RNA structure and heterologous recombination in the double-stranded RNA bacteriophage phi 6.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA, designated L, M, and S. A 1.2-kbp kanamycin resistance gene was inserted into segment M but was shown to be genetically unstable because of a high recombination rate between segment M and the 3' ends of segments S and L. The high rate of recombination is due to complementary homopolymer tracts bounding the kan gene. Removal of one arm of this potential hairpin stabilizes the insertion. The insertion of a 241- or 427-bp lacZ' gene into segment M leads to a stable Lac+ phage. The insertion of the same genes bounded by complementary homopolymer arms leads to recombinational instability. A stable derivative of this phage was shown to have lost one of the homopolymer arms. Several other conditions foster recombination. The truncation of a genomic segment at the 3' end prevents replication, but such a damaged molecule can be rescued by recombination. Similarly, insertion of the entire 3-kb lacZ gene prevents normal formation of virus, but the viral genes can be rescued by recombination. It appears that conditions leading to the retardation or absence of replication of a particular genomic segment facilitate recombinational rescue.     

In vitro packaging and replication of individual genomic segments of bacteriophage phi 6 RNA.

The genome of bacteriophage phi 6 contains three segments of double-stranded RNA. Procapsid structures whose formation was directed by cDNA copies of the large genomic segment are capable of packaging the three viral message sense RNAs in the presence of ATP. Addition of UTP, CTP, and GTP results in the synthesis of minus strands to form double-stranded RNA. In this report, we show that procapsids are capable of taking up any of the three plus-strand single-stranded RNA segments independently of the others. In manganese-containing buffers, synthesis of the corresponding minus strand takes place. In magnesium-containing buffers, individual message sense viral RNA segments were packaged, but minus-strand replication did not take place unless all three viral single-stranded RNA segments were packaged. Since the conditions of packaging in magnesium buffer more closely resemble those in vivo, these results indicated that there is no specific order or dependence in packaging and that replication is regulated so that it does not begin until all segments are in place.     

RNA secondary structures of the bacteriophage phi6 packaging regions

Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.     

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

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.     

Nonspecific nucleoside triphosphatase P4 of double-stranded RNA bacteriophage phi6 is required for single-stranded RNA packaging and transcription

Bacteriophage phi6 has a segmented double-stranded RNA genome. The genomic single-stranded RNA (ssRNA) precursors are packaged into a preformed protein capsid, the polymerase complex, composed of viral proteins P1, P2, P4, and P7. Packaging of the genomic precursors is an energy-dependent process requiring nucleoside triphosphates. Protein P4, a nonspecific nucleoside triphosphatase, has previously been suggested to be the prime candidate for the viral packaging engine, based on its location at the vertices of the viral capsid and its biochemical characteristics. In this study we were able to obtain stable polymerase complex particles that are completely devoid of P4. Such particles were not able to package ssRNA segments and did not display RNA polymerase (either minus- or plus-strand synthesis) activity. Surprisingly, a mutation in P4, S250Q, which reduced the level of P4 in the particles to about 10% of the wild-type level, did not affect RNA packaging activity or change the kinetics of packaging. Moreover, such particles displayed minus-strand synthesis activity. However, no plus-strand synthesis was observed, suggesting that P4 has a role in the plus-strand synthesis reaction also.     

Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase.

The RNA polymerase in the nucleocapsid of Pseudomonas phaseolicola bacteriophage phi 6 transcribed large, medium, and small single-stranded RNA from the viral double-stranded RNA genome by a semiconservative (displacement) mechanism. Approximately 23%, 63%, and 65% of the nucleocapsid particles in the assay mixture synthesized at least one round of large, medium, and small single-stranded RNA molecules, respectively. Some of these particles reinitiated synthesis such that an average of 1.5 large, 33 medium, and 24 small single-stranded RNAs were synthesized from each double-stranded RNA.     

RNA dependence of the bacteriophage phi 29 DNA packaging ATPase.

