Autophagy has recently been demonstrated in both cultured cells infected with varicella- zoster virus (VZV) as well as in a human zoster vesicle. The presence of punctate autophagosomes in a zoster skin vesicle firmly established that autophagy (macroautophagy) is a relevant biological process during the natural history of disease caused by this herpesvirus. Of importance, we have carried out sufficient preliminary studies to document that autophagy induced by VZV infection does not resemble HSV-induced autophagy. First of all, the VZV genome lacks a homolog of the HSV ICP 34.5 gene. Secondly, we have now shown by two different methods that VZV infection quickly induces marked ER stress, an event not known to occur in the HSV system. Therefore, the re-stated central hypothesis for this proposal is that ER stress is a critical component of VZV infection, that ER stress is related to an over abundance of misfolded VZV glycoproteins in the ER, and that ER-associated degradation and autophagy are consequences that relieve ER stress. These events allow the infected cell to survive longer, to avoid apoptosis, and in that sense can be considered pro-viral. The Research Plan to test this hypothesis involves three Specific Aims. Aim 1 is called Induction of autophagy as a consequence of VZV induced ER stress. Aim 2 is called Activation of the unfolded protein response (UPR) by VZV induced ER stress. Aim 3, which involves the A. Arvin laboratory, includes Studies of autophagy and ER stress in the SCID-hu mouse model, following infection with various recombinant mutated VZV genomes. In our Research Plan, we will continue prior investigations with an expanded experimental approach directed toward the three sensor pathways that lead from ER stress to the UPR (IRE1, ATF6 and PERK). We have already detected four proteins associated with the UPR in VZV infected cells, including BiP (HSPA5), HSPA8, HSPD1 and PPIA: peptidyl-propyl-cis-trans-isomerase). More recently we have detected the spliced form of XBP1 (X-Box binding protein) in VZV infected cells, another marker of the UPR. The final approach will include animal experiments with already prepared recombinant viruses containing numerous gE mutations. The phenotypes of these mutant viruses have already been determined in both cell culture and the SCID mouse model. Thus the above experimental plan will determine the relative importance of ER stress and autophagy in the cellular response to VZV infection. The results are relevant not only to our understanding of varicella vaccination but also the debilitating zoster-related illness called post-herpetic neuralgia.