Surface Structure of Vesicular Stomatitis Virus

134

We have determined the COOH-terminal and NHa-terminal amino acid sequences of the vesicular stomatitis virus (VSV) glycoprotein (G) A sequence of 122 COOH-terminal amino acids was deduced from the complete sequence of a cloned DNA insert carrying 470 nucleotides derived from the 3' end of the G mRNA. Evidence presented indicates that this portion of the poyptide includes the domains of G that reside inside the virion an span the lipid bilayer of the virion. This seems clear because a partial amino acid sequence of a fragment of G that remains associated with the membrane of the virion after exhaustive proteolytic digestions can be located unambiguously in the predicted sequence. This predicted sequence contains an uninterrupted hydrophobic domain beginning 49 amino acids and ending 30 amino acids from the COOH terminus. This region presumably spans the lipid bilayer. The COOH-terminal portion of 29 amino acids contains a high proportion of basic residues and resides inside the virion. The COOH-terminal portion of the VSV G protein therefore resembles in structure that of glycophorin, an erythrocyte membrane protein well characterized previously. Enveloped viruses, such as vesicular stomatitis virus (VSV) serve as simple and useful model systems for the study of the structure, biosynthesis, and function of membrane proteins. VSV virions contain a single glycoprotein, G, which forms spikes on the surface of the virion. This 70,000-dalton glycoprotein contains two asparagine-linked complex oligosaccharides (1, 2) and is positioned in the virion such that almost 90% of the polypeptide chain is external to the lipid bilayer (3,4). G plays two roles in the life cycle of the virus. First, it is responsible both for the binding of the virus to susceptible host cells and for inducing the uptake of the virus by the cell (5, 6). Second, during virus maturation the interaction between the internal components of the virion and the portion of G exposed on the cytoplasmic face of the plasma membrane probably directs envelopment and virus budding.The G protein is inserted into the rough endoplasmic reticulum (RER) as a nascent polypeptide chain (7,8). Both ends of the molecule appear to have critical roles. Like the majority of the secreted and membrane proteins, the nascent G polypeptide has a short hydrophobic NH2-terminal signal or leader peptide (9-12), which appears to initiate association of the nascent polypeptide-mRNA-ribosome complex with the membrane of the endoplasmic reticulum and which is removed prior to chain termination (13). Unlike secreted proteins, however, membrane proteins such as G are not discharged completely across the microsomal membrane and instead become anchored stably in the membrane. Preliminary studies have suggested that G is bound to microsomal membranes at a site very near to its COOH terminus (12,14). Hence, this region of G must differ from the COOH termini of secreted proteins in some fundamental but as yet not understood way so as to halt extrusion into the lumen of the RER.A know...

Section: Resultssupporting

confidence: 86%

mentioning

confidence: 99%

Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus

Rose

Welch

Sefton

et al. 1980

134

“…virus to the host cell and therefore is essential for infectivity (2,3). Antibodies directed against G-protein have been shown to neutralize virus infectivity (4).…”

mentioning

confidence: 99%

“…Spikes on the surface of the VSV virion are composed of a single species of glycoprotein (G-protein). G-protein functions in the binding of virus to the host cell and therefore is essential for infectivity (2,3). Antibodies directed against G-protein have been shown to neutralize virus infectivity (4).…”

mentioning

confidence: 99%

Conditional expression of the vesicular stomatitis virus glycoprotein gene in Escherichia coli.

Rose¹,

Shafferman²

1981

Bacterial plasmids that directed expression ofthe vesicular stomatitis virus glycoprotein (G-protein) gene under control ofthe tryptophan operon regulatory region were constructed. A plasmid directing the synthesis of a G-protein-like protein (containing the NH2-terminal segment of seven amino acids encoded by the trpE gene fused to the complete G-protein sequence lackdng only its NH2-terminal methionine) could be transformed into trpR+ (repressed) but not into trpR-(derepressed) cells. This result suggested initially that derepressed synthesis ofthe G-proteinlike protein encoded by this plasmid was lethal in Echerichia coi. Deletion ofthe sequence encoding the large hydrophobic segment near the COOH terminus of G-protein did not overcome this lethality. Lethality of derepressed synthesis was overcome by deletion of the G-protein gene region encoding 10 amino acids in the hydrophobic NH2-terminal domain (signal peptide). Tryptic peptide mapping demonstrated that the G-protein-like protein and some truncated proteins encoded by the plasmid contained G-protein protein sequences. Antisera to vesicular stomatitis virus precipitated the G-protein-like protein, showing that it shares antigenic determinants with the authentic G-protein protein.Vesicular stomatitis virus (VSV), an enveloped animal virus, is the prototype of the rhabdovirus family (1). Although VSV causes a relatively mild disease of cattle, other members of the family such as rabies virus cause serious disease. Spikes on the surface of the VSV virion are composed of a single species of glycoprotein (G-protein). G-protein functions in the binding of virus to the host cell and therefore is essential for infectivity (2,3). Antibodies directed against G-protein have been shown to neutralize virus infectivity (4). In the VSV virion, G-protein is anchored in the lipid envelope by a 20-amino acid hydrophobic transmembrane domain and a basic domain near the COOH terminus (5), leaving the NH2-terminal 95% of G-protein exposed on the virion surface. Two complex oligosaccharides are apparently linked to asparagines at amino acid residues 178 and 335 (6-8).As part of a determination of the nucleotide sequence of the VSV genome, we have isolated and determined the sequence of a cDNA clone containing the entire sequence coding for the VSV G-protein. This nucleotide sequence predicts a protein sequence of 511 amino acids, including a signal peptide of 16 amino acids that is not present on the mature protein (6).Expression ofeukaryotic viral glycoprotein sequences in bacteria may allow synthesis oflarge quantities ofviral antigens that could be useful as vaccines. VSV should prove to be an especially useful model system for such expression studies because: (i) large amounts of viral glycoprotein can be prepared from virions, and its immunogenicity can be compared with that of the protein produced in bacteria; (ii) antibody neutralization of virus infectivity can be examined in a rapid plaque assay; and (iii) immunity to lethal VSV infection can be assayed rea...

“…(i) The G protein (envelope glycoprotein) constitutes the basic unit of surface projections of the virus particles emerging from the lipid-coated membrane protein(s) and is the antigen capable of binding virus neutralizing antibodies and of eliciting their formation (8)(9)(10). This surface antigen is also responsible for agglutination of erythrocytes by certain rhabdoviruses (11) and for the adsorption of the virus particles onto the surface of the host cells.…”

mentioning

confidence: 99%

Structure-function relationships and mode of replication of animal rhabdoviruses.

Sokol¹,

Koprowski²

1975