The activity of the DNA packaging adenosine triphosphatase (ATPase) of the Bacillus subtilis bacteriophage phi 29 is dependent upon prohead RNA. The 174 nucleotide viral-encoded RNA is positioned on the head-tail connector at the portal vertex of the phi 29 precursor shell (prohead). Here, the RNA interacts with the ATP-binding gene 16 product (gp16) to constitute the DNA-packaging ATPase and initiate DNA packaging in vitro. Both the prohead connector (gene 10 product, gp10) and gp16 may utilize an RNA recognition motif characteristic of a number of RNA-associated proteins, and the binding of gp16 by proheads shields the prohead RNA from RNase A. The ATPase activity of gp16 is stimulated fourfold by RNA and tenfold by proheads with RNA. RNA is needed continuously for the gp16/RNA ATPase activity and is essential for the gp16/prohead ATPase activity. The prohead, with its connector, RNA and associated gp16 in an assembly-regulated configuration, hydrolyzes ATP and drives phi 29 DNA translocation.     

Role of RNA in bacteriophage phi 29 DNA packaging

A novel bacteriophage phi 29 RNA of 174 nucleotides is essential for the in vitro packaging of the DNA-terminal protein complex into proheads. The RNA, bound to the prohead portal vertex (connector), participates in assembly and function of the DNA translocating ATPase and in recognition of the DNA left-end during the course of the packaging reaction. The RNA is present in related phages and varies widely in primary sequence, but its secondary structure, as deduced by phylogenetic analysis, is both highly conserved and unique among small RNAs.     

Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6

In nature, synthesis of both minus- and plus-sense RNA strands of all the known double-stranded RNA viruses occurs in the interior of a large protein assembly referred to as the polymerase complex. In addition to other proteins, the complex contains a putative polymerase possessing characteristic sequence motifs. However, none of the previous studies has shown template-dependent RNA synthesis directly with an isolated putative polymerase protein. In this report, recombinant protein P2 of double-stranded RNA bacteriophage phi6 was purified and demonstrated in an in vitro enzymatic assay to act as the replicase. The enzyme efficiently utilizes phage-specific, positive-sense RNA substrates to produce double-stranded RNA molecules, which are formed by newly synthesized, full-length minus-strands base paired with the plus-strand templates. P2-catalyzed replication is also shown to be very effective with a broad range of heterologous single-stranded RNA templates. The importance and implications of these results are discussed.     

Structure of the bacteriophage phi6 nucleocapsid suggests a mechanism for sequential RNA packaging

Bacteriophage phi6 is an enveloped dsRNA virus with a segmented genome. Phi6 specifically packages one copy of each of its three genome segments into a preassembled polymerase complex. This leads to expansion of the polymerase complex, minus and plus strand RNA synthesis, and assembly of the nucleocapsid. The phi6 in vitro assembly and packaging system is a valuable model for dsRNA virus replication. The structure of the nucleocapsid at 7.5 A resolution presented here reveals the secondary structure of the two major capsid proteins. Asymmetric P1 dimers organize as an inner T = 1 shell, and P8 trimers organize as an outer T = 13 laevo shell. The organization of the P1 molecules in the unexpanded and expanded polymerase complex suggests that the expansion is accomplished by rigid body movements of the P1 monomers. This leads to exposure of new potential RNA binding surfaces to control the sequential packaging of the genome segments.     

Characterization of the small RNA of the bacteriophage phi 29 DNA packaging machine.

The prohead connector of the bacteriophage luminal diameter 29 DNA packaging machine was reconstructed with the small RNA that regulates DNA packaging in vitro. The complete sequence of the 120 nucleotide RNA proved its origination from the promoter PE1(A1) of the left early region of phi 29 DNA, the end packaged first during assembly. The prohead RNA was clearly distinct from eubacterial 5S rRNA in sequence and composition.     

Intermediates in the assembly pathway of the double-stranded RNA virus phi6.

The double-stranded RNA bacteriophage phi6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E. coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the phi6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures.     

In vitro synthesis of late bacteriophage phi 29 RNA.

A crude P-100 fraction prepared from Bacillus subtilis 21 min after infection with wild-type phage phi 29 supported the in vitro synthesis of late phi 29 RNA by added RNA polymerase. Synthesis of late RNA was also detected when purified phi 29 DNA was transcribed by RNA polymerase in the presence of an S-150 fraction obtained by lysis of phi 29-infected cells in the presence of 1 M NaCl. Late phi 29 RNA was not synthesized when either the P-100 or the S-150 fraction was prepared from cultures infected with phi 29 having a mutation in gene 4.