The template specificity of bacteriophage Phi6 RNA polymerase.

Bacteriophage Φ6 contains three double-stranded RNA (dsRNA) genomic segments, L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The virion RNA polymerase in the core particle transcribes segments M and S in vitro. Segment L is transcribed poorly because its transcript starts with GU instead of GG found on segments S and M. Transcription in vivo is modified by the binding of host protein YajQ to the outside the core particle so that segment L is transcribed well. This mechanism is the determinant of the temporal control of gene expression in Φ6. Mutants of Φ6 have been isolated that are independent of YajQ for transcription of segment L. The mutations are found in the gene of the viral polymerase or the major capsid protein or both. These mutants are capable of transcribing segment L with the GU start or GA or GC. The same is found to be true when YajQ is added to wild-type particles. Minus-strand synthesis has restrictions that are different from that of plus-strand synthesis, and YajQ or mutations to independence do not modify minus-strand synthesis behavior. Purified polymerase P2 is able to transcribe dsRNA, but transcription behavior of segment L by both wild-type and mutant polymerases is different from that seen in capsid structures. Adding YajQ to purified polymerase does not change its transcription specificity.

Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage 6 procapsid.

Bacteriophage 6 is a double-stranded RNA (dsRNA) virus whose genome is packaged sequentially as three single-stranded RNA (ssRNA) segments into an icosahedral procapsid which serves as a compartment for genome replication and transcription. The procapsid shell consists of 60 copies each of P1(A) and P1(B), two nonequivalent conformers of the P1 protein. Hexamers of the packaging ATPase P4 are mounted over the 5-fold vertices, and monomers of the RNA-dependent RNA polymerase (P2) attach to the inner surface, near the 3-fold axes. A fourth protein, P7, is needed for packaging and also promotes assembly. We used cryo-electron microscopy to localize P7 by difference mapping of procapsids with different protein compositions. We found that P7 resides on the interior surface of the P1 shell and appears to be monomeric. Its binding sites are arranged around the 3-fold axes, straddling the interface between two P1(A) subunits. Thus, P7 may promote assembly by stabilizing an initiation complex. Only about 20% of the 60 P7 binding sites were occupied in our preparations. P7 density overlaps P2 density similarly mapped, implying mutual occlusion. The known structure of the 12 homolog fits snugly into the P7 density. Both termini-which have been implicated in RNA binding-are oriented toward the adjacent 5-fold vertex, the entry pathway of ssRNA segments. Thus, P7 may promote packaging either by interacting directly with incoming RNA or by modulating the structure of the translocation pore.

Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage Phi2954 infection.

Bacteriophage Phi2954 contains three dsRNA genomic segments, designated L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The core particle is covered by a shell of protein P8, and this structure is enclosed within a lipid-containing membrane. We have found that normal infection of the host Pseudomonas syringae is dependent on the action of a host protein, glutaredoxin 3 (GrxC). GrxC removes the P8 shell from the infecting particle and binds to the inner core. Removal of P8 activates the transcription of segments S and M, whereas binding of GrxC to the core particle activates the transcription of segment L. The differences in transcription behavior are due to the preference of the polymerase for G as the first base of the transcript. Transcripts of segments S and M begin with GCAA, whereas those of segment L begin with ACAA. The binding of GrxC to the particle results in changes in polymerase activity. Mutations resulting in independence of GrxC are found in the gene for protein P1, the major structural protein of the inner core particle.

Packaging in dsRNA viruses.

Several families of viruses have segmented genomes with 3-12 chromosomes. They are capable of packaging these segments in a precise manner so that each virus particle contains one each of the genomic segments. The Cystoviridae are a family of bacteriophages that contain three genomic segments of dsRNA. During infection, the virus produces empty dodecahedral core particles that have the ability to specifically package plus strand transcripts of the genomic segments. The program of packaging makes use of the conformational changes in the surface of the particle as each transcript is packaged. The particles have complexes of a hexameric NTPase that serve as motors to bring the transcripts into the particle, and they have polymerase molecules in the interior that synthesize minus and plus strand copies of the genomic segments.

Interaction of a host protein with core complexes of bacteriophage phi6 to control transcription.

Bacteriophages of the family Cystoviridae have genomes consisting of three double-stranded RNA (dsRNA) segments, L, S, and M, packaged within a polyhedral capsid along with RNA polymerase. Transcription of genomic segment L is activated by the interaction of host protein YajQ with the capsid structure. Segment L codes for the proteins of the inner capsid, which are expressed early in infection. Green fluorescent protein (GFP) fusions with YajQ produce uniform fluorescence in uninfected cells and in cells infected with viruses not dependent on YajQ. Punctate fluorescence develops when cells are infected with YajQ-dependent viruses. It appears that the host protein binds to the infecting particles and remains with them during the entire infection period.

