Taxonomic Structure of the Family
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Picornaviridae |
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Virions consist of a capsid, with no envelope, surrounding a core of ssRNA. Hydrated native particles are 30 nm in diameter, but vary from 22-30 nm in electron micrographs due to drying and flattening during preparation. Electron micrographs reveal no projections, the virion appearing as an almost featureless sphere (Fig. 1). The capsid is composed of 60 identical units (protomers), each consisting of three surface proteins, 1B, 1C and 1D, of Mr 24-41 103, and, in most picornaviruses, an internal protein, 1A of Mr 5.5-13.5 103. Total Mr of protomer is 80-97 103. Proteins 1A, 1B, 1C and 1D are also commonly named VP4, VP2, VP3, and VP1, respectively. Proteins 1B, 1C and 1D each possess a core structure comprising an eight-stranded beta-sandwich (“-barrel”). The -barrels pack together in the capsid with T = 1, pseudo T = 3, icosahedral symmetry. (These structural features are shared by certain plant viruses that exhibit T = 3, or pseudo T = 3, symmetry, e.g., Sobemovirus and Comoviridae, respectively.) Genera differ in the external loops that interconnect the strands. These loops account for differences in surface relief of each genus (Fig. 2) and in thickness of the capsid wall. Assembly occurs via pentameric intermediates (pentamer = five protomers). Proteins within each pentamer are held together by an internal network formed from the N-termini of the three major capsid proteins (CP), the C-termini lying on the outer capsid surface. Empty capsids, which are produced by some picornaviruses, are very similar to virions, except that 1A and 1B are normally replaced by the uncleaved precursor, 1AB.
Physicochemical and Physical Properties
Virion Mr is 8-9 106, S20w is 140-165S (empty particle S20w is 70-80S). Buoyant density in CsCl is 1.33-1.45 g/cm3, depending on the genus. Some species are unstable below pH 7; many are less stable at low ionic strength than at high ionic strengths. Virions are insensitive to ether, chloroform, or non-ionic detergents. Viruses are inactivated by light when grown with, or in the presence of photodynamic dyes such as neutral red or proflavin. Virions are stabilized by divalent cations. Thermal stability varies with the genus.
Virions contain one molecule of positive sense, ssRNA, 7-8.5 kb in size, containing a single long ORF. A poly(A) tract, heterogenous in length, is located after the 3-terminal heteropolymeric sequence. A small protein, VPg (Mr ± 2.4 103, is linked covalently to the 5-terminus. The non-translated regions (NTR) at both termini contain regions of secondary structure which are essential to genome function. The very long 5-NTR (0.6-1.5 kb) includes a 5-terminal domain involved in replication (e.g., the poliovirus “clover-leaf”) and an internal ribosome entry site (IRES) of 400-450 nts upstream of the translational start site; each picornaviral IRES element can be assigned to one of two types, according to its secondary structure. Between the 5-terminal domain and the IRES there may be one, or more, pseudoknots and/or a poly(C) tract (Fig. 3). The 3-NTR, which may also contain a pseudoknot, ranges from 40 to 165 nts in length. The overall sequence identity between viruses of different genera is typically less than 40%.
In addition to the major CPs, 1A, 1B 1C and 1D, and 3BVPg, described above, small amounts of 1AB (VP0) are commonly seen in lieu of one or more copies of 1A and 1B. Protein 1A is small in hepatoviruses, and appears to be absent (i.e., 1AB is uncleaved) in parechoviruses. Traces of other proteins, including the viral RNA-dependent RNA polymerase, 3Dpol, may also be present in purified virus preparations.
Some picornavruses carry a sphingosine-like molecule (“pocket factor”) in a cavity (“pocket”) located inside 1D. Protein 1A, where present, has a molecule of myristic acid covalently attached to the amino terminal glycine.
None of the viral proteins are glycosylated.
Genome Organization and Replication
The virion RNA is infectious and serves as both the genome and the viral messenger RNA. Gene maps are shown in Fig. 3. Initiation of protein synthesis is stimulated by the IRES. Translation of the single ORF produces the polyprotein precursor (Mr 240-250 103) to the structural proteins (derived from the P1 region of the genome) and the nonstructural proteins (P2, P3 regions). In some viruses P1 is preceded by a leader protein (L). The polyprotein is cleaved to functional proteins by specific proteases contained within it. Intermediates are denoted by letter combinations (e.g., 3CD, the uncleaved precursor of 3C and 3D). The viral proteases are as follows: Protease 3Cpro, a serine-like cysteine protease encoded by all picornaviruses, performs most of the cleavages. In most genera, 2A is also associated with proteolytic activity; the 2Apro of cardioviruses and aphthoviruses acts only in cis. The leader protein of aphthoviruses has proteolytic activity (Lpro) while that of cardioviruses does not. Some intermediates are stable and serve functions distinct from those of their cleavage products (e.g., cleavage of poliovirus P1 by 3CDpro, not by 3Cpro). The cleavage of 1AB, which accompanies RNA encapsidation, is thought to be autocatalytic.
Replication of viral RNA occurs in complexes associated with cytoplasmic membranes. These complexes contain proteins derived from the whole of the 2BC-P3 region of the polyprotein, including the polymerase (3Dpol, an RNA chain-elongating enzyme), and 2C (an ATPase containing a nucleotide binding sequence motif). The poliovirus 3Cpro component has been shown to be required for binding to the 5-terminal RNA cloverleaf. Many compounds that specifically inhibit replication have been described. Mutants resistant to, or dependent on drugs have been reported. Genetic recombination, complementation, and phenotypic mixing, occur. Defective particles, carrying deletions in the CPs or L, have been produced experimentally but have not been observed in natural virus populations.
Serotypes are classified by cross-protection, neutralization of infectivity, complement-fixation, specific ELISA using a capture format or immunodiffusion. Some serotypes can be identified by haemagglutination. Antigenic sites, defined by mutations that confer resistance to neutralization by monoclonal antibodies, typically number three or four per protomer. Neutralization by antibody follows first-order inactivation kinetics.
Most picornaviruses are specific for one, or a very few host species (exceptions are Foot-and-mouth disease virus (FMDV) and Encephalomyocarditis virus, (EMCV). Most species can be grown in cell culture. Resistant host cells (e.g., mouse cells in the case of the primate-specific polioviruses) can often be infected (single round) by transfection with naked, infectious RNA. Transmission is horizontal, mainly by fecal-oral, fomite or airborne routes. Transmission by arthropod vectors is not known, although EMCV has been isolated from mosquitoes and ticks.
Infection is generally cytolytic, but persistent infections are common with some species and reported with others. Poliovirus infected cells undergo extensive vacuolation as membranes are reorganised into viral replication complexes. Infection may be accompanied by rapid inhibition of cap-dependent translation of cellular mRNAs (2Apro of poliovirus and Lpro of aphthovirus are each powerful inhibitors), messenger RNA synthesis, and the cellular secretary pathway (poliovirus 2B and 3A have been implicated).
A picornavirus species is a polythetic class of phylogenetically related serotypes or strains which would normally be expected to share (1) a limited range of hosts and cellular receptors, (2) a significant degree of compatibility in proteolytic processing, replication, encapsidation and genetic recombination, and (3) essentially identical genome maps.
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