Taxonomic Structure of the Family
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Rhabdoviridae |
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Virions are 100-430 nm long and 45-100 nm in diameter. Defective virus particles are proportionately shorter. Viruses infecting vertebrates are bullet-shaped or cone-shaped; viruses infecting plants mostly appear bacilliform when fixed prior to negative staining; in unfixed preparations they may appear bullet-shaped or pleomorphic. Some putative plant rhabdoviruses lack envelopes. The outer surface of virions (except for the quasiplanar end of bullet-shaped viruses) is covered with projections (peplomers) 5-10 nm long and about 3 nm in diameter. They consist of trimers of the viral glycoprotein (G). A honeycomb pattern of peplomers is observed on the surface of some viruses. Internally, the nucleocapsid, about 30-70 nm in diameter, exhibits helical symmetry and can be seen as cross-striations (spacing 4.5-5 nm) in negatively stained and thin-sectioned virus particles. The nucleocapsid consists of an RNA and N protein complex together with L and P proteins. A lipid envelope containing the G glycoprotein interacts with nucleocapsids via the M protein. The nucleocapsid contains transcriptase activity and is infectious. Uncoiled it is filamentous, about 700 nm long and 20 nm in diameter (Fig. 1).
Physicochemical and Physical Properties
Virion Mr is 300-1,000 106 and S20w is 550-1,045S (plant rhabdoviruses have larger S20w values). Virion buoyant density in CsCl is 1.19-1.20 g/cm3; in sucrose it is 1.17-1.19 g/cm3. Virus infectivity is rapidly inactivated at 56°C, or following UV- or X-irradiation, or exposure to lipid solvents.
Viruses contain a single molecule of linear, negative-sense ssRNA (Mr 4.2-4.6 106, about 11-15 kb in size). The RNA represents about 1-2% of particle weight. The RNA has a 5-terminal triphosphate and is not polyadenylated. The ends have inverted complementary sequences. Defective RNAs, usually significantly shorter than full-length RNA (less than half size), may be identified in RNA recovered from virus populations. They are usually negative sense, however hairpin RNA forms are also found. Defectives only replicate in the presence of homologous and, occasionally, certain heterologous helper rhabdoviruses. They may contain functional genes. Full-length positive-strand RNA may constitute up to 5% of a viral RNA population.
Viruses generally have 5 structural polypeptides (designated L, G, N, P and M; see Table 1 for summary of their location, sizes and functions). The functions of other proteins (including additional glycoproteins) of certain rhabdoviruses are not known. The structural proteins represent 65-75% of the virus dry weight. For certain viruses, other nomenclature has previously been used for the P protein (NS, M1 or M2) and the M protein (M1 or M2). For Vesicular stomatitis Indiana virus (VSIV) the numbers of molecules per infectious virus particle is estimated as: L (20-50); G (500-1,500); N (1,000-2,000); P (100-300); and M (1,500-4,000). The enzymes identified in virions include the RNA transcriptase (L and P proteins), a 5 capping enzyme, guanyl and methyl transferases, a protein kinase (viral-, possibly host-coded), a nucleoside triphosphatase and a nucleoside diphosphate kinase. These activities may be functions of L.
Virions are composed of about 15-25% lipids; their composition reflecting the host cell membrane where virions bud. Generally phospholipids represent about 55-60%, and sterols and glycolipids about 35-40% of the total lipids. G protein has a covalently associated fatty acid proximal to the lipid envelope.
Virions are composed of about 3% carbohydrate by weight. The carbohydrates are present as N-linked glycan chains on G protein and as glycolipids. In mammalian cells, the oligosaccharide chains are generally of the complex type, in insect cells they are of the non-complex types.
Genome Organization and Replication
Viruses contain at least 5 ORFs in the negative-sense genome in the order 3-N-P-M-G-L-5 (e.g., for VSIV), or the equivalent. For certain viruses additional genes are interposed. Genes are transcribed processively (from the 3 to 5 of the template virus RNA and in decreasing molar abundances) as 5-capped, 3-polyadenylated and generally monocistronic mRNAs (Fig. 2). Polycistronic mRNAs have been identified for some species. A short uncapped, unpolyadenylated and untranslated “leader” RNA, corresponding to the complement of the 3-terminus of the viral RNA (i.e., preceding the N mRNA), is also transcribed. Unlike mRNA species, it has a 5 triphosphate terminus (Fig. 2). Leader RNA has been identified in the nucleus of infected cells. For individual viruses and for different viruses, the mRNAs generally have common 5-terminal sequences (generally m7Gppp(m)AmA(m)CA). Intergenic sequences are generally short but may be up to ~ 50 nts in some species. In certain cases the 5-end of an mRNA overlaps the 3-end of the preceding gene. The untranslated region following the L gene is longer than the sequence that precedes N at the other end of the genome.
