Isolation and analysis of mutants of double-stranded-RNA bacteriophage phi6 with altered packaging specificity

Abstract

The genomes of bacteriophage phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented double-stranded RNA genomes into preformed polyhedral structures called procapsids or inner cores. This packaging requires hydrolysis of nucleoside triphosphates and takes place in the order S-M-L. Packaging is dependent on unique sequences of about 200 nucleotides near the 5' ends of plus strand transcripts of the three genomic segments. Changes in the pac sequences lead to loss of packaging ability but can be suppressed by second-site changes in RNA or amino acid changes in protein P1, the major structural protein of the procapsid. It appears that P1 is the determinant of the RNA binding sites, and it is suggested that the binding sites overlap or are conformational changes of the same domains.

FIG. 1.

Physical map of the genomic segments of bacteriophage φ6.

FIG. 2.

Secondary structure of the 5′ end of the transcript of genomic segment M according to Zuker et al. (13).

FIG. 3.

Secondary structure of the 5′ end of the transcript of genomic segment S according to Zuker et al. (13).

FIG. 4.

Packaging of radioactive plus strands of genomic segment S. The wild-type pac sequence (w) is packaged by both. Altered pac sequence GC99AU (m) is only packaged by mutant procapsid (pc). A pac sequence, pLM1299 (d), that is missing four nucleotides at position 62 is not packaged by either.

FIG. 5.

Minus strand synthesis to demonstrate packaging. Wild-type procapsids do not make minus strands when incubated with only M and L (a). With S, M, and L, they do make minus strands (b). With M, L, and an altered S pac sequence, there is no minus strand synthesis (c). There is minus strand synthesis with L and a chimeric segment that includes normal S and M (SM) (d). If the chimera has an altered S pac gene, there is no minus strand synthesis (e). A mutant procapsid that suppresses the poor packaging of the altered S pac site is able to package the altered chimera (j) and to synthesize minus strands as well as it does with the wild-type sequence (i). However; the mutant procapsid is also able to package segment M in the absence of segment S (f). The mutant procapsid shows competition between the plus strand of M and that of S. In lane g, synthesis of the minus strand of S is greatly reduced; however, the response is similar with the plus strand carrying the GC99AU alteration in the pac sequence (h). The band labeled sm is the plus strand transcript of chimeric segment SM.