Rhabdoviridae are membrane-enveloped viruses that are widely distributed in nature where they infect vertebrates, invertebrates, and plants. The Rhabdoviridae are divided into 6 genera, wherein Vesicular stomatitis virus (VSV) serves as a prototype for the vesiculovirus genus. Rhabdoviridae possess non-segmented, negative-strand RNA genomes of 11-12,000 nucleotides. The virus particles contain a helical, nucleocapsid core composed of the genomic RNA and protein. Generally, three proteins, termed N (nucleocapsid, which encases the genome tightly), P (formerly termed NS, originally indicating nonstructural), and L (large) are found to be associated with the nucleocapsid. An additional matrix (M) protein lies within the membrane envelope, perhaps interacting both with the membrane and the nucleocapsid core. A single glycoprotein (G) species spans the membrane and forms the spikes on the surface of the virus particle. The G protein is responsible for binding to host cells and facilitating membrane fusion. Because the Rhabdoviral genome is negative sense [i.e., complementary to host cell RNA sequences (positive sense) that comprise host mRNA], for transcription and translation of Rhabdoviral genes to occur, the virus must encode and package an RNA-dependent RNA polymerase in its virion. The RNA-dependant RNA polymerase is composed of the P and L proteins, which transcribes genomic RNA to subgenomic mRNA's encoding the 5-6 Rhabdoviral proteins and also replicates full-length positive and negative sense RNAs. Rhabdoviral genes are transcribed sequentially, starting at the 3′ end of the genome.
Due to its broad host range, simple genetic organization and rapid growth in cell culture, VSV has been used widely to study various aspects of Rhabdoviral entry, assembly and release. The virus enters cells via receptor-mediated endocytosis. Approximately 1200 VSV glycoprotein (G) molecules, organized as homotrimeric spikes anchored in the viral envelope, are responsible for virus attachment as well as for mediating fusion of the viral envelope with the endosomal membrane of the host cell following endocytosis. Low endosomal pH causes a conformational change in the G protein facilitating fusion of the viral envelope with the endosomal membrane (9, 2, 26). Fusion of the two membranes results in the release of the viral nucleocapsid into the host cell cytoplasm where viral replication then occurs.
VSV G protein differs from the prototypic viral fusion protein, influenza hemagglutinin (HA), in that G protein does not require proteolytic processing to become fusion-competent (33, 17). Also unlike influenza HA, the N-terminus of G, apart from the signal sequence, is not particularly hydrophobic and there is no obvious region in the amino acid sequence that can be defined as a “classical” fusion peptide (51). It was postulated that the VSV G fusion peptide is internal and that the region between amino acids (aa) 118-139 could be the putative fusion domain (33). Mutational analysis has provided evidence that the region between aa 118-136 corresponds to the G protein fusion peptide (14, 25, 53, 55).
Other regions of G protein have also been shown to be important for its fusion activity. Insertion of a 3 aa linker in the membrane-proximal domain at positions 410 and 415 abolished membrane fusion activity, indicating that this region may be important for fusion (25). Substitution of amino acids in the region between 395 and 418 also affected the fusogenic activity of G protein (47). When mutations in the fusion peptide were combined with point mutations in the membrane-proximal region between amino acids 395-418 fusion activity was inhibited additively. However, one double mutant G131A-G404A was more fusogenic than the two individual mutations alone, suggesting that these regions may interact during fusion (46).
The membrane proximal region of the VSV G protein is highly conserved among vesiculovirus members (41). Structure predictions for the region between aa 385 and 444 of the VSV GIND serotype glycoprotein have indicated that this region has a propensity to form α-helices suggestive of its direct interaction with cellular membranes (16). Recently the Applicants have demonstrated that the membrane proximal cctodomain of the “stem” region of the VSV G protein (G-stem or GS) is responsible for efficient VSV budding (41), a finding which may perhaps be technically exploited for the development of the next generation of recombinant VSV yectors. There is, therefore, an advantage in developing recombinant VSV vectors containing the membrane proximal “stem” region of the VSV G protein ectodomain as these may provide accelerated fusion activity, a result providing a multiple of biologically relevant applications.
USPTO Backgrounds
USPTO Backgrounds