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Alphaviruses have enveloped, 65–70 nm spherical virions with an icosahedral core.
Alphaviruses virions are spherical, enveloped particles about 70 nm in diameter. The particle consists of a nucleocapsid core surrounded by a lipid bilayer that is embedded with glycoprotein spikes (Cheng et al., 1995, Zhang et al., 2011).
The nucleocapsid core comprises 240 copies of capsid protein that surround the viral genome. The genome is a single-strand of positive-sense RNA that is capped and has a poly-A tail. Some studies have shown host factors are incorporated into the nucleocapsid core, but their quantities are host-dependent (Sokoloski et al., 2013). The lipid bilayer is host-derived from the site of budding. Alphaviruses bud from the plasma membrane although, in arthropods, budding into internal vesicles has also been observed (Gliedman et al., 1975, Lu and Kielian 2000, Strauss and Strauss 1994). Eighty trimeric glycoprotein spikes cover the surface of the alphavirions (Cheng et al., 1995, Zhang et al., 2011). Each trimeric spike is composed of three E1-E2 heterodimers. However, in some alphaviruses, the E3 glycoprotein remains non-covalently associated to these spikes.
E1 and E2 each contain a single transmembrane domain. E1 has a short cytoplasmic tail that has been shown to be dispensable. E2 has a long (greater than 30 amino acid) cytoplasmic domain that interacts with a hydrophobic pocket in the capsid protein (Lee et al., 1996, Owen and Kuhn 1997, Skoging et al., 1996, Wilkinson et al., 2005, Jose et al., 2012). This interaction between the external glycoprotein spikes and the internal nucleocapsid core is rare in enveloped virions. Both the nucleocapsid core and the trimeric glycoprotein spikes are arranged with T=4 icosahedral symmetry (Zhang et al., 2011). The interaction between the E2 cytoplasmic domain and capsid protein is thought to mediate this organization.
The capsid, E2, and E1 proteins are the minimal proteins required for an infectious virion but the alphaviruses also translate two further proteins, the 6K protein and its frameshifting product,TF (for TransFrame) (Firth et al., 2008). The TF protein is present in virions of at least some species; very rarely is the 6K protein found in the released particle (Snyder et al., 2013, Ramsey and Mukhopadhyay 2017, Ramsey et al., 2017). TF is found in sub-stoichiometric amounts compared to the other structural proteins. Because of its small size and low quantities, its location and organization in the particle is not known.
The sedimentation coefficient of alphaviruses is 280S. Alphavirus particles have a buoyant density is between 1.15–1.22 g cm-3 in sucrose gradients (Kuhn 2013, Sokoloski et al., 2013) and between 1.18–1.19 g cm-3in tartrate gradients (Hernandez et al., 2005). In addition to the methods of denaturing and decreasing infectivity as described in the family section, the infectivity of alphaviruses can also be decreased by exposure to low pH in the absence of lipid vesicles.
The genome of alphaviruses ranges from 9.7–11.8 kb. The genome RNA consist of a 5ʹ-cap (viral type 0 7meGpppA) and non-coding region followed by genes for the non-structural and structural proteins, a 3ʹ-non-coding region and polyadenylation (Ahola and Kaariainen 1995, Strauss and Strauss 1994). The negative-sense RNA of the replication intermediate is neither capped nor contains a poly-A tail. A subgenomic RNA encoding the structural proteins is transcribed from the negative-sense RNA and consists of a 5′-cap and poly-A tail.
The genome of alphaviruses encodes 10 different proteins. The non-structural proteins (gene order: nsP1-nsP4) are important for replicating the viral genome, and the structural proteins (gene order: capsid, E3, E2, 6K, TF, and E1) function in virus assembly. The non-structural proteins are not incorporated into virion particles (Strauss and Strauss 1994).
The structural proteins are translated as a polyprotein in the order capsid-E3-E2-6K-E1 from the subgenomic mRNA. As soon as capsid is produced it auto-proteolytically cleaves itself from the rest of the polyprotein and forms nucleocapsid cores in the cytoplasm (Choi et al., 1991). E3 contains an ER signal sequence and translocates the polyprotein into the ER. The entire polyprotein crosses the ER membrane (going between lumen and cytoplasm) several times until host proteases cleave it into the individual structural proteins. At a frequency of 10–30%, a ribosomal frameshift event occurs resulting in the production of E3, E2, and TF; E1 is not translated. The host protease signalase cleaves the individual proteins from the polyprotein. The E3 and E2 proteins (pE2 or p62) form heterodimers with E1. E1 has been shown to undergo disulphide rearrangement during assembly, and the same is predicted for E2. E3 stabilizes the E2/E1 heterodimers and trimers during spike assembly by preventing dissociation of the complex as it transits through the secretory pathway to the plasma membrane. E3 is cleaved by the host protease furin in the trans-Golgi which converts the spikes to a metastable, fusion competent state, in which form they are transported to the plasma membrane. TF and E2 are palmitoylated. E3, E2, and E1 are glycosylated.
