In vivo processing and isolation of furin protease-sensitive alphavirus glycoproteins: a new technique for producing mutations in virus assembly.
Sindbis virus particles are composed of three structural proteins (Capsid/E2/E1). In the mature virion the E1 glycoprotein is organized in a highly constrained, energy-rich conformation. It is 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 with detergent results in disulfide-bridge rearrangement and the collapse of the protein to a number of low-energy, non-native configurations. In a new approach to the production of membrane-free 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 during transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane. Processing of the proteins also impacts the envelopment of the nucleocapsid in the modified plasma membrane. 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.
Nelson
Steevenson
S
Department of Molecular and Structural Biochemistry, North Carolina State University, Campus Box 7622, Raleigh NC 27695-7622, USA.
Hernandez
Raquel
R
Ferreira
Davis
D
Brown
Dennis T
DT
D000818:Animals; D036022:Capsid Proteins / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D045683:Furin / Q000494:pharmacology*; D007668:Kidney; D016297:Mutagenesis, Site-Directed; D009153:Mutagens; D012845:Sindbis Virus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics
D036022:Capsid Proteins; D009153:Mutagens; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; C064950:glycoprotein E2, Sindbis virus; D045683:Furin; D005033:Ethylmaleimide; D004229:Dithiothreitol