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

Abstract

Bacteriophage phi6 has a genome of three segments of double-stranded RNA. Each virus particle contains one each of the three segments. Packaging is effected by the acquisition, in a serially dependent manner, of the plus strands of the genomic segments into empty procapsids. The empty procapsids are compressed in shape and expand during packaging. The packaging program involves discrete steps that are determined by the amount of RNA inside the procapsid. The steps involve the exposure and concealment of binding sites on the outer surface of the procapsid for the plus strands of the three genomic segments. The plus strand of segment S can be packaged alone, while packaging of the plus strand of segment M depends upon prior packaging of S. Packaging of the plus strand of L depends upon the prior packaging of M. Minus-strand synthesis begins when the particle has a full complement of plus strands. Plus-strand synthesis commences upon the completion of minus-strand synthesis. All of the reactions of packaging, minus-strand synthesis, and plus-strand synthesis can be accomplished in vitro with isolated procapsids. Live-virus constructions that are in accord with the model have been prepared. Mutant virus with changes in the packaging program have been isolated and analyzed.

FIG. 1

The φ6 virion is composed of a procapsid containing proteins P1, P2, P4, and P7, which is covered by a shell of protein P8 to form the nucleocapsid. The nucleocapsid is enveloped in a lipid-containing membrane composed of phospholipids and proteins P9, P10, P13, P3, and P6. P3 specifies the host range of the virus. P511 is a lytic endopeptidase that is associated with the surface of the nucleocapsid (4, 43).

FIG. 2

Life cycle of φ6. The virion attaches to a pilus and is brought into contact with the outer membrane (om). The viral membrane fuses with the outer membrane to place the nucleocapsid in the periplasmic space. The murein (cw) is digested by viral protein P5, and the filled procapsid (fpc) penetrates the inner membrane (im) and enters the cell, leaving P8 behind. The procapsid transcriptase synthesizes complete copies of the three genomic segments. The L mRNA is translated to produce P1, P2, P4, and P7, which constitute the procapsid (pc). This is filled with dsRNA and continues transcription until it is covered by P8 to form nucleocapsids (nc). Membrane proteins are placed in the host membrane and then transferred to the virion (mv) along with host lipids. The membrane formation or translocation is dependent upon protein P12.

FIG. 3

Physical and genetic maps of the genomic segments of φ6. Genes are identified below the segments (6, 43). The sequences necessary for packaging are located in the 5′ noncoding regions (left). The sequences necessary for minus-strand synthesis are at the 3′ ends.

FIG. 4

Reconstructions of the structures of empty procapsids and filled viral cores of φ6 determined from cryoelectron micrographs of procapsid particles containing proteins P1 and P4 (A and B) and cores visualized from viral nucleocapsids that have been stripped of the shell of protein P8 (C and D) (3). The structures in panels C and D are cut-open representations showing the inner surfaces of the structures viewed down a twofold axis of symmetry. The structures in panels A and B resemble particles that have not packaged RNA, while those in panels C and D resemble those that have packaged RNA and carried out minus-strand synthesis.

FIG. 5

In vitro packaging of radioactive plus strands of exact copies of genomic segments S and M and of a truncated segment L. Radioactive transcripts of plasmids pLM659 (s), pLM656 (m), and pLM1157 (l) were incubated with procapsids, treated with RNase I, and applied to a 2% agarose gel (67). An l strand of reduced size is used because its packaging is more efficient than that of normal-sized segment l. Note the dependence relationships in packaging.

FIG. 6

Diagram of the packaging of normal RNA and a hairpin structure by procapsids. The diagram is not to scale, and the small secondary structure is the normal 3′ end of the genomic segments. The upper pathway is that of a normal segment, and the lower pathway is that of a hairpin structure (68).

FIG. 7

Secondary structure of the pac region of segment M of φ6. The first 305 nucleotides of segment M can be packaged by procapsids (24). The first 268 nucleotides cannot be packaged. Deletions of nucleotides 11 to 18, nucleotides 17 to 36, or nucleotides 23 to 43 does not prevent packaging, but deletion of nucleotides 11 to 43 does prevent packaging. Deletion of nucleotides 11 to 33 is not packaged but competes for packaging with normal segment M. The bases shown with arrows are all the changes in this region between φ6 and the related bacteriophage, φ7. It is notable that most of the changes found in proposed hairpin stems are compensated by changes that maintain base pairing.

FIG. 8

Secondary structure of the pac region of segment S of φ6. The first 270 nucleotides are sufficient for packaging (24). The first 234 nucleotides cannot be packaged. Deletion of nucleotides 11 to 23 did not prevent packaging, but deletion of nucleotides 11 to 32 resulted in an RNA that competed with normal RNA for packaging but was not packaged itself. Deletion of nucleotides 11 to 43 resulted in RNA that neither competed nor packaged.

FIG. 9

Secondary structure of the pac region of segment L of φ6. The first 205 nucleotides are sufficient for packaging (24).

FIG. 10

dsRNA extracted from virions with deletions in segment M. Lanes: v, wild-type virus, a, virus missing gene 3 of segment M; b and c, virus as in lane a that has acquired transcript RNA as a fourth segment. M_t is the segment formed from the transcript, and M_Δ is the segment missing gene 3.

FIG. 11

(A) Packaging model (69). The procapsid shows only binding sites for segment S at the beginning. After a full-sized S is packaged, the S sites disappear and M sites appear. After a full-sized M is packaged, the M sites disappear and L sites appear. After a full-sized L is packaged, minus-strand synthesis commences. After minus-strand synthesis is completed, plus-strand synthesis commences. (B) If segment S is of a size equal to the sum of both S and M, the S sites will disappear after packaging of the 7-kb segment, the L sites will appear, and segment L will be packaged without segment M.

FIG. 12

dsRNA of virions with one or two genomic segments (62). Lanes: v, RNA from wild-type virus; a, RNA from a virus construct with a deletion in segment M; b, RNA from a virus containing normal segment S, normal segment L, and a chimera of M and S in which the pac sequence is that of M; c, RNA from a virus with normal L and the MS chimera and a segment S that has no genes; d, RNA from a two-segment virus in which there is normal segment L and a chimera of S and M with the pac sequence of S.

FIG. 13

dsRNA from a virion that has the entire genome in one 14-kbp segment with the pac sequence of S. Lanes: v, RNA from wild type virus; a, RNA from the virus with the entire genome in one segment (62).

FIG. 14

Minus-strand synthesis reactions of normal and mutant procapsids (61). Procapsids were prepared from cells producing proteins containing the mutation of ERA12 that results in a carrier-state infection where segment S is missing from the procapsids. Nonradioactive plus-strand RNA was added alone (s, m, l) or in combinations (sm, sl, ml, sml) to procapsids under conditions for minus-strand synthesis with radioactive nucleotides. Note that procapsids containing mutant P1 do not need segment S for minus-strand synthesis of segments M and L.