FIG. 3.

(A) Expression of the γ₁34.5 protein. HeLa cells were mock infected or infected with HSV-1(F), R3616 (in which the coding region of the γ₁34.5 gene was deleted), or H9813 (in which Vla¹⁹³Glu and Phe¹⁹⁵Leu substitutions were made in the γ₁34.5 gene) at 10 PFU per cell). At 15 h postinfection, cells were harvested, washed with phosphate-buffered saline, and resuspended in disruption buffer containing 50 mM Tris-HCl (pH 7.0), 5% 2-mercaptoethanol, 2% SDS, and 2.75% sucrose. Samples were then electrophoretically separated on denaturing 12% polyacrylamide gels and transferred to a nitrocellulose membrane. The blot was probed with anti-γ₁34.5 antibody (1). (B) eIF-2α phosphatase activity in HeLa cells which were mock infected or infected with the indicated viruses. ³²P-labeled eIF-2α, prepared as described in Materials and Methods, was incubated with lysates of HeLa cells which were mock infected or infected with indicated viruses at 34°C. After incubation for 2 min, the reaction was stopped by the addition of disruption buffer, and samples were separated electrophoretically on a denaturing 12% polyacrylamide gel, transferred to a nitrocellulose membrane, and subjected to autoradiography (21). Lanes 1, ³²P-labeled eIF-2α and GST-PKR not reacted with cell lysates; lanes 2 to 5, ³²P-labeled eIF-2α reacted with lysates of cells which were mock infected or infected with the indicated viruses. (C) Immunoblot of the autoradiogram in panel B.