Taxonomic Structure of the Family
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Togaviridae |
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Virions are 70 nm in diameter, spherical, with a lipid envelope containing heterodimeric glycoprotein spikes composed of two virus glycoproteins. For alphaviruses, the heterodimers are organized in a T = 4 icosahedral lattice consisting of 80 trimers (Fig. 1). The envelope is tightly organized around an icosahedral nucleocapsid that is 40 nm in diameter. The nucleocapsid is composed of the capsid protein, organized in a T = 4 icosahedral symmetry, and the genomic RNA. The one-to-one relation between glycoprotein heterodimers and nucleocapsid proteins is believed to be important in virus assembly. The three dimensional structure of rubella virions has not been determined. Virions of Rubella virus (RUBV) are pleomorphic in nature, indicating that the capsid-glycoprotein interaction is not as tight as it is in virions of alphaviruses.
Physicochemical and Physical Properties
Virion Mr is about 52 106. Alphaviruses have a buoyant density in sucrose of 1.22 g/cm3 and an S20w of 280S. Rubella virus has a buoyant density of 1.18-1.19 g/cm3 and a similar S value. Alphaviruses are stable between pH 7 and 8, but are rapidly inactivated by very acidic pH. Virions have a half-life at 37°C of about 7 hours in culture medium. Most alphaviruses are rapidly inactivated at 58°C with a half-life measured in minutes. Rubella virions more heat labile than alphaviruses, with a half-life at 37°C of 1 to 2 hours and a half-life at 58°C of 5-20 minutes. Generally, togaviruses are sensitive to organic solvents and detergents which solubilize their lipoprotein envelopes. Sensitivity to irradiation is directly proportional to the size of the viral genome.
The genome consists of a linear, positive sense, ssRNA molecule 9.7-11.8 kb in size. The viral RNA is capped (7-methylguanosine) at the 5 terminus and polyadenylated at the 3 terminus.
The structural proteins of togaviruses include a basic capsid protein (CP, Mr 30-33 103) and two envelope glycoproteins (E1 and E2, Mr 45-58 103). Some alphaviruses may contain a third envelope protein, E3 (Mr 10 103). The four nonstructural proteins, which are present in infected cells but not found in virions, are called nsP1-4. Their functions are described below.
Lipids comprise about 30% of the dry weight of virions. They are derived from the host-cell membrane from which budding occurs: the plasma membrane for alphaviruses, both intracellular membranes and the plasma membrane for Rubella virus. Their composition depends upon the cells in which the virus was grown. Phospholipids (including phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, and sphingomyelin) and cholesterol are present in a molar ratio of about 2:1 for alphaviruses, 4:1 for Rubella virus, presumably because the latter matures primarily at intracellular membranes.
Both high mannose and complex N-linked glycans are found on the envelope glycoproteins. In addition, Rubella virus E2 protein contains O-linked glycans.
Genome Organization and Replication
The genomic RNA serves as the mRNA for the non-structural proteins of the virus (Fig. 2). In alphaviruses, the polyprotein precursor is cleaved by a viral-encoded protease in nsP2 to produce four final products, nsP1, nsP2, nsP3 and nsP4 (Fig. 3). In eight of ten alphaviruses sequenced, there is a termination codon (UGA) between nsP3 and nsP4 genes which is read-through with moderate efficiency (5-20%), whereas in the two other alphaviruses this codon has been replaced by a codon for arginine (CGA). Polyproteins containing nsP2 are enzymes and function primarily in trans to produce the cleaved non-structural proteins. In Rubella virus the polyprotein precursor is cleaved into two products, P150 and P90. The protease mediating this cleavage is located near the C-terminus of P150, and can function in either cis or trans.
