Evidence for the existence of protomers in the assembly of encephalomyocarditis virus

Abstract

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with 3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: a, y, and a. This (e,y,a)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (8,'y,a)s, and 5S, (B,y,a), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A) ; 14S, (e, y,a),; 6S, (w,y,a). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: a, 36,000; a, 32,000; ,, 29,500; y, 26,500; and 6, 7,800. With the exception of the b-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A)-(A)5 _ (,y,a)s-(b,f,y,a)o n(evy,a)n where n is the number of immature protomers per virion. Picomaviruses contain four nonidentical polypeptide chains here designated a, ,S, y, and 6 (29). All four chains are generated by posttranslational cleavage of a single, large (mol wt 100,000) precursor, the A chain (3, 13, 17, 22). The virions also frequently contain traces of a fifth polypeptide, a, which is an uncleaved precursor of # and 6 chains (3, 13). The cardioviral (encephalomyocarditis [EMC ] virus, mouse Elberfeld [ME ] virus, mengovirus) chains A, a, a, ,B, y, and 6 correspond to the polioviral chains NCVP-la, VP-0, VP-1, VP-2, VP-3, and VP-4, respectively (13, 32). We have adopted the cardiovirus nomenclature which lends itself more readily to the description of oligomeric structures. Cytoplasmic extracts of poliovirus-infected HeLa cells contain two capsid-related structures, one sedimenting at 148 and the other at 73S (25-27). Both contain the capsid peptides

In vitro synthesis and assembly of picornaviral capsid intermediate structures

“…In contrast to pentamers isolated from infected cells (9), no capsid precursors (Al, A, B, Dl) have ever been observed in 14S peaks formed in lysates, even when short-term translation samples were fractioned. This indicates that complete cleavage (to e,y,a) precedes or occurs simultaneously with assembly in vitro.…”

Picornaviral structure and assembly.

“…In fact, such alterations may lead to increased capsid stability (68). Most studies indicate the presence of 60 copies of each of the four capsid polypeptides (143,150,215), although discrepancies have been reported. A bovine enterovirus was shown to contain one-half (ca.…”

“…When exposed to mild acid in the presence of C1or Br-, mengovirus or Maus-Elberfeld virus yields particles that sediment at 13.4S and are composed of five molecules each of VP1, VP2, and VP3 [i.e., (VP1-VP2-VP3)5] (66,144). VP4 precipitates under these conditions (150) (Fig. 2).…”

“…2). In the presence of 2 M urea, which disrupts hydrogen bonds and hydrophobic interactions, the 13.4S pentamers are degraded into 5S components containing only one molecule of each protein (143,150). In an electron microscope, the 13.4S pentamers appear as concave disks or skullcaps measuring 17 by 14 nm, and the 5S structures appear as 6.8-nm spheres (150).…”

Protease required for processing picornaviral coat protein resides in the viral replicase gene

“…However, an unanswered question raised by purification of the -y-like chain is why it did not also contain the other coat proteins with which it is normally tightly associated in the coat pro-tomer (19). The possibility that the protease might be some other still unrecognized protein was reinforced by recent reports (23,31) that coat precursor proteins synthesized in cell-free extracts fail to be cleaved unless translation of the viral RNA is continued beyond the region coding for coat protein.…”