Aromatic and polar residues spanning the candidate fusion peptide of the Andes virus Gc protein are essential for membrane fusion and infection.
Hantaviruses infect human cells through cell attachment and subsequent fusion of viral and cellular membranes at low pH. This largely unknown entry process is mediated by the Gn and Gc glycoproteins, anchored at the viral envelope membrane. Performing bioinformatic analysis and peptide-liposome-binding assays we suggested in a former report that Gc of Andes virus (ANDV) and other hantaviruses corresponds to the viral fusion protein sharing characteristics with class II fusion proteins. To gain insights into the fusion protein of hantaviruses, residues within the previously predicted fusion peptide of ANDV Gc were substituted and mutant proteins tested in fusion and infection assays. To ensure proper folding of mutant proteins, they were first characterized for trafficking to the plasma membrane and incorporation on to ANDV Gn/Gc-pseudotyped lentiviral particles. Cell attachment of these particles was assessed using a newly developed binding assay and their subsequent entry properties determined by FACS analysis of transduced cells expressing the GFP reporter gene. Furthermore, a three-colour-based cell-cell fusion assay of ANDV Gn/Gc expressing cells was performed. The results indicate an essential role of conserved Gc residues W115 and N118 in membrane fusion. Conversely, substitutions of the non-conserved Gc residue G116 did not considerably affect fusion and infection. Altogether, the findings are fully consistent with our earlier prediction suggesting Gc residues 115-121 as an internal fusion peptide and further emphasize the importance of aromatic and polar residues in hantavirus-cell membrane fusion.
Fundación Ciencia para la Vida, Santiago, Chile.
Nicolás
N
Cifuentes-Muñoz
Gonzalo P
GP
Barriga
Pablo D T
PD
Valenzuela
Nicole D
ND
Tischler
D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D002522:Chlorocebus aethiops; D005434:Flow Cytometry / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006476:Orthohantavirus / Q000472:pathogenicity*; D006801:Humans; D008561:Membrane Fusion; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D013194:Staining and Labeling / Q000379:methods; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D053586:Virus Internalization
D000596:Amino Acids; D050505:Mutant Proteins; D014760:Viral Fusion Proteins; D049452:Green Fluorescent Proteins