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
Cc. Smith et al., 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, J VIROLOGY, 74(22), 2000, pp. 10417-10429
We used a herpes simplex virus type 2 (HSV-2) mutant with a deletion in the
RR1 (ICP10) PK domain (ICP10 Delta PK) and an MEK inhibitor (PD98059) to e
xamine the role of ICP10 PK in virus growth. In HSV-2-infected cells, ICP10
PK binds and phosphorylates the GTPase activating protein Pas-CAP. In vitr
o binding and peptide competition assays indicated that Ras-GAP N-SH2 and P
H domains, respectively, bind ICP10 at phosphothreonines 117 and 141 and a
WD40-like motif at positions 160 to 173. Binding and phosphorylation did no
t occur in cells infected with ICP10 Delta PK GTPase activity was significa
ntly lower in HSV-2 than in ICP10 Delta PK-infected cells. Conversely, the
levels of activated Pas and mitogen-activated protein kinase (MAPK), and th
e expression and stabilization of the transcription factor c-Fos were signi
ficantly increased in cells infected with HSV-2 or a revertant virus [HSV-2
(R)] but not with ICP10 Delta PK. PD98059 inhibited MAPK activation and ind
uction-stabilization of c-Pos. Expression from the ICP10 promoter was incre
ased in cells infected with HSV-2 but not with ICP10 Delta PK, and increase
d expression was ablated by PD98059. ICP10 DNA formed a complex with nuclea
r extracts from HSV-2-infected cells which was supershifted by c-Fos antibo
dy and was not seen,vith extracts from ICP10 Delta PK-infected cells. Compl
ex formation was abrogated by PD98059. Onset of HSV-2 replication was signi
ficantly delayed by PD98059 (14 h versus 2 h in untreated cells), a delay s
imilar to that seen for ICP10 Delta PK. The data indicate that Pas-CAP phos
phorylation by ICP10 PK is involved in the activation of the Ras/MEK/MAPK m
itogenic pathway and c-Fos induction and stabilization. This results in inc
reased ICP10 expression and the timely onset of HSV-2 growth.