Mutations in the poliovirus P1 capsid precursor at arginine residues VP4-ARG34, VP3-ARG223, and VP1-ARG129 affect virus assembly and encapsidation of genomic RNA

Abstract

To begin to identify poliovirus capsid protein determinants required for assembly and RNA encapsidation, we have addressed the functional significance of three arginine residues of the poliovirus capsid in virus assembly and encapsidation of genomic RNA. These studies were conducted by using a recently described system in which recombinant vaccinia viruses are used to supply poliovirus capsid proteins in trans to a poliovirus subgenomic replicon [D. C. Ansardi, D. C. Porter, and C. D. Morrow (1993) J. Virol. 67, 3684-3690]. Two of the arginine residues, located at position 34 of VP4 (VP4-R034) and position 129 of VP1 (VP1-R129), are located within a cavity on the poliovirus capsid interior, whereas the third arginine, residue 223 of VP3 (VP3-R223), is located at a promoter-protomer interface. Five mutants were constructed by site-directed mutagenesis of poliovirus P1 capsid precursor cDNA to separately encode lysine or glutamine substitutions at VP4-R034 (VP4-R034K, VP4-R034Q), lysine or glutamine substitutions at residue 129 of VP1 (VP1-R129K, VP1-R129Q), or a lysine substitution at residue 223 of VP3 (VP3-R223K). Processed capsid proteins derived from the VP3-R223K, VP1-R129K, and VP1-R129Q mutant precursors were unstable and failed to assemble subviral particles or virions at 37 degrees. The assembly defect for cleavage products of the VP3-R223K precursor was partially overcome at 33 degrees, as empty capsids, but not mature virions, assembled from the mutant capsid subunits at the lower temperature. With regard to the third arginine residue analyzed, VP4-R034, processed capsid proteins derived from both the VP4-R034K and the VP4-R034Q mutant precursors assembled 155S virions at 37 degrees; however, capsid proteins derived from the VP4-R034Q precursor were temperature-sensitive for virion formation at 39.5 degrees. The reduced virion formation at 39.5 degrees was apparently a reflection of a defect in forming assembly competent subunits which also prevented accumulation of surplus VP4-R034Q subunits as empty capsids. By using graphics to display the poliovirus three-dimensional structure, the locations of these residues on the poliovirus capsid interior and their interactions with adjacent amino acids were visualized to provide structural explanations for the observed assembly defects which highlight the important role these residues play in capsid assembly and RNA encapsidation.