Figure 2.

Inactivation of 4E-BP1 in HSV-1-infected cells requires the virus-encoded Us3 Ser/Thr kinase. (A) Growth-arrested NHDFs were mock-infected (Mock) or infected (MOI = 5) with wild-type HSV-1 (WT), a UL13-deficient mutant (ΔUL13), a Us3-deficient virus (ΔUs3), or a virus where the Us3 deficiency was repaired (ΔUs3 REPAIR). At 18 hpi, total protein was isolated, fractionated by SDS-PAGE (17.5% gel), and analyzed by immunoblotting using anti-4E-BP1. Slow-migrating hyperphosphorylated (hyper) and fast-migrating hypophosphorylated (hypo) 4E-BP1 forms are noted on the right. (B) As in A, except total protein was fractionated using a 7.5% gel, which does not resolve phosphorylated 4E-BP1 isoforms. (C) As in A, except a virus expressing a catalytically inactive Us3 protein was included (Us3 K220A contains a single Lys-to-Ala substitution at residue 220). (D) Soluble extracts prepared from NHDFs described in A were incubated with m⁷GTP-Sepharose and analyzed as described in Figure 1C.