Regulation of translation during herpes simplex virus-1 infection

In an effort to better understand the regulation of viral and cellular gene expression during HSV-1 infection, I examined several mechanisms employed by the virus to regulate translation. I described an element in the 5' leader of the pol transcript that was necessary and sufficient to inhibit translation in vitro and in transfected cells. This inhibitory element was characterized by RNase structure mapping and mutagenic analyses. Deleting this element from HSV-1 resulted in an increase in the translation of Pol and also resulted in a replication defect relative to a control virus with wild type pol sequence, indicating the importance of this inhibitory element for viral replication. To better understand the RNA binding activity, and thus potentially the gene regulatory activity, of HSV-1 US11 during infection, I investigated the interaction between US11 and in vitro selected aptamers. I found that US11 bound the selected aptamers specifically with high affinity. Analysis of the selected sequences revealed a consensus sequence that was protected from hydroxyl radical cleavage upon US11 binding. Interestingly, US11 may alter the conformation of RNA ligands because it was observed to induce regions of RNA to become hypersensitive to hydroxyl radical cleavage upon binding. US11 also bound double stranded RNA, but with lower affinity than it bound the selected aptamers and this binding seemed to be non-specific. Additionally, I investigated the antiherpesviral activity of the small molecule salubrinal. Salubrinal treatment increases phosphorylation of the translation initiation factor eIF2[alpha] by inhibiting GADD34 mediated dephosphorylation of eIF2[alpha]. Since HSV-1 encodes a homologue of GADD34, ICP34.5, we investigated whether sal could inhibit ICP34.5 mediated dephosphorylation and whether sal had antiherpesviral activity. Sal did inhibit ICP34.5 mediated eIF2[alpha] dephosphorylation and had antiviral activity in cell culture and the mouse ocular model of infection. Sal exhibited reduced antiviral activity in a mutant cell line containing non-phosphorylatable eIF2[alpha], relative to wild-type control cells, indicating that eIF2[alpha] phosphorylation is critical for the activity of the compound. ICP34.5 seemed to be necessary for the full activity of sal during HSV-1 infection. This work establishes regulation of translation during HSV-1 infection as a novel drug target.