This proposal is to characterize the endomembrane trafficking between transitional endoplasmic reticulum and cis Golgi apparatus in lentivirus coat protein processing as a strategy for target identification in antiviral drug design using cultured cells infected with feline immunodeficiency virus (FIV). FIV is a lentivirus that causes lymphadenopathy and increased susceptibility to opportunistic infection in domestic cats. Like its human counterpart, the disease culminates eventually in the death of the infected animals. The virus shares many structural features with HIV. As such, feline cells infected with FIV provide a readily manipulated model for cell biological studies of endomembrane trafficking of envelope proteins of lentiviruses. Endomembrane trafficking of virus envelope glycoproteins is a complex multi-step process involving membrane budding, translocation, targeting and fusion. It is a major, though largely unexploited, target for antiviral drug therapy. In the work proposed, principal focus will be on the formation, migration and fusion of the 50-70 nm vesicles, termed transition vesicles, that function in the transfer of envelope proteins from sites of synthesis in endoplasmic reticulum to sites of processing in the Golgi apparatus. Our laboratory has recently reconstituted these events in a completely cell-free system and has developed procedures to isolate and concentrate the vesicle intermediate. These developments, plus the introduction of permeabilized cells by other groups, allow unprecedented opportunities to identify and characterize functional and regulatory molecules in the transport process. Using infected Crandall feline kidney cells, we will compare FIV transport between endoplasmic reticulum in situ and in the cell-free systems. Donor transitional endoplasmic reticulum, acceptor Golgi apparatus and the transition vesicle intermediate will be isolated, characterized and utilized in studies to identify and characterize specific molecules and steps in the transport process. One example will involve conjugated retinoids targeted to a ca. 27 kD GTP-binding-protein. The hypothesis to be tested is that certain of these proteins such as the 27 kD GTP-binding protein have potential as drug targets for management of lentivirus infections.
