Plasma Membrane Microdomains Containing Vesicular Stomatitis Virus M Protein Are Separate from Microdomains Containing G Protein and Nucleocapsids

Abstract

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.

Investigation of the Vesicular Stomatitis Virus Matrix Protein: Uncoating and Assembly

“…and then M (membrane bound green circles) is incorporated to these microdomains (IV.) as recently shown (195). The role for P, in particular residues 191 to 200, in assembly has been reported recently and it may be involved in steps III.…”

“…It has been shown that cytosolic and membrane bound M can bind to RNPs but the proteins have a higher affinity for RNPs isolated from virus versus cytosolic RNPs (57). These observations suggested that M binding to RNPs is dependent on a change in cytosolic RNPs at the plasma membrane and recent evidence has led to a model where G and RNPs associate in microdomains at budding sites and then M is incorporated (195). Treating purified VSV virions with detergent at low ionic strength solubilizes the viral membrane and G protein liberating the highly condensed, tightly coiled skeleton which consists of the RNP and M protein (153,154).…”

“…and the efficient condensation of RNPs at the plasma membrane ( Fig. 5-2, V.) as evidenced by the build up of N protein, hyper-transcription of viral mRNA in rVSV-D125A infected cells and the increased ability of the RNPs from virions to exchange with introduced M. This model also includes the most recent model for how budding sites are formed, where RNPs and G initially associate at microdomains before incorporation of M at these sites (195) (Fig. 5-2, III.).…”

“…The plasma membrane is where M and RNPs interact with each other, suggesting that condensation of RNPs does not begin until they associate with the membranes right before budding (146,157). Also, it has been proposed that an additional priming event is needed in order to initiate the assembly process and may involve specific M nucleation sites (67) or modification of the RNPs by viral or host factors (57,130,195).…”

“…This observation suggests the interaction between the cytoplasmic tails of each virion's G protein with their respective skeleton is different between these two viruses found in Rhabdoviridae. Recently, it has been shown that G and N may interact and this interaction allows for the RNP to associate with the M protein at the budding sites (195).…”