Probing, by Self-Assembly, the Number of Potential Binding Sites for Minor Protein Subunits in the Procapsid of Double-Stranded RNA Bacteriophage φ6

Abstract

The double-stranded RNA bacteriophage 6 is an extensively studied prokaryotic model system for virus assembly. There are established in vitro assembly protocols available for the 6 system for obtaining infectious particles from purified protein and RNA constituents. The polymerase complex is a multifunctional nanomachine that replicates, transcribes, and translocates viral RNA molecules in a highly specific manner. The complex is composed of (i) the major structural protein (P1), forming a T‫1؍‬ icosahedral lattice with two protein subunits in the icosahedral asymmetric unit; (ii) the RNA-dependent RNA polymerase (P2); (iii) the hexameric packaging nucleoside triphosphatase (NTPase) (P4); and (iv) the assembly cofactor (P7). In this study, we analyzed several 6 virions and recombinant polymerase complexes to investigate the relative copy numbers of P2, P4, and P7, and we applied saturated concentrations of these proteins in the self-assembly system to probe their maximal numbers of binding sites in the P1 shell. Biochemical quantitation confirmed that the composition of the recombinant particles was similar to that of the virion cores. By including a high concentration of P2 or P7 in the self-assembly reaction mix, we observed that the numbers of these proteins in the resulting particles could be increased beyond those observed in the virion. Our results also suggest a previously unidentified P2-P7 dependency in the assembly reaction. Furthermore, it appeared that P4 must initially be incorporated at each, or a majority, of the 5-fold symmetry positions of the P1 shell for particle assembly. Although required for nucleation, excess P4 resulted in slower assembly kinetics.

Rescue of Maturation Off-Pathway Products in the Assembly of Pseudomonas Phage ϕ6

“…The light-scattering zones of the gradients were collected using a BioComp gradient fractionator, and the protein compositions of the collected fractions were analyzed by SDS-PAGE. The estimation of relative protein amounts was based on the known relationship between the protein band intensity in a Coomassie brilliant blue-stained SDS-polyacrylamide gel and relative protein quantity; relative protein quantities were converted to copy numbers by use of the known molecular weights of the proteins and the copy number 120 for P1 (16).…”

“…During particle assembly, a P4 hexamer is incorporated at each, or at a majority, of the 5-fold symmetry positions of the P1 shell (16). To probe the pathway for P4 association on the potential 11 empty P4 hexamer binding sites in the P4-deficient particles, we titrated the amount of P4 in the reassociation reaction mixtures from 1/4ϫ to 4ϫ, where "1ϫ" refers to a molar ratio of 11:1 between P4 hexamers and P4-deficient particles.…”

“…Three derivatives of recombinant 6 PC particles were used in this study ( Fig. 1): wild-type PCs that have a protein composition similar to that of the viral cores (16,24) (Fig. 1, lane 2) and two types of P4-deficient particles, OG-PC and S250Q, which have equivalent amounts of P4 to form one P4 hexamer per particle (12,25) and thus 11 potential empty P4 hexamer binding sites (Fig.…”

“…P4 hexamers nucleate PC assembly and are efficiently incorporated at the 12 potential binding sites on the PC shell during the course of the in vitro assembly reaction (1,16). However, the interaction of the P4 hexamer with the PC is not stable (35): a majority (ϳ90%) of P4 hexamers dissociate from the PC surface if the particles are exposed to detergents (12).…”

“…It requires NTP and a divalent metal ion (Mg 2ϩ or Ca 2ϩ ) for hexamerization (14), is incorporated into the PC as a preformed hexamer during the assembly of the particle (1), and remains associated with the PC shell during its maturation (11,15). Of the 12 potential P4 hexamer binding sites, approximately 11 are occupied in the virion (16). The PC framework is formed by 120 copies of protein P1 (17).…”