Vesicular Stomatitis Virus Glycoprotein Containing the Entire Green Fluorescent Protein on Its Cytoplasmic Domain Is Incorporated Efficiently Into Virus Particles

Virology. 2001 Jan 20;279(2):414-21. doi: 10.1006/viro.2000.0736.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Electrophoresis, Polyacrylamide Gel
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism*
  • Green Fluorescent Proteins
  • Immunoblotting
  • Luminescent Proteins / metabolism*
  • Microscopy, Electron
  • Microscopy, Fluorescence
  • Precipitin Tests
  • Vesicular stomatitis Indiana virus / genetics
  • Vesicular stomatitis Indiana virus / metabolism*
  • Virus Assembly

Substances

  • Luminescent Proteins
  • Green Fluorescent Proteins
  • GTP-Binding Proteins