In situ cross-linking of vesicular stomatitis virus proteins with reversible agents

Abstract

Several reversible cross-linking agents were tested for their ability to cross-link the proteins of intact vesicular stomatitis virus (VSV). Results were analyzed by two-dimensional acrylamide gel electrophoresis in the sodium dodecyl sulfate-discontinuous buffer system. Formaldehyde cross-linking was found to be reversible by heat, and gave results similar to the reagent dimethyl-3,3′-dithio-bisproprionimidate (DTBP), cleavable by reduction of its internal disulfide bond. These two reagents caused the formation of cross-linked polypeptides with molecular weights consistent with the species N:M, G:M, and G:N, where N, M, and G stand for the VSV nucleoprotein, membrane protein, and glycoprotein, respectively. Other agents tested included o-phenanthroline in the presence of Cu 2+ and O 2, and the heavy metal ions Cu 2+ and Ni 2+, all reversible by β-mercaptoethanol. These agents gave similar results: No cross-linking of G proteins was observed, and of the possible heterodimers, only N:M was found. NS, the phosphopolypeptide of VSV, was found in complexes corresponding in molecular weight to homodimer and homotetramer species, possibly indicating a tetrameric configuration of the native NS protein. The apparent molecular weights of cross-linked polypeptides were found to be about 20% greater than for linear polypeptides of the same molecular weight, when the mobility in acrylamide gels of a number of cross-linked species from native proteins, enzymes, and viral nucleocapsids was compared to the mobility of linear polypeptides. The accessibility of sulfhydryl groups in native and SDS-disupted VSV was assayed by reaction with radioactive iodoacetic acid; it was found that the G protein contained no free sulfhydryl groups, explaining its failure to react with o-phenanthroline/Cu 2+ and heavy metal ions. Removal of the G proteins from the virus with nonionic detergent prior to cross-linking with DTBP showed the coordinate loss of both the G and the N spots corresponding in apparent molecular weight to the G:N heterodimer. This result provides evidence that the glycoprotein of VSV may be capable of interacting in some manner with the core nucleoprotein.