The capsid protein consists of two domains, a highly-charged, N-terminal domain that interacts with the viral RNA in the interior of the nucleocapsid core and a C-terminal domain that has a chymotrypsin-like fold (Choi et al., 1991). The spikes formed by E2 and E1 are responsible for viral entry (Kielian 2014, Kielian et al., 2010, Vaney et al., 2013). The E2 protein binds to the host-cell receptor and interacts with the capsid protein. The E1 protein is a class II fusion protein that mediates fusion between the virus membrane and the host cell membrane in the endosome (Kielian 2014, Kielian et al., 2010, Vaney et al., 2013). There are some reports of fusion occurring at the plasma membrane (Kononchik et al., 2011, Vancini et al., 2015). The atomic structures of E3, E2, and E1 have been determined at both neutral and low pH providing insight to the conformational changes that occur during the entry process (Li et al., 2010, Voss et al., 2010). The exact roles of 6K and TF are not known but 6K has been shown to have viroporin activity (Sanz et al., 1994, Gonzalez and Carrasco 2003) and TF has been shown to be a virulence factor (Snyder et al., 2013).
Alphaviruses contain a host-derived lipid membrane that is acquired during budding from the plasma membrane. In arthropods, budding into internal vesicles has also been observed (Gliedman et al., 1975, Lu and Kielian 2000). In vertebrate cells, alphaviruses bud from regions rich in cholesterol and sphingomyelin but analysis of lipid membrane from purified particles shows a relatively similar lipid composition to that of the plasma membrane (Marquardt et al., 1993, Kielian et al., 2000, Kalvodova et al., 2009).
The glycoproteins E3, E2, and E1 have N-linked glycosylation in most alphaviruses (Sefton 1977). However, the locations and numbers of glycosylation are not conserved between members of different alphavirus species. The carbohydrate moieties incorporated differ depending on the host; virus produced in invertebrates incorporate different carbohydrate residues than virus produced in vertebrate cells. Mutation of glycosylation sites affects virus infectivity and affects the interferon response (Shabman et al., 2008).
See discussion under family description.
Species demarcation criteria in the genus include:
Species demarcation considers a combination of each of the criteria listed above. Whereas members of most species show at least 10% difference in amino acid sequence identity over entire coding regions, there is not a clear cutoff of sequence divergence to provide absolute species demarcation. For example, for Venezuelan equine encephalitis virus (VEEV), there are multiple subtypes with amino acid identities as low as 85%. However, although Everglades, Tonate, and Mucambo viruses are phylogenetically located within the VEEV cluster (Figure 4) and have 94–97% amino acid identity with each other or the closest related VEEV strains, they are considered to belong to different individual species due to differences in several of the traits listed above. For example, the justification for Everglades virus remaining as a distinct species is based upon its avirulence for equids, as well as a different rodent host and mosquito vector usage in Southern Florida.
The reason that some very divergent strains such as those of VEEV and Sindbis virus are considered subtypes but not species reflects the lack of phenotypic information. Madariaga virus, previously considered to comprise 3 subtypes within the species Eastern equine encephalitis virus, was assigned to the species Madariaga virus based on major differences in vector usage and human virulence for most strains (Arrigo et al., 2010).
Aura virus and Una virus: names of small rivers in Belém, Brazil where the viruses were originally isolated (Causey et al., 1963).
Chikungunya virus: chikungunya derives from a word in the Kimakonde language, meaning “to become contorted”, and describes the stooped appearance of sufferers with joint pain (arthralgia).
getah virus: Isolated near rubber plantations; Getah means rubber in Malay.
Madariaga virus: first isolates from General Madariaga Partido, Buenos Aires Province, Argentina.
Mosso das Pedras virus: named after locality in Brazil (Calisher et al., 1982).
onyong-nyong virus: onyong-nyong means "weakening of the joints" in the Nilotic language
Trocara virus: after Trocará, Brazil.
Taï Forest alphavirus
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