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.     

A symmetry mismatch at the site of RNA packaging in the polymerase complex of dsRNA bacteriophage phi6

The polymerase complex of the enveloped double-stranded RNA (dsRNA) bacteriophage phi6 fulfils a similar function to those of other dsRNA viruses such as Reoviridae. The phi6 complex comprises protein P1, which forms the shell, and proteins P2, P4 and P7, which are involved in RNA synthesis and packaging. Icosahedral reconstructions from cryo-electron micrographs of recombinant polymerase particles revealed a clear dodecahedral shell and weaker satellites. Difference imaging demonstrated that these weak satellites were the sites of P4 and P2 within the complex. The structure determined by icosahedral reconstruction was used as an initial model in an iterative reconstruction technique to examine the departures from icosahedral symmetry. This approach showed that P4 and P2 contribute to structures at the 5-fold positions of the icosahedral P1 shell which lack 5-fold symmetry and appear in variable orientations. Reconstruction of isolated recombinant P4 showed that it was a hexamer with a size and shape matching the satellite. Symmetry mismatch between the satellites and the shell could play a role in RNA packaging akin to that of the portal vertex of dsDNA phages in DNA packaging. This is the first example of dsRNA virus in which the structure of the polymerase complex has been determined without the assumption of icosahedral symmetry. Our result with phi6 illustrates the symmetry mismatch which may occur at the sites of RNA packaging in other dsRNA viruses such as members of the Reoviridae.     

Characterization of subunit-specific interactions in a double-stranded RNA virus: Raman difference spectroscopy of the phi6 procapsid

The icosahedral core of a double-stranded (ds) RNA virus hosts RNA-dependent polymerase activity and provides the molecular machinery for RNA packaging. The stringent requirements of dsRNA metabolism may explain the similarities observed in core architecture among a broad spectrum of dsRNA viruses, from the mammalian rotaviruses to the Pseudomonas bacteriophage phi6. Although the structure of the assembled core has been described in atomic detail for Reoviridae (blue tongue virus and reovirus), the molecular mechanism of assembly has not been characterized in terms of conformational changes and key interactions of protein constituents. In the present study, we address such questions through the application of Raman spectroscopy to an in vitro core assembly system--the procapsid of phi6. The phi6 procapsid, which comprises multiple copies of viral proteins P1 (copy number 120), P2 (12), P4 (72), and P7 (60), represents a precursor of the core that is devoid of RNA. Raman signatures of the procapsid, its purified recombinant core protein components, and purified sub-assemblies lacking either one or two of the protein components have been obtained and interpreted. The major procapsid protein (P1), which forms the skeletal frame of the core, is an elongated and monomeric molecule of high alpha-helical content. The fold of the core RNA polymerase (P2) is also mostly alpha-helical. On the other hand, the folds of both the procapsid accessory protein (P7) and RNA-packaging ATPase (P4) are of the alpha/beta type. Raman difference spectra show that conformational changes occur upon interaction of P1 with either P4 or P7 in the procapsid. These changes involve substantial ordering of the polypeptide backbone. Conversely, conformations of procapsid subunits are not significantly affected by interactions with P2. An assembly model is proposed in which P1 induces alpha-helix in P4 during formation of the nucleation complex. Subsequently, the partially disordered P7 subunit is folded within the context of the procapsid shell.     

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.     

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.     

RNA packaging device of double-stranded RNA bacteriophages,possibly as simple as hexamer of P4 protein

