Fig 3.

The Us3 Ser/Thr kinase activity is required to suppress ERK activation. (A) Serum-deprived NHDFs were mock infected or infected (MOI = 5) with WT HSV-1, ΔUs3, a Us3-deficient virus in which the Us3 allele was restored (ΔUs3-Repair), or a virus expressing a kinase-deficient Us3 gene containing a K-to-A single amino acid substitution at residue 220 (K220A Us3). At 15 hpi, total protein was isolated and analyzed by immunoblotting with the indicated antisera. RhoGDI served as a loading control. (B) U2OS cells were mock infected or infected with WT HSV-1 or ΔU3 (MOI = 10), and at 9 hpi total protein was collected and analyzed as described for panel A. A modest reduction in Us11 accumulation was observed in cells infected with ΔUs3, as Us3-deficient viruses are unable to activate mTORC1 and display reduced viral protein synthesis (6, 7). (C) U2OS cells were transfected with the vector control plasmid, a K220A Us3-Flag-expressing plasmid, or a WT Us3-Flag-expressing plasmid. After 24 h, total protein was isolated and analyzed as described for panel A.