The role of host protein YajQ in the temporal control of transcription in bacteriophage Phi6.

Bacteriophage Phi6 contains three dsRNA genomic segments L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The virion RNA polymerase in the core particle transcribes segments M and S in vitro. Yet early in infection, the transcription of L is highly active. Late in infection, transcription of L is low, and that of M and S is high. A host protein encoded by yajQ is responsible for the activation of L transcription. Knockout mutants of yajQ do not support the replication of Phi6, although they do support the replication of distantly related members of the Cystoviridae. Phi6 can mutate to independence of YajQ. This requires two mutations in the gene for the RNA-dependent RNA polymerase. YajQ acts indirectly on the polymerase by binding to P1, the major structural protein of the core. Previous studies have shown that the activity of the polymerase in the core is controlled by the conformation of the core particle structure.

Cryo-electron tomography of bacteriophage phi6 procapsids shows random occupancy of the binding sites for RNA polymerase and packaging NTPase.

Assembly of dsRNA bacteriophage phi6 involves packaging of the three mRNA strands of the segmented genome into the procapsid, an icosahedrally symmetric particle with recessed vertices. The hexameric packaging NTPase (P4) overlies these vertices, and the monomeric RNA-dependent RNA polymerase (RdRP, P2) binds at sites inside the shell. P2 and P4 are present in substoichiometric amounts, raising the questions of whether they are recruited to the nascent procapsid in defined amounts and at specific locations, and whether they may co-localize to form RNA-processing assembly lines at one or more "special" vertices. We have used cryo-electron tomography to map both molecules on individual procapsids. The results show variable complements that accord with binomial distributions with means of 8 (P2) and 5 (P4), suggesting that they are randomly incorporated in copy numbers that simply reflect availability, i.e. their rates of synthesis. Analysis of the occupancy of potential binding sites (20 for P2; 12 for P4) shows no tendency to cluster nor for P2 and P4 to co-localize, suggesting that the binding sites for both proteins are occupied in random fashion. These observations indicate that although P2 and P4 act sequentially on the same substrates there is no direct physical coupling between their activities.

Temporal control of message stability in the life cycle of double-stranded RNA bacteriophage phi8.

The cystoviruses have genomes of three double-stranded RNA segments. The genes of the L transcript are expressed early in infection, while those of M and S are expressed late. In all cystovirus groups but one, the quantity of the L transcript late in infection is lower than those of the other two because of transcriptional control. In bacteriophage Phi8 and its close relatives, transcription of L is not controlled; instead, the L transcript is turned over rapidly late in infection. The three messages are produced in approximately equal amounts early in infection, but the amount of L is less than 10% of the amounts of the others late in infection. The decay of the Phi8 L message depends upon the production of protein Hb, which is encoded in segment L. It also depends upon a target site within the H gene region. Phage mutants lacking either the Hb gene or the target region do not show the late control of L message quantity. In addition to having a role as a negative regulator, Hb functions to neutralize the activity of protein J, encoded by segment S, which causes the degradation of all viral transcripts.

Characterization of Phi2954, a newly isolated bacteriophage containing three dsRNA genomic segments.

BACKGROUND: Bacteriophage Phi12 is a member of the Cystoviridae and is distinct from Phi6, the first member of that family. We have recently isolated a number of related phages and five showed high similarity to Phi12 in the amino acid sequences of several proteins. Bacteriophage Phi2954 is a member of this group. RESULTS: Phi2954 was isolated from radish leaves and was found to have a genome of three segments of double-stranded RNA (dsRNA), placing it in the Cystoviridae. The base sequences for many of the genes and for the segment termini were similar but not identical to those of bacteriophage Phi12. However, the host specificity was for the type IV pili of Pseudomonas syringae HB10Y rather than for the rough LPS to which Phi12 attaches. Reverse genetics techniques enabled the production of infectious phage from cDNA copies of the genome. Phage were constructed with one, two or three genomic segments. Phage were also produced with altered transcriptional regulation. Although the pac sequences of Phi2954 show no similarity to those of Phi12, segment M of Phi2954 could be acquired by Phi12 resulting in a change of host specificity. CONCLUSIONS: We have isolated a new member of the bacteriophage family Cystoviridae and find that although it shows similarity to other members of the family, it has unique properties that help to elucidate viral strategies for genomic packaging and gene expression.

In vivo studies of genomic packaging in the dsRNA bacteriophage Phi8.

BACKGROUND: Phi8 is a bacteriophage containing a genome of three segments of double-stranded RNA inside a polyhedral capsid enveloped in a lipid-containing membrane. Plus strand RNA binds and is packaged by empty procapsids. Whereas Phi6, another member of the Cystoviridae, shows high stringency, serial dependence and precision in its genomic packaging in vitro and in vivo, Phi8 packaging is more flexible. Unique sequences (pac) near the 5' ends of plus strands are necessary and sufficient for Phi6 genomic packaging and the RNA binding sites are located on P1, the major structural protein of the procapsid. RESULTS: In this paper the boundaries of the Phi8 pac sequences have been explored by testing the in vivo packaging efficacy of transcripts containing deletions or changes in the RNA sequences. The pac sequences have been localized to the 5' untranslated regions of the viral transcripts. Major changes in the pac sequences are either tolerated or ameliorated by suppressor mutations in the RNA sequence. Changes in the genomic packaging program can be established as a result of mutations in P1, the major structural protein of the procapsid and the determinant of RNA binding specificity. CONCLUSION: Although Phi8 is distantly related to bacteriophage Phi6, and does not show sequence similarity, it has a similar genomic packaging program. This program, however, is less stringent than that of Phi6.

Packaging, replication and recombination of the segmented genome of bacteriophage Phi6 and its relatives.

The genomes of bacteriophage Phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented dsRNA genomes into preformed polyhedral structures called procapsids or inner cores. The packaging requires hydrolysis of NTPs and takes place in the order S:M:L. Minus strand synthesis begins after the completion of the plus strand packaging. The packaging and replication reactions can be studied in vitro with purified components. A model has been presented that proposes that the program of serially dependent packaging is determined by the conformational changes at the surface of the procapsid due to the amount of RNA packaged at each step. The in vitro packaging and replication system has facilitated the application of reverse genetics and the study of recombination in the family of Cystoviridae.

Subunit folds and maturation pathway of a dsRNA virus capsid.

The cystovirus ϕ6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. ϕ6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an α-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.

Stepwise expansion of the bacteriophage ϕ6 procapsid: possible packaging intermediates.

The initial assembly product of bacteriophage ϕ6, the procapsid, undergoes major structural transformation during the sequential packaging of its three segments of single-stranded RNA. The procapsid, a compact icosahedrally symmetric particle with deeply recessed vertices, expands to the spherical mature capsid, increasing the volume available to accommodate the genome by 2.5-fold. It has been proposed that expansion and packaging are linked, with each stage in expansion presenting a binding site for a particular RNA segment. To investigate procapsid transformability, we induced expansion by acidification, heating, and elevated salt concentration. Cryo-electron microscopy reconstructions after all three treatments yielded the same partially expanded particle. Analysis by cryo-electron tomography showed that all vertices of a given capsid were either in a compact or an expanded state, indicating a highly cooperative transition. To benchmark the mature capsid, we analyzed filled (in vivo packaged) capsids. When these particles were induced to release their RNA, they reverted to the same intermediate state as expanded procapsids (intermediate 1) or to a second, further expanded state (intermediate 2). This partial reversibility of expansion suggests that the mature spherical capsid conformation is obtained only when sufficient outward pressure is exerted by packaged RNA. The observation of two intermediates is consistent with the proposed three-step packaging process. The model is further supported by the observation that a mutant capable of packaging the second RNA segment without previously packaging the first segment has enhanced susceptibility for switching spontaneously from the procapsid to the first intermediate state.

Initial location of the RNA-dependent RNA polymerase in the bacteriophage Phi6 procapsid determined by cryo-electron microscopy.

The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage Phi6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as single-stranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route. The RdRP (protein P2) is assembled within the procapsid, and it was thought that it should be located on the 5-fold axes near the RNA entry and exit channels. To determine the initial location of the RdRP inside the procapsid of bacteriophage Phi6, we performed cryo-electron microscopy of wild type and mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the 3-fold axes that are strong in a mutant that has approximately 10 copies of P2 per particle; faint in wild type, reflecting the lower copy number of approximately 3; and completely absent in a P2-null mutant. The dimensions and shapes of these densities match those of the known crystal structure of the P2 monomer. We propose that, during maturation, the P2 molecules rotate to occupy positions closer to adjacent 5-fold vertices where they conduct replication and transcription.

Heterologous recombination in the segmented dsRNA genome of bacteriophage Φ6.

The genome of bacteriophage Φ6 is composed of three unique segments of double-stranded RNA packaged within a procapsid. One segment can recombine with another in regions that share little sequence similarity. Although the recombination is therefore heterologous, the crossover points usually consist of two to six identical nucleotides. The frequency of recombinants is enhanced by conditions that prevent or hinder the minus strand synthesis of a single plus strand segment. Recombination serves as a repair system as well as a means of changing the genetic structure of the virus. The reaction can be studied in an in-vitro packaging and replication system involving purified procapsids and ssRNA. Although there are striking differences in the mechanisms of recombination in RNA viruses, there are also strong similarities. All seem to use a copy-choice template switching action for recombination. The Φ6 system is a useful model for the recombination of other segmented double-stranded RNA viruses such as the Reoviridae.

Analysis of specific binding involved in genomic packaging of the double-stranded-RNA bacteriophage phi6.

The genomes of bacteriophage phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus-strand transcripts of the segmented double-stranded-RNA genomes into preformed polyhedral structures called procapsids or inner cores. The packaging requires the hydrolysis of nucleoside triphosphates and takes place in the order segment S-segment M, segment L. Packaging is dependent upon unique sequences of about 200 nucleotides near the 5' ends of plus-strand transcripts of the three genomic segments. It appears that P1 is the determinant of the RNA binding sites. Directed mutation of P1 was used to locate regions that are important for genomic packaging. Specific binding of RNA to the exterior of the procapsid was dependent upon ATP, and a region that showed a high level of cross-linking to phage-specific RNA was located. Antibodies to peptide sequences were prepared, and their abilities to bind to the exterior of procapsids were determined. Sites sensitive to trypsin and to factor Xa were determined as well.

Construction of carrier state viruses with partial genomes of the segmented dsRNA bacteriophages.

The cystoviridae are bacteriophages with genomes of three segments of dsRNA enclosed within a polyhedral capsid. Two members of this family, Phi6 and Phi8, have been shown to form carrier states in which the virus replicates as a stable episome in the host bacterium while expressing reporter genes such as kanamycin resistance or lacalpha. The carrier state does not require the activity of all the genes necessary for phage production. It is possible to generate carrier states by infecting cells with virus or by electroporating nonreplicating plasmids containing cDNA copies of the viral genomes into the host cells. We have found that carrier states in both Phi6 and Phi8 can be formed at high frequency with all three genomic segments or with only the large and small segments. The large genomic segment codes for the proteins that constitute the inner core of the virus, which is the structure responsible for the packaging and replication of the genome. In Phi6, a carrier state can be formed with the large and middle segment if mutations occur in the gene for the major structural protein of the inner core. In Phi8, carrier state formation requires the activity of genes 8 and 12 of segment S.

Isolation and analysis of mutants of double-stranded-RNA bacteriophage phi6 with altered packaging specificity.

The genomes of bacteriophage phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented double-stranded RNA genomes into preformed polyhedral structures called procapsids or inner cores. This packaging requires hydrolysis of nucleoside triphosphates and takes place in the order S-M-L. Packaging is dependent on unique sequences of about 200 nucleotides near the 5' ends of plus strand transcripts of the three genomic segments. Changes in the pac sequences lead to loss of packaging ability but can be suppressed by second-site changes in RNA or amino acid changes in protein P1, the major structural protein of the procapsid. It appears that P1 is the determinant of the RNA binding sites, and it is suggested that the binding sites overlap or are conformational changes of the same domains.

Unique properties of the inner core of bacteriophage phi8, a virus with a segmented dsRNA genome.

The inner core of bacteriophage phi8 is capable of packaging and replicating the plus strands of the RNA genomic segments of the virus in vitro. The particles composed of proteins P1, P2, P4, and P7 can be assembled in cells of E. coli that carry plasmids with cDNA copies of genomic segment L. The gene arrangement on segment L was found to differ from that of other cystoviruses in that the gene for the ortholog of protein P7 is located at the 3' end of the plus strand rather than near the 5' end. In place of the normal location of gene 7 is gene H, whose product is necessary for normal phage development, but not necessary for in vitro genomic packaging and replication. Genomic packaging is dependent upon the activity of an NTPase motor protein, P4. P4 was purified from cell extracts and was found to form hexamers with little NTPase activity until associated with inner core particles. Labeling studies of in vitro packaging of phi8 RNA do not show serial dependence; however, studies involving in vitro packaging for the formation of live virus indicate that packaging is stringent. Studies with the acquisition of chimeric segments in live virus indicate that phi8 does package RNA in the order s/m/l. The inner core of bacteriophage phi8 differs from that of its relatives in the Cystoviridae in that the major structural protein P1 is able to interact with the host cell membrane to effect penetration of the inner core into the cell.