Picornaviral capsid assembly: similarity of rhinovirus and enterovirus precursor subunits

Abstract

Cytoplasmic extracts of rhinovirus lA-infected HeLa cells, pulsed 15 min with [3H]leucine, contained a 13S subunit which was rich in the capsid precursor, peptide 92. After a 30-min chase, most of the capsid-related protein sedimented in a 14S peak that contained equimolar amounts ofthe capsid peptides e, a, and y, and some residual chain 92. The 14S subunit could be dissociated at pH 4.8 into 6S subunits containing only E, a, and y chains in equal proportions, indicating that the 14S subunit is an oligomer of (Eya) protomers. These subunits resemble subunits previously identified in the assembly of enteroviruses. These observations support the idea that rhinovirus assembly is basically similar to that of enteroviruses. Comparative studies on the peptide stoichiometry of the virion and the capsid precursor subunits indicate that rhinovirus 1A can contain as many as 11 immature protomers per virion. The picornaviruses have been divided (8) into EMC virus. This finding supports the view of a two genera: the acid-stable enteroviruses and common assembly pattern for both rhinovithe acid-labile rhinoviruses. Both enteroviruses and enteroviruses. ruses and rhinoviruses appear to be composed of 60 protomers, where each protomer is a pro-MATERIALS AND METHODS tein subunit composed of four nonidentical Media. Medium A is Eagle minimal medium in polypeptides (83y a). In the case ofthe cardiovi-Earle saline supplemented with 0.1 mM nonessenrus group (enteroviruses), the virion dissociates tial amino acids and 2 x 10-5 M inositol (7). Medium as if the protomers were held together by two B is a modification of medium A lacking calcium types of bonding domains (2, 4, 6). One domain chloride and containing 0.1% Pluronic F68 (7). Mebonds protomers together to form a pentameric dium AH is medium A containing 25 mM N-2-hysubunit; the second domain bonds 12 pentamers droxyethylpiperazine-N'-2-ethanesulfonic (HEPES) together to form the 60 subunit capsid. acid buffer, pH 7.4 (1). Medium AL is medium AH It has been suggested that the assembly of lacking amino acids. Medium P5 consists of Eagle minimal medium supplemented with 0.1% bovine the picornaviral capsid is also controlled by two serum albumin, 40 mM magnesium chloride, and 75 bonding sites (4, 11). Theoretically, assembly of ,ug of diethylaminoethyl dextran per ml (3). a 60-subunit shell, from such "bivalent" proto-Buffers. PBSA, buffer IV, and reticulocyte stanmers, can occur via one of three possible inter-dard buffer have been described (4). mediates: a dimer, a trimer, or a pentamer (11). Cells. Rhinovirus-sensitive HeLa cells, desig-Recent work on poliovirus (10) and encephalo-nated H-HeLa, were obtained from V. V. Hamparmyocarditis (EMC) virus (4) indicates that cap-ian,-Ohio State University (7). They were propasid assembly proceeds through a 14S subunit. gated in suspension in medium B supplemented sid as.mbly. oe through a 14S......subunit. with 10% bovine serum by shaking 300-ml cultures This subunit has been tentatively identified as in siliconized Florence flasks ...

In vitro morphogenesis of foot-and-mouth disease virus

“…The smallest detectable capsid structures found in many picornavirus-infected cells are 13S particles, which are pentamers of the capsid protein precursor (16,17,20). The precursor protein pentamer is cleaved to yield 14S particles composed of five copies each of VP0, VP1, and VP3.…”

Picornaviral structure and assembly.

“…The capsid precursor NCVP la apparently can exist in infected cells in soluble and aggregate forms, a conclusion drawn both from in vivo experiments (151) and in vitro experiments (208; Phillips, unpublished data). The earliest detectable, organized structure containing NCVP la is the 13S particle found in EMC virus-infected cells (150) and in rhinovirus-infected cells (151); the relatively slow turnover of this particle in these cells permitted its isolation and partial characterization. In poliovirus-infected cells, indirect evidence for the presence of this particle was derived from pulse-labeling experiments in which a 14S particle with a relatively high concentration of NCVP la was found (217).…”

“…These results probably reflect contamination with other viral proteins, as well as the presence of immature or defective 14S particles or both (172). Therefore, the most likely composition of biologically active particles is (VPO-VP1-VP3)5 or five protomers, as suggested by Rueckert and co-workers (150,151). Until recently, procapsids were envisioned as empty shells composed of 12 14S particles (i.e., 60 protomers).…”

Toward an in vitro system for picornavirus assembly: purification of mengovirus 14S capsid precursor particles

“…Virus-specific structures having sedimentation coefficients of about 145 in sucrose density gradients have been detected in lysates of cells infected with poliovirus (39, 40), EMC virus (31), or human rhinovirus (32). Similarly, when L cells were infected with mengovirus in the presence of [35S]methionine, a peak of radioactive material sedimenting at about 14S was resolved by centrifuging crude lysates in sucrose gradients ( Fig.…”

“…Of the various subviral structures identified in picornavirus-infected cells, a 14S capsid precursor appears to be common and to play a * Corresponding author. key role in virion assembly (31,32,39,40,59). Three lines of evidence support this view.…”