Characterization of Furin Protease Sensitive Site Processing and Its Effects on Sindbis Virus Assembly and Budding

Abstract

Sindbis virus particles are composed of three structural proteins (C/E2/E1). The E1 glycoprotein is organized into a highly constrained, energy-rich conformation. Its hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from virus membranes results in disulfide-bridge rearrangement and the collapse of the protein to a low-energy, non-native configuration. In a new approach to the production of membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin sensitive sites undergo normal folding and assembly in the endoplasmic reticulum and only experience the consequence of the mutation after transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane from which they are assembled. This processing also impacts the envelopment of the nucleocapsid in the modified plasma membrane. E2 has been shown to be responsible for host receptor recognition and thus plays a critical role in the virus lifecycle. To expand on our previously characterize E1 furin mutant study, we installed furin protease recognition motifs at various positions in the ectodomain of E2. Mutants were analyzed for production of truncated proteins and the effect of the mutations on virus assembly and budding was also characterized. Processing of the E2 mutants by the enzyme furin results in the release of the truncated proteins from the membrane in a fashion that is similar to the processing observed in the E1 furin sensitive mutants. This processing was also observed to impact envelopment of the nucleocapsid with virus protein modified plasma membrane at a step consistent with an early event in the envelopment process. Overall, this technique provides a unique method for studying the mechanism of virus assembly and protein structure without altering crucial early events in protein assembly, folding and maturation.