Much interest exists in understanding the replication of vaccinia virus, as it represents the model system to understand poxviruses that include variola virus, the causative agent of smallpox. A key phase of viral replication involves the acquisition of membranes, as this event transforms non-infectious assembling viral cores into infectious virions. Accumulated evidence suggests that the virus acquires two set of membranes from organellar compartments of the host. The inner set of membranes is derived from the Endoplasmic Reticulum Golgi Intermediate Compartment (ERGIC) to form an intracellular mature virus (IMV), while the outer set of membranes is subsequently acquired from the trans-Golgi network to form an intracellular envelope virus. Viral proteins that participate in membrane wrapping are beginning to be identified, but host proteins that would also be predicted to play a key role during this process, as the membranes are derived from host organelles, have remained unknown. In preliminary studies, we have found that vaccinia virus specifically concentrates a host protein, a subunit of the COPI coat complex, on its inner set of membranes. Thus, we propose to elucidate how the COPI coat complex might play a role in viral biogenesis. First, we will determine the stage of viral biogenesis when COPI is first detected on viral membranes. Second, we will determine which viral stage might be arrested when COPI function is abrogated. Third, we will determine whether the virus usurps any of the known host regulatory proteins to recruit COPI onto viral membranes. Fourth, we will test whether any of the current known viral proteins that regulate viral morphogenesis affects COPI recruitment onto viral membranes. These sequential approaches also suggest a systematic way of identifying potential other host proteins that would be predicted to affect IMV formation, as it is the first stage that the virus becomes infectious. Thus, we will screen the currently known transport regulators that function at the ERGIC compartment to test whether any is similarly enriched on viral membranes. Subsequently, the function of candidate host proteins will be abrogated to determine whether viral biogenesis is affected. These efforts will likely not only provide a better understanding of how the virus interacts with its host during replication, but also suggest novel biochemical targets in the future rational design of therapeutic intervention against poxviruses.