Virus adsorption is mediated by G protein attachment to cell surface receptors and penetration of the cell is by endocytosis via coated pits. The identities of the receptors are not known. After penetration, the viral envelope is removed by lysosomal activity leading to deposition of the transcriptionally-active nucleocapsid (RNA, N, L, P) into the cytoplasm. Virus RNA is repetitively transcribed (primary transcription) by the virion transcriptase into capped and polyadenylated mRNAs that, apart from G mRNA, are translated in cytoplasmic polysomes. G mRNA translation occurs on membrane-bound polysomes. Transcription occurs in the presence of protein synthesis inhibitors indicating that it does not depend on de novo host protein synthesis. Following translation, RNA replication occurs in the cytoplasm (full-length positive and then full-length negative RNA synthesis) and depends on the prior translation of the viral mRNA species. Certain plant viruses may replicate RNA in the cell nucleus. Replication requires the newly synthesized N, P and L protein species and involves the formation of replicative intermediate nucleocapsids. It may require host factors. It has been proposed that binding of N protein to the 5 proximal (encapsidation) sequences of nascent positive or negative-sense viral RNA species prevents transcription and, by progressive addition of N, promotes replication, including read-through of transcription termination signals. Following replication, further rounds of transcription (secondary transcription), translation and replication ensue.
Post-translational trafficking and modification of G protein involves transportation across the membrane of the endoplasmic reticulum, removal of the amino-proximal signal sequence and step-wise glycosylation in compartments of the Golgi apparatus. Depending on the cell, the G protein may move to the plasma membrane, in particular, to the basolateral surfaces of polarized cells.
Viral nucleocapsid structures are assembled in association with M and lipid envelopes containing viral G protein. The site of formation of particles depends on the virus and host cell. For vesiculoviruses, lyssaviruses, ephemeroviruses and novirhabdoviruses, nucleocapsids are synthesized in the cytoplasm and viruses bud from the plasma membrane in most, but not all cells. Some lyssaviruses bud predominantly from intracytoplasmic membranes and in some cases prominent virus-specific cytoplasmic inclusion bodies containing N protein in infected cells (rabies inclusion bodies are called Negri bodies). Cytorhabdoviruses bud from intracytoplasmic membranes associated with viroplasms. None has been observed to bud from plasma membranes. Nucleorhabdoviruses bud from the inner nuclear membrane and accumulate in the perinuclear space.
Vesiculoviruses can replicate in enucleated cells, indicating that newly synthesized host gene products are not required. Depending on the virus and host cell type, virus infections may inhibit cellular macromolecular syntheses. The mechanism is not known.
Generally, 5 complementation groups of mutants have been defined by using temperature-sensitive mutants. Host range and temperature-sensitive mutants with altered polymerase functions have also been described. Complementation may occur between related viruses (e.g., between vesiculoviruses), but not between viruses representing distinct genera. Complementation is also reported to occur involving re-utilization of the structural components of UV-irradiated virus (VSIV). Recombination of genes between different virus isolates has not been demonstrated although recombination will occur during the formation of defective RNAs. Phenotypic mixing occurs between some animal rhabdoviruses and other enveloped animal viruses (e.g., paramyxoviruses, orthomyxoviruses, retroviruses, herpesviruses).
Six genera have been established, on the basis of significant differences in genome organization, amino acid sequence of structural proteins, replication site and host range. Phylogenetic relationships based on available N, G or L protein sequences support assignments of species to the identified genera (Fig. 3).
G protein is involved in virus neutralization and defines the virus serotype. N protein is a cross-reacting, complement-fixing (CF) antigen. Weak serological cross-reactions may occur between viruses in different genera. Protection follows vaccination with attenuated viruses, killed viruses, subunits consisting of G protein alone or G protein together with the ribonucleoprotein complex, and expression vectors (e.g., Vaccinia virus) or plasmid DNA that synthesize G and/or N.
Some species multiply only in mammals, or fish, or arthropods, or other invertebrates, many have both arthropod and vertebrate hosts (arboviruses), while some species infect plants and certain plant-feeding arthropods. Some of the viruses of vertebrates have a wide experimental host range. A diverse range of vertebrate and invertebrate cells are susceptible to vertebrate rhabdoviruses in vitro. The viruses of plants usually have a narrow host range among higher plants; some replicate in insect vectors and grow in insect cell cultures.
Sigma virus (SIGMAV) was recognized first as a congenital infection of Drosophila. No rhabdovirus is transmitted vertically in vertebrates, or plants. Some viruses are transmitted mechanically between plants. Vector transmission may involve mosquitoes, sandflies, mites, culicoides, aphids, lacewings, leafhoppers, or planthoppers, etc. Some viruses are transmitted mechanically in sap or from the body fluids of infected hosts. Mechanical transmission of viruses infecting vertebrates may be by contact, aerosol, bite, or venereal.
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