increased ICP10 expression and the timely onset of HSV-2 growth.
Smith
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Departments of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Nelson
J
J
Aurelian
L
L
Gober
M
M
Goswami
B B
BB
D020816:Amino Acid Motifs; D000818:Animals; D002522:Chlorocebus aethiops; D005246:Feedback; D015967:Gene Expression Regulation, Viral; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D006801:Humans; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D014779:Virus Replication; D020703:ras GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
D016760:Proto-Oncogene Proteins c-fos; D020703:ras GTPase-Activating Proteins; C100671:ICP10 protein, herpes simplex virus type 2; D012264:Ribonucleotide Reductases; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins