Heterogeneity of vesicular stomatitis virus particles: implications for virion assembly

This study demonstrates that the glycoprotein of vesicular stomatitis virus clusters in the plasma membrane of infected Chinese hamster lung cells during morphogenesis and suggests that viral nucleocapsids are required for this clustering. A mutant virus (ts E-1) which is temperature sensitive for the synthesis of viral nucleocapsids but not viral membrane proteins was used. The surface distribution of the viral glycoprotein in cells infected by this virus was determined by a specific indirect immunoferritin stain. Early in infection at permissive temperatures, the glycoprotein was randomly distributed on membrane ghosts. Later, clusters of ferritin the size and shape of virus particles were seen. In contrast, ghosts prepared from virus-infected cells maintained at a restrictive temperature always had a random distribution of viral glycoprotein.Integral membrane proteins span the membranes with which they are associated and may participate in significant molecular interactions on both sides of the membrane. In many cases, these proteins are freely diffusible in the plane of the membrane (37). An increasing number of membrane proteins, however, are found to be distributed over the surface of the cell in a nonrandom fashion. For example, Ash et al. (1) have shown that several antibody-patched integral membrane proteins are arranged in linear arrays on the surface of cultured fibroblasts, specific receptor molecules are frequently localized in defined regions of the membrane, and viral glycoproteins of lipid-containing viruses are localized in regions of the membrane active in virus assembly (8). The molecular interactions which lead to the nonrandom distribution of integral proteins are currently under active study in a number of laboratories. Evidence is accumulating which indicates that the linear arrays of membrane proteins visible on the surface of fibroblasts result from interactions of domains of these proteins present on the cytoplasmic surface with elements of the cytoskeleton (1, 14,22). In the case of the best-studied receptor system, that of the low-density lipoproteins, the molecules appear to be inserted into the membrane at random sites and then to migrate laterally in the plane of the membrane until they reach a "coated pit." A domain of the receptor presumed to be on the cytoplasmic side t Present address:

Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Assembly of vesicular stomatitis virus: distribution of the glycoprotein on the surface of infected cells

Jacobs¹,

Penhoet²

1982

“…It is clear from numerous studies that M protein is essential for assembly of VSV virions (26,27,49). Maturation of the condensed RNP-M complex at the infected cell membrane can take place in the absence of G protein, suggesting that interaction of M protein with the cell membrane is sufficient to promote envelopment and budding of the VSV virion.…”

Section: Discussionmentioning

confidence: 99%

Mapping regions of the matrix protein of vesicular stomatitis virus which bind to ribonucleocapsids, liposomes, and monoclonal antibodies

Ogden¹,

Pal²,

Wagner³

1986

The matrix (M) protein of vesicular stomatitis virus (VSV) appears to function as a bridge between the ribonucleocapsid (RNP) core and the envelope in assembly of the virion. Two such properties would necessitate at least one site for interaction with the nucleocapsid and one with the envelope. In this study M protein was found to mediate the in vitro binding to RNP cores of phospholipid vesicles, representing membrane structures. The M protein could bind initially to either the vesicles or the RNP cores to promote RNP-vesicle association. A trypsin-resistant fragment (MT) of M protein, missing the initial 43 amino acids from its amino terminus, reconstituted with acidic phospholipid vesicles with the same binding efficiency as did whole M protein, suggesting that the carboxy-terminal 81% retained those regions of the M protein which interact with a lipid bilayer. The MT protein, however, was considerably less efficient than intact M protein as an inhibitor of in vitro virus transcription; almost 2.5-fold more MT protein than intact M protein was required for 50% inhibition of VSV transcription, indicating that a site for interaction with the RNP core may have been lost. A monoclonal antibody which is able to reverse the in vitro inhibition of transcription by M protein did not react by immunoblotting with MT protein. Partial tryptic digests of the M protein probed with this monoclonal antibody indicated that epitope 1 lies between amino acid residues 18 and 43. This region appears to be a site that promotes interaction of the M protein with the RNP core of VSV. Monoclonal antibodies to epitopes 2 and 3, which exhibit some overlap in binding to M protein but do not reverse transcription inhibition, were mapped by cleavage with N-chlorosuccinimide at regions in a carboxy direction from epitope 1. * Corresponding author. t Present address: Biotherapeutics Incorporated, Franklin, TN 37064.G, and M) take independent paths to the site of virus assembly at the plasma membrane of the infected cell (1,2,6,16,22,23,34); synthesis of M protein is the rate-limiting step in this maturation process (53). Removal of M protein from the RNP core yields a relaxed, highly extended nucleocapsid structure, while condensation of the RNP into a tightly coiled skeleton is mediated through the association of M protein with the nucleocapsid (20,37,38). Appearance of M protein at the cell membrane causes decreased mobility of the already-inserted G protein (46). Protein cross-linking studies with intact virions revealed the formation of G-M and M-N but not G-N heterodimers (18). These studies suggest that M protein functions as a bridge between the cytoplasmic RNP structures and those regions of the cellular membrane into which the G protein has been inserted.The strong interaction between M protein and the RNP core enables M protein to act as an endogenous inhibitor of viral transcription. Transcription reactions with detergentdisrupted whole virions demonstrate a virus-concentrationdependent regulatory effect which was attributed to an ...

“…Nucleocapsids appear to be necessary for the production of authentic bacilliform particles; the formation of abnormal particles which lack a nucleocapsid is very rare in wild-type virus infections but does occur with certain temperature-sensitive viral mutants (43). The viral envelope contains a variable amount of the transmembrane protein G (29). In differentiated epithelial cells, G protein is transported predominantly to the basolateral plasma membrane, and VSV buds almost exclusively from this membrane domain (38,39).…”

mentioning

confidence: 99%

Stereo images of vesicular stomatitis virus assembly

Odenwald¹,

Arnheiter²,

Dubois‐Dalcq³

et al. 1986

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.