mRNA decay during HSV infections: Interactions between a viral nuclease and cellular translation factors

During lytic infections, the virion host shutoff protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps control the levels and kinetics of viral and cellular gene expression. In vivo, UL41 shows a strong preference for mRNAs, as opposed to non-messenger RNAs, and initiates cleavage at or near regions of translation initiation. To define the mechanism behind the viral strategy, I searched for cellular proteins that interact with UL41 using the yeast two-hybrid system. The UL41 polypeptide was found to interact with a eukaryotic translation factor, eIF4H. This interaction was confirmed by GST pull-down experiments as well as by co-immunoprecipitation of the UL41 polypeptide and eIF4H from extracts of transfected mammalian cells. The interaction was abolished by several UL41 point mutations that abrogate the mRNA degradative activity of the protein in vivo, indicating the significance of this interaction. In addition, the difference in Vhs activity observed between HSV-1 and HSV-2 strains correlates with difference in the binding affinities of the UL41 polypeptides for eIF4H.; Biochemical evidence indicates that eIF4H interacts functionally with eIF4A. Indeed, eIF4H was found to interact physically with eIF4A in the yeast two-hybrid system as well as in GST pulldown experiments, and the two proteins could be co-immunoprecipitateed from transfected mammalian cells.; A putative Vhs-binding domain of eIF4H was characterized to span about 60 residues. Three key residues within the putative Vhs-binding domain of eIF4H were identified by site-directed mutagenesis, since alteration of these residues abolished or substantially reduced the eIF4H-Vhs interaction.; Subsequently, UL41 also was found to interact with eIF4A. The direct physical interaction was demonstrated by GST pull-down and co-immunoprecipitation experiments, and further confirmed by several mutations of UL41 that abolished the eIF4H-UL41 interaction but maintained its ability to interact with eIF4A.; It is postulated that the UL41 polypeptide, the viral mRNase of HSV, is targeted to mRNAs, and to regions of translation initiation, through interactions with the cellular translation factors, eIF4H and/or eIF4A. Taken together, this study further elucidates the mechanism leading to the accelerated mRNA decay during lytic infection of HSV.