Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junín arenavirus envelope glycoprotein.
The G2 fusion subunit of the Junín virus envelope glycoprotein GP-C contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of Class I viral fusion proteins. We have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. All the mutant glycoproteins were expressed and transported to the cell surface. Proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (SKI-1/S1P) cleavage site was observed in all but two of the mutants. Among the adequately cleaved mutant glycoproteins, four positions in the N-terminal region (I333, L336, L347 and L350) and two positions in the C-terminal region (R392 and W395) were shown to be important determinants of cell-cell fusion. Taken together, our results indicate that alpha-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. These findings support the inclusion of the arenavirus GP-C among the Class I viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever.
York
Joanne
J
Montana Biotechnology Center, The University of Montana, Science Complex Room 221, Missoula, MT 59812, USA. joanne.york@umontana.edu
Agnihothram
Sudhakar S
SS
Romanowski
Victor
V
Nunberg
Jack H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018051:Junin virus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D008561:Membrane Fusion; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics*; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics*
D021122:Protein Subunits; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins