Bacteriophage Φ29 Research Articles

Locating the minor components of double-stranded RNA bacteriophage φ6 by neutron scattering

The polymerase core of double-stranded (ds) RNA virus provides the molecular machinery for RNA packaging and replication. Procapsid of bacteriophage φ6 constitutes the best studied model of such dsRNA-processing machine. A total of 120 copies of protein P1 form the procapsid framework to which other proteins are attached. Due to their low abundance minor procapsid constituents, P2 (RNA polymerase) and P7 (packaging factor), have not up to now been localized. We have applied small-angle neutron scattering (SANS) and contrast variation in order to localize the two proteins. Procapsids containing deuterated P2 or P7 were produced in vitro and SANS was collected at several contrast levels. Radial positions of labeled proteins were obtained and modeled within the electron density of the procapsid. P2 monomers reside at each five-fold vertex just under the RNA packaging complex. P7 was detected at a distance of 160 Å from the procapsid center indicating localization on the inner surface of P1 framework.

Mechanisms of coexistence of a bacteria and a bacteriophage in a spatially homogeneous environment

AbstractWhen bacteriophage are added to laboratory bacteria populations, bacteria mutants that are resistant to the phage quickly dominate the population. The phage will only persist in the long‐term if there are sufficient bacteria in the population that show susceptibility to the phage. We investigated the mechanisms allowing for coexistence by adding the virulent bacteriophage φ6 to cultures of the bacterium Pseudomonas syringae pv. phaseolicola in a spatially homogeneous environment. We saw large differences between replicate cultures, in particular when one or both of the species persisted. These differences can be explained by variation in the timing of the appearance of various resistant phenotypes in the bacteria populations before the phage were added, which determines their relative frequencies within the populations. Although these resistant phenotypes have similar fitnesses in the presence and in the absence of the phage, they have a profound effect on the persistence of the phage. Our results give a clearer understanding of the ecological mechanisms that lead to the coexistence of bacteria and virulent phage in environments where there are no spatial refuges available to the bacteria population.

Escape from Prisoner's Dilemma in RNA phage phi6.

We previously examined competitive interactions among viruses by allowing the RNA phage phi6 to evolve at high and low multiplicities of infection (ratio of infecting viruses to bacterial cells). Derived high-multiplicity phages were competitively advantaged relative to their ancestors during coinfection, but their fixation caused population fitness to decline. These data conform to the evolution of lowered fitness in a population of defectors, as expected from the Prisoner's Dilemma of game theory. However, the generality of this result is unknown; the evolution of viruses at other multiplicities may alter the fitness payoffs associated with conflicting strategies of cooperation and defection. Here we examine the change in matrix variables by propagating the ancestor under strictly clonal conditions, allowing cooperation the chance to evolve. In competitions involving derived cooperators and their selfish counterparts, our data reveal a new outcome where the two strategies are predicted to coexist in a mixed polymorphism. Thus, we demonstrate that the payoff matrix is not a constant in phi6. Rather, clonal selection allows viruses the opportunity to escape the Prisoner's Dilemma. We discuss mechanisms that may afford selfish genotypes an advantage during intrahost competition and the relevance in our system for alternative ecological interactions among viruses.

φ29 DNA Polymerase Residue Phe128 of the Highly Conserved (S/T)Lx 2h Motif is Required for a Stable and Functional Interaction with the Terminal Protein

Bacteriophage φ29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication. By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx 2h motif, and a glutamate belonging to the Exo III motif. Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of φ29 DNA polymerase. Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As a consequence, very few initiation products were obtained, and essentially no transition products were detected. Interestingly, φ29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules. These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps.

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.

Modulation of Hepatitis C Virus RNA-dependent RNA Polymerase Activity by Structure-based Site-directed Mutagenesis

The hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (NS5B), which is indispensable for the viral genome replication. Although structural comparison among HCV NS5B, poliovirus 3D-pol, and human immunodeficiency virus-reverse transcriptase RNA-dependent polymerase reveals the canonical palm, fingers, and thumb domains, the crystal structure of HCV NS5B highlights the presence of a unique A1-loop, which extends from the fingers to the thumb domain (amino acids 12-46), providing many contact points for the proposed "closed" conformation of the enzyme. The polymerase also possesses a tunnel, which starts at the active site and terminates on the back surface of the enzyme. This tunnel of 19 A contains five basic amino acids, which may be engaged in NTP trafficking. In the present study, we exploited the crystal structure of the enzyme to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis. As predicted, the replacement of leucine 30 located in the Lambda 1-loop is detrimental to the NS5B activity. Heparin-Sepharose column chromatography and analytical ultracentrifugation experiments strongly suggest a local alteration in the structure of the Leu-30 mutant. An analysis of amino acid substitutions in Arg-222 and Lys-151 within the putative NTP tunnel indicates that Arg-222 was critical in delivering NTPs to the active site, whereas Lys-151 was dispensable. Interestingly, the substitution of lysine 151 for a glutamic acid resulted in an enzyme that was consistently more active in de novo synthesis as well as by "copy-back" mechanism of a self-primed substrate when compared with the wild type NS5B enzyme. Burst kinetic analyses indicate that the gain in function of K151E enzyme was primarily the result of the formation of more productive pre-initiation complexes that were used for the elongation reaction. In contrast to the recent observations, both the wild type and mutant enzymes were monomeric in solution, whereas molecules of higher order were apparent in the presence of RNA template.

Implication of the prohead RNA in phage φ29 DNA packaging

RNA is an important component of many biological processes, including DNA encapsidation of bacteriophage φ29 of Bacillus subtilis. Interestingly, the prohead RNA is involved in this encapsidation, and was found in monomer, dimer, pentamer and hexamer conformations. This article presents and debates current knowledge about the prohead RNA structures, mechanisms, and roles in DNA encapsidation. A new dimer structure is presented, and its specific role in DNA encapsidation is discussed.

The in vivo function of phage φ29 nucleoid-associated protein p6 requires formation of dimers

The Bacillussubtilis phage φ29 nucleoid-associated protein p6 (103 amino acids) is essential for in vivo viral DNA replication and control of transcription, and it has been proposed to play a role in genome organization and compaction. This protein self-associates in vitro from preformed dimers forming high-molecular-weight oligomers and binds to double-stranded DNA giving rise to multimeric nucleoprotein complexes. Site-directed mutants, p6I8T and p6A44V, were completely or partially inactive, respectively, in an in vitro dimerization assay. In this paper, and by in vivo crosslinking, we have detected dimers of protein p6 either in phage-infected cells or in protein p6 producing B. subtilis or Escherichiacoli cells. Therefore, this self-association does not require viral DNA. We also show that mutants p6I8T and p6A44V are deficient in dimer formation, and they do not support phage DNA replication in a trans-complementation assay with φ29sus6 mutant-infected B. subtilis cells. We conclude that dimeric protein p6 is the active form of the protein in vivo, required for viral DNA replication.

Note to the Paper by Guasch et al. (2002) Detailed Architecture of a DNA Translocating Machine: The High-resolution Structure of the Bacteriophage φ29 Connector Particle

The recent paper by Guasch et al.1 describing the structure of the ϕ29 bacteriophage head–tail connector at 2.1 A resolution (PDB code 1h5w), makes reference to the original 3.2 A resolution structure (PDB code 1fou) published by Simpson et al.2 and used by Guasch et al. to solve the 1h5w structure by molecular replacement. The Guasch paper states that there are marked differences between the 1h5w and 1fou structures in the chain tracing and the side-chain positioning in the wide domain. As these and a number of other statements in the Guasch paper may have led to the impression that these differences are larger than they really are, we give here Tables summarizing the similarities and differences (Tables 1 and ​and22). Table 1 Statistical comparison of the ϕ29 connector wide domains in different structures Table 2 Superposition of the entire monomers in the four reported structures Another paper by Simpson et al.,3 which was accidentally omitted from the text of the Guasch et al. paper, reports two additional structures of the ϕ29 connector: at 3.2 A resolution and at 2.9 A resolution (PDB codes 1jnb and 1ijg, respectively). The 2.9 A resolution structure shows no significant differences from the structure subsequently reported by Guasch et al. (Figure 1; Tables 1 and ​and22). Figure 1 Stereo diagram showing the superposition of the Cα back-bone of monomer A in the 2.1 A resolution structure 1h5w (green) and the 2.9 A resolution 1ijg structure (red).

Characterization of φ12, a Bacteriophage Related to φ6: Nucleotide Sequence of the Large Double-Stranded RNA

The isolation of additional bacteriophages besides φ6 containing segmented double-stranded RNA genomes (dsRNA) has expanded the Cystoviridae family to nine members. Comparing the genomic sequences of these viruses has allowed evaluation of important genetic as well as structural motifs. These comparative studies are resulting in greater understanding of viral evolution and the role played by genetic and structural variation in the assembly mechanisms of the cystoviruses. In this regard, the large double-stranded RNA genomic segment of bacteriophage φ12 was copied as cDNA and its nucleotide sequence determined. This genome's organization is similar to that of the large segment of bacteriophages φ6, φ8, and φ13. In the amino acid sequence of the viral RNA-dependent RNA polymerase (P2), similarity was found to the comparable proteins of φ6, φ8, and φ13. Amino acid sequence similarity was also noted in the nucleotide triphosphate phosphorylase (P4) to the comparable proteins of φ8 and φ13.

Characterization of φ12, a Bacteriophage Related to φ6: Nucleotide Sequence of the Small and Middle Double-Stranded RNA

The isolation of additional bacteriophages containing segmented double-stranded RNA genomes has expanded the Cystoviridae family to nine members. Comparing the genomic sequences of these viruses has allowed evaluation of important genetic as well as structural motifs. These comparative studies are resulting in greater understanding of viral evolution and the role played by genetic and structural variation in the assembly mechanisms of the cystoviruses. In this regard, the small and middle double-stranded RNA genomic segments of bacteriophage φ12 were copied as cDNA and their nucleotide sequences determined. This genome's organization is similar to that of the small and middle segments of bacteriophages φ6, φ8, and φ13. Although there is little similarity in the nucleotide sequences, similarity exists in the amino acid sequence of the lysis cassette proteins to those of φ6. The host cell attachment proteins are found to have marked similarity to the φ13 attachment proteins.

The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.

Detailed architecture of a DNA translocating machine: the high-resolution structure of the bacteriophage φ29 connector particle

The three-dimensional crystal structure of the bacteriophage φ29 connector has been solved and refined to 2.1 Å resolution. This 422 kDa oligomeric protein connects the head of the phage to its tail and translocates the DNA into the prohead during packaging. Each monomer has an elongated shape and is composed of a central, mainly α-helical domain that includes a three-helix bundle, a distal α/β domain and a proximal six-stranded SH3-like domain. The protomers assemble into a 12-mer, propeller-like, super-structure with a 35 Å wide central channel. The surface of the channel is mainly electronegative, but it includes two lysine rings 20 Å apart. On the external surface of the particle a hydrophobic belt extends to the concave area below the SH3-like domain, which forms a crown that retains the particle in the head. The lipophilic belt contacts the non-matching symmetry vertex of the capsid and forms a bearing for the connector rotation. The structure suggests a translocation mechanism in which the longitudinal displacement of the DNA along its axis is coupled to connector spinning.

Bacteriophage φ29 DNA packaging

This chapter discusses the DNA packaging mechanisms of bacteriophage ϕ29. The tailed double-stranded DNA phages share many common features of the packaging process and use a common mechanism. They all package their viral DNA unidirectionally into a preformed capsid precursor called “the prohead.” The DNA substrate for packaging is either a concatemer or a unit-length molecule, depending on the replication strategy of the particular phage. The phages have two packaging proteins, which are responsible for the maturing and the translocation of the DNA. The smaller protein of the pair binds to the DNA while the larger protein interacts with the prohead and binds the smaller subunit to link the prohead and DNA. A prevailing theory of the mechanism is that DNA packaging utilizes the symmetry mismatch between the 5-fold-symmetric icosahedral shell and the 12-fold-symmetric head–tail connector. The ability to transform free energy into motion––a ubiquitous property of biological systems––is manifest dramatically in the translocation of ϕ29 DNA into the prohead. The ϕ29 DNA packaging cascade involves protein, RNA, and DNA conformational changes and movement that are comparable to allosteric regulation in enzyme–substrate interactions.

Analysis of early promoters of the Bacillus bacteriophage GA-1.

Bacteriophage GA-1, which infects Bacillus sp. strain G1R, is evolutionarily related to phage phi29, which infects Bacillus subtilis. We report the characterization of several GA-1 promoters located at either end of its linear genome. Some of them are unique for GA-1 and drive the expression of open reading frames that have no counterparts in the genome of phi29 or related phages. These unique promoters are active at early infection times and are repressed at late times. In vitro transcription reactions revealed that the purified GA-1-encoded protein p6 represses the activity of these promoters, although the amount of p6 required to repress transcription was different for each promoter. The level of protein p6 produced in vivo increases rapidly during the first stage of the infection cycle. The protein p6 concentration may serve to modulate the expression of these early promoters as infection proceeds.

Domain architecture of a high mobility group A-type bacterial transcriptional factor.

Myxococcus xanthus transcriptional factor CarD participates in carotenogenesis and fruiting body formation. It is the only reported prokaryotic protein having adjacent "AT-hook" DNA-binding and acidic regions characteristic of eukaryotic high mobility group A (HMGA) proteins. The latter are small, unstructured, nonhistone nuclear proteins that function as architectural factors to remodel DNA and chromatin structure and modulate various DNA binding activities. We find CarD to be predominantly dimeric with two stable domains: (a) an N-terminal domain of defined secondary and tertiary structure which is absent in eukaryotic HMGA proteins; (b) a C-terminal domain formed by the acidic and AT-hook segments and lacking defined structure. CarD, like HMGA proteins, binds specifically to the minor-groove of AT-rich DNA present in two appropriately spaced tracts. As in HMGA proteins, casein kinase II can phosphorylate the CarD acidic region, and this dramatically decreases the DNA binding affinity of CarD. The acidic region, in addition to modulating DNA binding, confers structural stability to CarD. We discuss how the structural and functional plasticity arising from domain organization in CarD could be linked to its role as a general transcriptional factor in M. xanthus.

Repression of Bacteriophage φ29 Early Promoter C2 by Viral Protein p6 Is Due to Impairment of Closed Complex

Bacillus subtilis phage phi 29 encodes a very abundant protein, p6, which is a non sequence-specific DNA-binding protein. Protein p6 has the potential to bind cooperatively to the phage genome, forming a nucleoprotein complex in which the DNA adopts a right-handed toroidal conformation winding around a protein core. The formation of this complex at the right end of the phage genome where the early promoter C2 is located affects local topology, which may contribute to the promoter repression, although the underlying molecular mechanism of this repression is not presently known. In this study, we analyzed the effect of the p6 nucleoprotein complex on the formation of transcription complexes at the C2 promoter. The results obtained indicate that the nucleoprotein complex does not occlude promoter C2 to RNA polymerase because both proteins can bind to the same DNA molecule. Protein p6 binds along the fragment including the sequence adjacent to the bound polymerase, altering the structure of the transcriptional complex and affecting specifically the stability of the closed complex. The findings presented might help to answer some of the open questions about the concerted molecular mechanisms of histone-like proteins as transcriptional silencers.

Composition and Mass of the Bacteriophage φ29 Prohead and Virion

The protein composition of the Bacillus subtilis bacteriophage φ29 prohead and virion was determined by combustion of gel bands of 3H-labeled proteins. Copy numbers of individual proteins were calculated relative to the 12 copies of the head–tail connector protein. The mean numbers of copies of the major capsid protein in the prohead and virion were 241 and 218, respectively, approaching the 235 copies determined previously by cryoelectron microscopy. The mean numbers of copies of the dimeric head fiber on the prohead and virion were 24 and 31, respectively, demonstrating partial occupancy of the 55 fiber binding sites. Measured copies of neck and tail proteins in the virion included 11 of the lower collar, 58 of the appendage, and 9 of the tail; if the true copies of these proteins are 12, 60, and 9, respectively, the entire neck and tail of φ29 has quasi-sixfold symmetry. The mass of the fiberless prohead with pRNA was about 14.2 MDa, and the mass of the prohead determined by scanning transmission electron microscopy was consistent with the biochemical data. The mass of the fiberless virion containing the 12.8-MDa DNA genome was about 30.4 MDa. A full complement of dimeric fibers on the prohead or virion would increase the mass of the particle by about 3.2 MDa. The data complement studies relating the structure of φ29 components to dynamic functions in morphogenesis and infection.

Phi29 family of phages.

Continuous research spanning more than three decades has made the Bacillus bacteriophage phi29 a paradigm for several molecular mechanisms of general biological processes, such as DNA replication, regulation of transcription, phage morphogenesis, and phage DNA packaging. The genome of bacteriophage phi29 consists of a linear double-stranded DNA (dsDNA), which has a terminal protein (TP) covalently linked to its 5' ends. Initiation of DNA replication, carried out by a protein-primed mechanism, has been studied in detail and is considered to be a model system for the protein-primed DNA replication that is also used by most other linear genomes with a TP linked to their DNA ends, such as other phages, linear plasmids, and adenoviruses. In addition to a continuing progress in unraveling the initiation of DNA replication mechanism and the role of various proteins involved in this process, major advances have been made during the last few years, especially in our understanding of transcription regulation, the head-tail connector protein, and DNA packaging. Recent progress in all these topics is reviewed. In addition to phi29, the genomes of several other Bacillus phages consist of a linear dsDNA with a TP molecule attached to their 5' ends. These phi29-like phages can be divided into three groups. The first group includes, in addition to phi29, phages PZA, phi15, and BS32. The second group comprises B103, Nf, and M2Y, and the third group contains GA-1 as its sole member. Whereas the DNA sequences of the complete genomes of phi29 (group I) and B103 (group II) are known, only parts of the genome of GA-1 (group III) were sequenced. We have determined the complete DNA sequence of the GA-1 genome, which allowed analysis of differences and homologies between the three groups of phi29-like phages, which is included in this review.

Primer-independent RNA sequencing with bacteriophage phi6 RNA polymerase and chain terminators.

Here we propose a new general method for directly determining RNA sequence based on the use of the RNA-dependent RNA polymerase from bacteriophage phi6 and the chain terminators (RdRP sequencing). The following properties of the polymerase render it appropriate for this application: (1) the phi6 polymerase can replicate a number of single-stranded RNA templates in vitro. (2) In contrast to the primer-dependent DNA polymerases utilized in the sequencing procedure by Sanger et al. (Proc Natl Acad Sci USA, 1977, 74:5463-5467), it initiates nascent strand synthesis without a primer, starting the polymerization on the very 3'-terminus of the template. (3) The polymerase can incorporate chain-terminating nucleotide analogs into the nascent RNA chain to produce a set of base-specific termination products. Consequently, 3' proximal or even complete sequence of many target RNA molecules can be rapidly deduced without prior sequence information. The new technique proved useful for sequencing several synthetic ssRNA templates. Furthermore, using genomic segments of the bluetongue virus we show that RdRP sequencing can also be applied to naturally occurring dsRNA templates. This suggests possible uses of the method in the RNA virus research and diagnostics.