Spatial relationships of the proteins of vesicular stomatitis virus: induction of reversible oligomers by cleavable protein cross-linkers and oxidation

Abstract

To delineate the proximity and spatial arrangement of the major structural proteins of intact vesicular stomatitis (VS) virions, protein complexes formed by oxidation or by bivalent cross-linkers were analyzed by 2-dimensional electrophoresis on polyacrylamide slab gels. H2O2 oxidation of VS virions produced an N-polypeptide dimer (MW .simeq. 110,000) on a 1st-dimension gel that could be reduced to N monomers (MW .simeq. 50,000). Proteins extracted from unreduced and unoxidized VS virions contained dimeric and trimeric forms of M-protein complexes and a heterodimer of M and N protein. Qualitatively similar VS viral protein complexes were generated by exposing VS virions to the reversible protein cross-linkers methyl-4-mercaptobutyrimidate (MMB), tartryl diazide (TDA) and dithiobis(succinimidyl proprionate) (DTBSP); cross-linked complexes on 1st-dimension gels were cleaved by reduction with 2-mercaptoethanol (MMB or DTBSP cross-linked) or by periodate oxidation (TDA cross-linked). Besides covalently linked homodimers of M and N proteins and a protein M-N heterodimer, the protein cross-linkers also generated homo-oligomers of G protein and a G-M heterodimer. The glycoprotein spike of VS virus is composed of > 1 G protein. The existence of N-M and G-M heterodimers indicates that the matrix (M) protein may serve as a bridge between the G and N proteins in assembly of the VS virion.