The Role of Host Translation Initiation Factors eIF4H and eIF4A and the Exon Junction Complex in the Control of mRNA turnover and Translation by the Virion Host Shut off Protein of Herpes Simplex Virus
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During lytic infections, the HSV Vhs endonuclease (UL41) degrades many viral and cellular mRNAs. This endonuclease activity is dependent upon the ability of Vhs to bind the host translation initiation factors eIF4AI/II and eIF4H. eIF4AI and 4AII are isoforms of an ATP-dependent RNA helicase that unwinds mRNA secondary structure near the 5’ cap to allow the binding and subsequent scanning of the 40S ribosomal subunit. eIF4H binds and stimulates the helicase activity of eIF4AI/II. Si-RNA mediated knockdown of both eIF4AI/AII isoforms significantly impeded Vhs activity. Furthermore, hippuristanol, a selective eIF4AI/II inhibitor, blocked Vhs-mediated mRNA decay both in vitro and in vivo. In addition, we can restore Vhs activity in cells depleted of their endogenous eIF4H protein levels by transfecting an expression vector that encodes wild-type eIF4H but lacks the sequences recognized by the siRNA. We used this assay to screen a library of mutant eIF4H polypeptides for their ability to participate in Vhs cleavage and assayed their ability to bind eIF4A. These experiments revealed that both binding and stimulating eIF4A helicase activity are required for Vhs activity. In addition, we showed that the splicing history of an mRNA can affect its expression levels and its sensitivity to cleavage by Vhs. In uninfected cells, spliced mRNAs bound by the exon junction complex (EJC) are translated more efficiently than unspliced mRNAs containing an identical primary sequence. Whereas most cellular mRNAs are spliced, most viral mRNAs are not except a few immediate-early and late genes. During HSV-1 infections, spliced mRNAs containing a luciferase reporter were much less sensitive to Vhs-mediated degradation and expressed significantly more RLuc protein per molecule of mRNA, than did the unspliced mRNAs. The intron translational stimulatory effect could be replicated by artificially tethering various EJC components to an intronless RLuc reporter. Furthermore, knocking down the expression of endogenous eIF4AIII, an essential component of the EJC, restores Vhs cutting of spliced transcripts to the same level as unspliced mRNAs. The data suggest that the exon junction complex plays an important role in gene expression during HSV infections and can temporarily protect spliced mRNAs from Vhs endonuclease activity.
Table of Contents
Introduction -- Materials and methods -- Results -- Discussion