Research into the molecular interactions between viruses and their hosts has revealed that viruses manipulate a multitude of cellular pathways to facilitate their dissemination. This is certainly in evidence for the class I antigen presentation pathway. Virus-encoded molecules have been described that affect this pathway at essentially every step: from peptide generation by the proteasome, early assembly events in the ER, trafficking through the secretory pathway, and cell surface half-life. The fact that viruses have evolved such diverse mechanisms to target this pathway highlights the importance of class I antigen presentation in the host defense against viruses. An effective mechanism of preventing antigen presentation is to target nascent, ER-resident class I molecules for rapid degradation in the cytosol. It is now clear that immune evasion proteins US2 and US11 of HCMV and mK3 of gammaHV68 co-opt and greatly expedite ERAD, a normal physiologic pathway for destruction of overexpressed, misfolded, or unassembled host proteins . The molecular mechanism of ERAD is the subject of intense investigation due to its fundamental role in regulating protein expression and in human disease. However the molecular mechanisms of ERAD are largely unknown or remain controversial particularly in mammals. For example salient questions remain unanswered including how misfolded proteins are recognized, how they are retro-translocated or dislocated from the ER, and how chaperones might direct these processes. Furthermore, substrate ubiquitination clearly plays a key role in ERAD, however its specific role in the detection of substrates in the ER lumen and their dislocation to the cytosol are unknown. In this grant proposal we have the unique ability to use the mK3 immune evasion protein to define how proteins implicated in the quality control of class I MHC expression elicit ERAD of misassemble MHC-I proteins. These investigations will also define the precise mechanism of substrate ubiquitination and its role in substrate dislocation and the kinetics of degradation.