Double-stranded RNA bacteriophage phi 6 protein P4 is an unspecific nucleoside triphosphatase activated by calcium ions.

Double-stranded RNA bacteriophage phi 6 has an envelope surrounding the nucleocapsid (NC). The NC is composed of a surface protein, P8, and proteins P1, P2, P4, and P7, which form a dodecahedral polymerase complex enclosing the segmented viral genome. Empty polymerase complex particles (procapsids) package positive-sense viral single-stranded RNAs provided that energy is available in the form of nucleoside triphosphates (NTPs). Photoaffinity labelling of both the NC and the procapsid has earlier been used to show that ATP binds to protein P4 and that the NC hydrolyzes NTPs. Using the NC and the NC core particles (NCs lacking surface protein P8) and purified protein P4, we demonstrate here that multimeric P4 is the active NTPase. Isolation of multimeric P4 is successful only in the presence of NTPs. The activity of P4 is the same in association with the viral particles as it is in pure form. P4 is an unspecific NTPase hydrolyzing ribo-NTPs, deoxy NTPs, and dideoxy NTPs to the corresponding nucleoside diphosphates. The Km of the reaction for ATP, GTP, and UTP is around 0.2 to 0.3 mM. The NTP hydrolysis by P4 absolutely requires residual amounts of Mg2+ ions and is greatly activated when the Ca2+ concentration reaches 0.5 mM. Competition experiments indicate that Mg2+ and Ca2+ ions have approximately equal binding affinities for P4. They might compete for a common binding site. The nucleotide specificity and enzymatic properties of the P4 NTPase are similar to the NTP hydrolysis reaction conditions needed to translocate and condense the viral positive-sense RNAs to the procapsid particle.     

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.     

Dependence of minus-strand synthesis on complete genomic packaging in the double-stranded RNA bacteriophage phi 6.

Bacteriophage phi 6 has a segmented genome consisting of three pieces of double-stranded RNA (dsRNA). The viral procapsid is the structure that packages plus strands, synthesizes the complementary negative strands to form dsRNA, and then transcribes dsRNA to form plus-strand message. The minus-strand synthesis of a particular genomic segment is dependent on prior packaging of the other segments. The 5' end of the plus strand is necessary and sufficient for packaging, while the normal 3' end is necessary for synthesis of the negative strand. We have now investigated the ability of truncated RNA segments which lack the normal 3' end of the molecules to stimulate the synthesis of minus strands of the other segments. Fragments missing the normal 3' ends were able to stimulate the minus-strand synthesis of intact heterologous segments. Minus-strand synthesis of one intact segment could be stimulated by the presence of two truncated nonreplicating segments. The 5' fragments of each single-stranded genomic segment can compete with homologous full-length single-stranded genomic segments in minus-strand synthesis reactions, suggesting that there is a specific binding site in the procapsid for each segment.     

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 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.     

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.     

Protein P4 of double-stranded RNA bacteriophage phi 6 is accessible on the nucleocapsid surface: epitope mapping and orientation of the protein.

Protein P4, an early protein of double-stranded RNA bacteriophage phi 6, is a component of the virion-associated RNA polymerase complex and possesses a nucleoside triphosphate (NTP) phosphohydrolase activity. We have produced and characterized a panel of 20 P4-specific monoclonal antibodies. Epitope mapping using truncated molecules of recombinant P4 revealed seven linear epitopes. The accessibility of the epitopes on the phi 6 nucleocapsid (NC) surface showed that at least the C terminus and an internal domain, containing the consensus sequence for NTP binding, protrude the NC shell. Four of the NC-binding antibodies distorted the integrity of the NC by releasing protein P4 and the major NC surface protein P8. This finding suggests a close contact between these two proteins. The dissociation of the NC led to the activation of the virion-associated RNA polymerase. The multimeric status of the recombinant P4 was similar to that of the virion-associated P4, indicating that no accessory virus proteins are needed for its multimerization.     

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.     

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.     

Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity.

Bacteriophage phi 6 contains three segments of double-stranded RNA. The procapsid consists of proteins P1, P2, P4, and P7, which are encoded by the viral L segment. cDNA copies of this segment have been cloned into plasmids that direct the production of these proteins, which assemble into polyhedral procapsids. These procapsids are capable of packaging plus-sense phi 6 RNA in the presence of nucleoside triphosphate and synthesizing the complementary minus strand to form double-stranded RNA. In this article, we report the presence of a nucleotide-binding site in protein P4. The viral procapsid and nucleocapsid exhibit a nucleoside triphosphate phosphohydrolase activity that converts nucleoside triphosphates into nucleoside diphosphates.     

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 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.