The nonstructural proteins, as individual entities and as polyproteins, are required to replicate viral RNA and probably act in association with cellular proteins. The alphavirus nsP1 protein is thought to be involved in capping of viral RNAs and in initiation of negative-strand RNA synthesis. The nsP2 functions as a protease to process the non-structural proteins and is believed to be a helicase required for RNA replication. Protein nsP4 is believed to be the viral RNA polymerase. Protein nsP3 is also required for RNA replication; P123 and nsP4 form the replicase complex for minus strand synthesis, whereas efficient plus-strand synthesis requires cleavage of P123. In RUBV, P150 contains a methyl transferase motif of unknown function that is also present in nsP3 of alphaviruses. RUBV P90 contains both helicase and replicase motifs. These motifs are in a different order than in the alphavirus genome. The difference in processing and order of nonstructural protein motifs between alphaviruses and RUBV suggests that evolution of these two genera was more complicated than simple divergence from a common ancestor. In RNA replication, a negative-strand copy is produced that is used as template in the synthesis of both genome-sized RNA as well as a subgenomic 26S mRNA that corresponds to the 3 third of the viral genome and encodes the viral structural proteins. This mRNA is capped and polyadenylated. It is translated as a polyprotein, which is processed in alphaviruses by a combination of an autoprotease activity present in the CP and cellular organelle-bound proteases, to produce the viral structural proteins. The RUBV CP lacks autoprotease activity and all of the cleavages of this precursor are mediated by a cellular signal endopeptidase.
Cis-acting regulatory elements in the 5 and 3 non-translated regions of the genomic RNA are required to produce alphavirus minus strands and to copy the minus strand into plus strands. There are believed to be other cis-acting regulatory elements within the viral RNA as well. For alphaviruses, the promoter for the production of the 26S sgRNA is a stretch of 24 nts that span the start point of the sgRNA. This minimal 24 nts sequence element is upregulated by upstream sequences. The RUBV subgenomic promoter is 50 nts upstream from the sgRNA start site. RUBV and alphaviruses share homology in the cis-acting elements at the 5-end of the genome and subgenomic promoter region.
The non-structural proteins function in the cytoplasm of infected cells, although some alphavirus nsP2 is translocated to the nucleus. The CP assembles with the viral RNA to form the viral nucleocapsids in the cytosol. Glycoproteins inserted into the endoplasmic reticulum during translation are translocated via the Golgi apparatus to the plasma membrane for alphaviruses; for RUBV they are also found at intracellular membranes. Assembled nucleocapsids bud through these membranes acquiring a lipid envelope containing the two integral membrane glycoproteins. For Rubella virus, the glycoproteins are retained in the Golgi apparatus, the preferred site of budding. Unlike alphaviruses, rubella capsids are not pre-assembled in the cytosol and only form during the budding process. Late in infection, the rubella glycoproteins also accumulate in the plasma membrane and budding also occurs at this site.
Member viruses of the genus Alphavirus were originally defined on the basis of serological cross-reactions. Thus, all alphaviruses are antigenically related to each other. They share a minimum amino acid sequence identity of about 40% in the more divergent structural proteins and about 60% in the non-structural proteins. Alphaviruses can be grouped into 7 antigenic complexes based on serologic cross-reactivity: the eastern, Venezuelan and western equine encephalitis, Middelburg, Ndumu, Semliki Forest and Barmah Forest complexes. Rubella virus is serologically distinct from alphaviruses and no structural protein amino acid sequence homology can be detected between Rubella virus and the alphaviruses.
Alphaviruses are transmitted biologically between vertebrates by mosquitoes and other hematophagous arthropods. Alphaviruses have a wide host range and nearly worldwide distribution. The infection of cells of vertebrate origin is cytolytic and involves the shutdown of host-cell macromolecular synthesis. In mosquito cells, alphaviruses usually establish a non-cytolytic infection in which the cells survive and become persistently infected. Cytopathology has been described in the midguts of mosquitoes infected with Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV). The assembly of virions in mosquito cells appears to differ from that for vertebrate cells in that most, and perhaps all, virus assembly occurs in association with intracellular membranes rather than by budding through the plasma membrane. The details may differ in different types of cells. In contrast, humans are the only known host for Rubella virus, which is spread via the respiratory route. Rubella virus replicates in a number of mammalian cell culture lines, including lines from humans, monkeys, rabbits and hamsters. The virus is not cytopathic in most of these lines and has a propensity to initiate persistent infections.
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