Novel second-site suppression of a cold-sensitive defect in phage P22 procapsid assembly

Abstract

The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalton gene 1 protein. The assembly of this dodecameric complex at a unique capsid vertex requires scaffolding subunits. The mechanism that ensures the location of the 12-fold symmetrical portal at only one of the 12 5-fold vertices of an icosahedral virus capsid presents a unique assembly problem, which, in some viruses, is solved by the portal also acting as initiator of procapsid assembly. Phage P22 procapsids, however, are formed in the absence of the portal protein. The 1 − csH137 mutation prevents the incorporation of the portal protein into procapsids. In a mixed infection with cs + phage, the mutant subunits are able to form functional portals, suggesting that the cold-sensitivity does not affect portal-portal interactions, but affects the interaction of portal subunits with some other molecular species involved in the initiation of portal assembly. Interestingly, the cs defect is suppressed by temperature-sensitive folding mutations at four sites in the P22 tailspike gene 9. The suppression is allelespecific; other tailspike tsf mutations fail to suppress the cs defect. Translation through a suppressor site is required for suppression. This observation is unexpected, since analysis of nonsense mutations in this gene indicates that it is not required for procapsid assembly. Examination of the nucleic acid sequences in the neighborhood of each of the suppressor sites shows significant sequence similarity with the scaffolding gene translational initiation region on the late message. This supports a previously proposed model, in which procapsid assembly is normally initiated in a region on the late messenger RNA that includes the gene 8 start site. By this model, the suppressor mutations may be acting through protein-RNA interactions, changing sequences that identify alternative or competing sites at which the mutant portal subunits may be organized for assembly into the differentiated vertex of the phage capsid.