Virion-derived vhs accelerates decay of ectopically expressed dsRNA and concomitantly reverses PKR activation.

Abstract

A) HeLa cells were transfected with fwd/rev and either pEGFP or pEGFP-US11 for 20 hours. Cells were mock infected or infected with HSV-1 (KOS) and HSV-2 WT and Vhs- at MOI 20 for 4 hours with continuous actD treatment. Total RNA was extracted, digested by DNaseI and RNaseA/T1 (high salt conditions) and analyzed by Northern blot with a probe specific for the inserted SRPRα fragment. B) Quantification of full-length SRPRα dsRNA from 3 (HSV-2) or 6 (HSV-1) independent experiments. P-values vs. mock: * p-: ns (HSV-2/pEGFP), pC) Cells were transfected with fwd/rev and pEGFP-US11 for 20 hours and infected as described in A). Whole cell lysates were analyzed by Western blot with the indicated antibodies. Results shown are representative of 3 independent experiments. D) Virion-derived vhs reverses PKR activation by preformed dsRNA. Cells were transfected with fwd/rev and pEGFP for 20 hours and infected as described in A). Whole cell lysates were analyzed by Western blot with the indicated antibodies. E) Quantification of P-PKR levels from 3 independent experiments. P-values vs. fwd/rev/mock: *p- p<0.0001, HSV-1 WT vs. Vhs- p<0.02.</p