Genomes of complex viruses have been demonstrated, in many cases, to be packaged into preformed empty capsids (procapsids). This reaction is performed by molecular motors translocating nucleic acid against the concentration gradient at the expense of NTP hydrolysis. At present, the molecular mechanisms of packaging remain elusive due to the complex nature of packaging motors. In the case of the double-stranded RNA bacteriophage phi 6 from the Cystoviridae family, packaging of single-stranded genomic precursors requires a hexameric NTPase, P4. In the present study, the purified P4 proteins from two other cystoviruses, phi 8 and phi 13, were characterized and compared with phi 6 P4. All three proteins are hexameric, single-stranded RNA-stimulated NTPases with alpha/beta folds. Using a direct motor assay, we found that phi 8 and phi 13 P4 hexamers translocate 5' to 3' along ssRNA, whereas the analogous activity of phi 6 P4 requires association with the procapsid. This difference is explained by the intrinsically high affinity of phi 8 and phi 13 P4s for nucleic acids. The unidirectional translocation results in RNA helicase activity. Thus, P4 proteins of Cystoviridae exhibit extensive similarity to hexameric helicases and are simple models for studying viral packaging motor mechanisms.     

Immunological cross-reactions and interactions between the Drosophila melanogaster ref(2)P protein and sigma rhabdovirus proteins.

The ref(2)P gene is one of the Drosophila melanogaster genes involved in the inhibition of sigma rhabdovirus multiplication. The partial restriction of viral replication varies according to the ref(2)P alleles and virus strains and involves intracellular interactions between parasite and host products. We identified the protein encoded by the ref(2)P gene and produced polyclonal antibodies directed against the whole ref(2)P protein obtained from a recombinant baculovirus and against a part of the protein expressed as a fusion protein. These antibodies were used to study the interactions with sigma virus proteins by different immunoprecipitation techniques. We showed that the native ref(2)P protein shared conformation-dependent common epitopes with the viral structural genome-associated N protein. Furthermore, the cellular protein was found to be associated in complexes with the viral P protein required for RNA polymerase activity. The significance of these observations in the control of sigma virus multiplication by its host is discussed.     

Mutational Analysis of the Role of Nucleoside Triphosphatase P4 in the Assembly of the RNA Polymerase Complex of Bacteriophage φ6

Bacteriophage 6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the Reoviridae. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.     

The nucleocapsid of the lipid-containing double-stranded RNA bacteriophage phi 6 contains a protein skeleton consisting of a single polypeptide species.

The nucleocapsid of the enveloped double-stranded RNA bacteriophage phi 6 was isolated by extraction with the nonionic detergent Triton X-114 and subjected to disruption analysis with chelating and protein-denaturing agents. The subnucleocapsid particles were separated in rate-zonal sucrose gradients, and their ultrastructure and protein composition were analyzed. The role of divalent cations in the nucleocapsid structure was studied by using a precipitation assay of the isolated nucleocapsid proteins. The phi 6 nucleocapsid had a cagelike skeleton consisting of a single polypeptide species (P1). Two other proteins (P2 and P4) were associated with the P1 cage. These three early proteins were previously known to be involved in the RNA synthesis machinery of the virus. The stability of the nucleocapsid surface lattice consisting of protein P8 was dependent on Ca2+ ions.     

In vitro translation of the three bacteriophage phi 6 RNAs.

In vitro translation of the three single-stranded RNAs transcribed in vitro by bacteriophage phi 6 RNA polymerase revealed that the large RNA codes for phage proteins P1, P2, P4, and P7, the medium RNA codes for P3, P6, and P10, and the smaller RNA for P5, P8, and P9.     

Long-term replication of Sendai virus defective interfering particle nucleocapsids in stable helper cell lines.

An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.     

Characterization of Lactococcus lactis phage antigens.

Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail). In order to develop an immunoenzymatic phage detection test, fusion proteins containing beta-galactosidase fused to epitopes of phage phi 197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector. Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac+ phenotype on Escherichia coli. Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined. Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope. This epitope was very immunogenic. Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter. Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope. Antibody cross-reactivity of phages related to phi 197 showed that this epitope is well conserved in this genetic group.     

Characterization of Lactococcus lactis phage antigens.

Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail). In order to develop an immunoenzymatic phage detection test, fusion proteins containing beta-galactosidase fused to epitopes of phage phi 197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector. Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac+ phenotype on Escherichia coli. Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined. Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope. This epitope was very immunogenic. Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter. Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope. Antibody cross-reactivity of phages related to phi 197 showed that this epitope is well conserved in this genetic group.     

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.