siRNAs that Deplete the Cellular Translation Factor eIF4H Impede mRNA Degradation by the Virion Host Shutoff (Vhs) Protein of Herpes Simplex Virus

School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, MO 64110

^* To whom correspondence should be addressed. Email: readgs{at}umkc.edu.

	Abstract

The herpes simplex virus (HSV) virion host shutoff (Vhs) protein is an endoribonuclease that accelerates decay of many host and viral mRNAs. Purified Vhs does not distinguish mRNAs from non-messenger RNAs and cuts target RNAs at many sites; yet within infected cells it is targeted to mRNAs, and cleaves those mRNAs at preferred sites including, for some, regions of translation initiation. This targeting may result, in part, from Vhs binding to the translation initiation factor eIF4H; in particular, several mutations in Vhs that abrogate its binding to eIF4H also abolish its mRNA degradative activity, even though the mutant proteins retain endonuclease activity. To further investigate the role of eIF4H in Vhs activity, HeLa cells were depleted of eIF4H or other proteins by transfection with siRNAs 48 hours prior to infection or mock infection in the presence of Actinomycin D. Cellular mRNA levels were then assayed 5 hours after infection. In cells transfected with an siRNA for the housekeeping enzyme GAPDH, wild-type HSV infection reduced -actin mRNA levels to between 20 and 30% of those in mock infected cells, indicative of a normal Vhs activity. In contrast, in cells transfected with any of three eIF4H siRNAs, -actin mRNA levels were indistinguishable in infected and mock infected cells, suggesting eIF4H depletion impeded Vhs-mediated degradation. Depletion of the related factor eIF4B did not affect Vhs activity. The data suggest eIF4H binding is required for Vhs-induced degradation of many mRNAs, perhaps by targeting Vhs to mRNAs and to preferred sites within mRNAs.