Stereo images of vesicular stomatitis virus assembly

Abstract

Viral assembly was studied by viewing platinum replicas of cytoplasmic and outer plasma membrane surfaces of baby hamster kidney cells infected with vesicular stomatitis virus. Replicas of the cytoplasmic surface of the basilar plasma membrane' revealed nucleocapsids forming bullet-shaped tight helical coils. The apex of each viral nose cone was anchored to the memtrane and was free of uncoiled nucleocapsid, whereas tortuous nucleocapsid was attached to the base of tightly coiled structures. Using immunoelectron microscopy, we identified the nucleocapsid (N) viral protein as a component of both the tight-coil and tortuous nucleocapsids, whereas the matrix (M) protein was found only on tortuous nucleocapsids. The M protein was not found on the membrane. Using immunoreagents specific for the viral glycoprotein (G protein), we found that the amount of G protein per virion varied. The G protein was consistently localized at the apex of viral buds, whereas the density of G protein on the shaft was equivalent to that in the surrounding membrane. These observations suggest that G-protein interaction with the nucleocapsid, via its cytoplasmic domain may be necessary for the initiation of viral assembly. Once contact is established, nucleocapsid coiling proceeds with nose cone formation followed by formation of the helical cylinder. M protein may function to induce a nucleocapsid conformation favorable for coiling or may cross-link adjacent turns in the tight coil or both.

Electron Tomography Reveals the Steps in Filovirus Budding

“…For example, budding VSV, a rhabdovirus, can be found protruding perpendicular to cell membranes in infected cells, but the NC can also be seen underneath the cell membrane, with which it associates laterally along one side [35]. The rounded end of the virus, easily distinguished in VSV, seems to associate more tightly with the membrane [43], and buds first. After budding, under certain preparation conditions, the rear of the virus is seen to show small membrane blebs or instabilities [41] similar in appearance to those described here for MARV.…”

The M protein of vesicular stomatitis virus associates specifically with the basolateral membranes of polarized epithelial cells independently of the G protein.

“…The mechanism of this targeting remains unknown. Perhaps the most appealing hypothesis had been that the M protein does not interact directly with membranes, but simply binds to the cytoplasmic domain of the G protein at the target membrane (42) or that it binds indirectly to the G protein via nucleocapsids (41). Support for these models came from cross-linking studies in which the M protein could be specifically cross-linked to the G protein in virions, thus demonstrating a specific interaction between M and G (15,40).…”

Function of Membrane Rafts in Viral Lifecycles and Host Cellular Response

“…VSV budding is known to occur at membrane microdomains containing the viral envelope G glycoproteins, some of which are 100-150 nm in size and smaller than the virus envelope (approximately 100-150 nm) and others of which extended in size to a maximum of 300-400 nm from the tip of the virus budding site [203,204]. However, immunoelectron microscopy observation did not confirm that gold-labeled G protein-containing microdomains are equivalent to lipid-enriched membrane rafts.…”