Vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles.
The envelope glycoprotein (G) of vesicular stomatitis virus (VSV) contains a short cytoplasmic domain of 29 amino acids. To determine whether VSV particle assembly could accommodate a G protein with a large cytoplasmic domain, we constructed a gene called G/GFP encoding the VSV G protein with the 27-kDa green fluorescent protein linked to its cytoplasmic domain. This gene was inserted into the infectious clone of VSV and we recovered a recombinant virus expressing G/GFP from this extra gene. This VSV-G/GFP virus grew to titers equivalent to that of wild-type virus and was stable upon passaging. The G/GFP protein formed mixed trimers containing an average of two wild-type G proteins and one G/GFP protein. This heterotrimeric protein was expressed on the cell surface, and was incorporated into virus particles with almost the same efficiency as wild-type VSV G protein. These results indicate that there is substantial space available between the viral membrane and the nucleocapsid that can accommodate such a large cytoplasmic domain. The green fluorescent virus particles were readily visualized by fluorescence microscopy and had a normal morphology by electron microscopy. To determine whether virus assembly could occur efficiently when all G proteins contained the GFP cytoplasmic domain, a VSV recombinant in which the G gene was completely replaced by the VSV-G/GFP gene was recovered. This virus rapidly lost expression of the GFP protein sequence through introduction of a stop codon within the sequence encoding the G cytoplasmic domain, indicating strong selection against homotrimeric G protein bearing such a large cytoplasmic domain.
Dalton
K P
KP
Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, Connecticut 06510-3218, USA.
Rose
J K
JK
D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D011233:Precipitin Tests; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins