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 part icle 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 recombinan t 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 expr essed on the cell surface, and was incorporated into virus particles with a lmost the same efficiency as wild-type VSV G protein. These results indicat e 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 m icroscopy and had a normal morphology by electron microscopy. To determine whether Virus assembly could occur efficiently when all G proteins containe d the GFP cytoplasmic domain, a VSV recombinant in which the G gene was com pletely replaced by the VSV-G/GFP gene was recovered. This virus rapidly lo st expression of the GFP protein sequence through introduction of a stop co don within the sequence encoding the G cytoplasmic domain, indicating stron g selection against homotrimeric G protein bearing such a large cytoplasmic domain. (C) 2001 Academic Press.