Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage phi6

Abstract

A model that explains the stoichiometric packaging of the chromosomes of phi6, a bacteriophage with a genome of three unique double-stranded RNA segments, is proposed and supported. Ordered switches in packaging specificity and RNA synthesis are determined by the amount of RNA within the procapsid. The plus strand of segment S binds to one of several sites on the outside of the empty procapsid. The RNA enters and the procapsid expands so that the S sites are lost and M sites appear. Packaging of segment M results in the loss of the M sites and the appearance of the L sites. Packaging of L readies the particle for minus-strand synthesis. If any of the segments is less than normal size, packaging of that class of segments continues until the normal content of RNA for that segment is packaged and the binding sites then change.

Figure 1

Restriction maps of the cDNA copies of the genomic segments of Φ6, L, M, and S as well as diagrams of the chimeric constructions used in this study. The sequences are all embedded in plasmid pT7T319U and the transcripts produced by T7 RNA polymerase have the correct base sequence at the 5′ ends. pLM1725 was prepared by ligating the 5′ end of segment S cut at the EcoRV site to a blunted SgrAI site of segment L. The aim was to prepare a transcript with the pac site of S, containing no sequence from M, but having a size close to that of the sum of S and M. pLM1827 was prepared by ligating segment M cut at the PstI site to segment S cut at the BamHI site to form an MS chimera (pLM1822) and then inserting the complete lacZ gene from plasmid 1871 into the ApaI site of M with PstI linkers. The aim was to prepare a transcript with the pac site of M, containing no sequence from L, but having a size close to that of the sum of M and L. pLM1809 contains the sequence of the three segments in the order S, M, L with the addition of a 427-base lacZα fragment to attain the size of the complete genome. The aim was to prepare a transcript with the pac site of S that was of the same size as the total Φ6 genome.

Figure 2

Autoradiogram of gel analysis. In vitro packaging of labeled plus strands showing competition for packaging by RNA that is not packaged. Equal amounts of ³²P-labeled transcripts of plasmids pLM658 and pLM656 were used for segments S and M, respectively. Plasmid pLM1157, which yields a 1.5-kb transcript, was used for segment L because it packages better than normal segment L. The packaging of the three segments is illustrated in lane 1, which shows an autoradiogram of an agarose gel analysis of a packaging mixture that had been treated with RNase I to degrade any unpackaged RNA. The conditions of packaging were as described (12). The effects of competition on packaging are shown in lanes 2 and 3, where a 20-fold excess of competitor RNA is added. In lane 2 the RNA is a normal segment M truncated at the SalI site at 1192. In lane 3 the RNA is derived from plasmid 794 also truncated at 1192. Plasmid pLM794 produces a transcript with a deletion of nucleotides 11–43. In lanes 4 and 5 the packaging of the competitor RNAs are shown in the absence of the normal plus strands.

Figure 3

(A) The packaging model. The procapsid shows only binding sites for S at the beginning. After a full size S is packaged, the S sites disappear and M sites appear. After a full size M is packaged, the M sites disappear and L sites appear. After a full size L is packaged, minus-strand synthesis commences. After minus-strand synthesis is completed, plus-strand synthesis commences. (B) If segment S is of the size equal to the sum of both S and M, the S sites will disappear and the L sites will appear and segment L will be packaged without segment M.

Figure 4

In vitro packaging of labeled plus strands showing that segment L can be packaged in the absence of M if segment S is made larger. The RNA in the packaging mixtures is shown below each lane. Segment S is derived from pLM658, segment M from 656, and segment L from pLM1157 as in Fig. 2. Segment S7 is the transcript of pLM1725 (Fig. 1) which is 6.8 kb. Note that segment S packages alone, whereas M and L package poorly alone. Segment M packages well in the presence of S and segment L packages well in the presence of S and M. Note that segment S7 packages alone (lane 4) and that segment L packages with segment S7 (lane 8) but not well with either S alone or M alone.

Figure 5

Minus-strand synthesis showing that segment L can be packaged in the presence of segment S7 and turn on minus-strand synthesis. Unlabeled plus-strand RNA was added to procapsids in the presence of labeled NTPs as described (12). Minus-strand synthesis normally occurs when all three segments are packaged. Segment S does not support packaging or minus-strand synthesis of segment L, but S7 does.

Figure 6

Minus-strand synthesis without segment L. Unlabeled RNA was added to procapsids along with labeled NTPs as described (12). Segment S alone or S and M together do not turn on minus-strand synthesis, but S, M, and L together do promote synthesis. Segment S and the transcript of pLM1827 (Fig. 1) do turn on minus-strand synthesis although the latter transcript contains no sequence of segment L. The pLM1827 transcript is 9.8 kb, which is close to the sum of M plus L, 10.5 kb. The transcript of pLM1822, which is 6.8 kb, does not turn on minus-strand synthesis. Lane 3, which has RNA of S, M, and L, is exposed for one-tenth the time of the other lanes. dsRNA, double-stranded RNA.

Figure 7

Plus-strand synthesis without segment L. Unlabeled RNA was added to procapsids along with labeled NTPs as described (12), but with 1 mM Mn²⁺ in addition to the 3 mM Mg²⁺ and 80 mM NH₄ acetate instead of 100 mM to promote plus-strand synthesis. In lane 1, segments L, M, and S served as templates for minus-strand synthesis (L, M, S) and for plus-strand synthesis (m, s). The plus strand of segment L is obscured by S. In lane 2 minus-strand synthesis occurs in the presence of intact segments S and M when packaged with segment L truncated at EcoRV (Fig. 1). In this case plus-strand synthesis does not occur as noted previously. Minus-strand synthesis is observed in lane 3 because Mn allows minus-strand synthesis without complete packaging (see Fig. 6). Plus-strand synthesis is seen in lane 4 for both segment S and the transcript of pLM1827, even though this transcript has no sequences of L. In lane 5 minus- and plus-strand synthesis is seen for the transcript of pLM1809 (Fig. 1), which has the pac site of S and the entire genetic complement of Φ6 and a size of 13.5 kb. Lanes 1 and 2 were exposed for one-tenth the time of the other lanes. dsRNA, double-stranded RNA; ssRNA, single-stranded RNA.