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Recombination and mutation of viral genomes represent major mechanisms for viral evolution and, in many cases, moderate pathogenicity. Segmented genome viruses frequently undergo reassortment of the genome via multiple infection of host organisms, with influenza and reoviruses being well-known examples. Specifically, major genomic shifts mediated by reassortment are responsible for radical changes in the influenza antigenic determinants that can result in pandemics requiring rapid preventative responses by vaccine modifications. In contrast, smaller mutational changes brought about by the error-prone viral RNA polymerases that, for the most part, lack a replication base mispairing editing function produce small mutational changes in the RNA genome during replication. Referring again to the influenza example, the accumulated mutations—known as drift—require yearly vaccine updating and rapid worldwide distribution of each new formulation. Coronaviruses with a large positive-sense RNA genome have long been known to undergo intramolecular recombination likely mediated by copy choice of the RNA template by the viral RNA polymerase in addition to the polymerase-based mutations. The current SARS-CoV-2 origin debate underscores the importance of understanding the plasticity of viral genomes, particularly the mechanisms responsible for intramolecular recombination. This review describes the use of the cystovirus bacteriophage as an experimental model for recombination studies in a controlled manner, resulting in the development of a model for intramolecular RNA genome alterations. The review relates the sequence of experimental studies from the laboratory of Leonard Mindich, PhD at the Public Health Research Institute—then in New York City—and covers a period of approximately 12 years. Hence, this is a historical scientific review of research that has the greatest relevance to current studies of emerging RNA virus pathogens.

Segmented RNA viruses are widespread in nature and include important human, animal and plant pathogens, such as influenza viruses and rotaviruses. Although the origin of RNA virus genome segmentation remains elusive, a major consequence of this genome structure is the capacity for reassortment to occur during co-infection, whereby segments are exchanged among different viral strains. Therefore, reassortment can create viral progeny that contain genes that are derived from more than one parent, potentially conferring important fitness advantages or disadvantages to the progeny virus. However, for segmented RNA viruses that package their multiple genome segments into a single virion particle, reassortment also requires genetic compatibility between parental strains, which occurs in the form of conserved packaging signals, and the maintenance of RNA and protein interactions. In this Review, we discuss recent studies that examined the mechanisms and outcomes of reassortment for three well-studied viral families - Cystoviridae, Orthomyxoviridae and Reoviridae - and discuss how these findings provide new perspectives on the replication and evolution of segmented RNA viruses.

Segmented double-stranded (ds)RNA viruses share remarkable similarities in their replication strategy and capsid structure. During virus replication, positive-sense single-stranded (+)RNAs are packaged into procapsids, where they serve as templates for dsRNA synthesis, forming progeny particles containing a complete equimolar set of genome segments. How the +RNAs are recognized and stoichiometrically packaged remains uncertain. While bacteriophages of the Cystoviridae family rely on specific RNA-protein interactions to select appropriate +RNAs for packaging, viruses of the Reoviridae instead rely on specific inter-molecular interactions between +RNAs that guide multi-segmented genome assembly. Although these families use distinct mechanisms to direct +RNA packaging, both yield progeny particles with a complete set of genomic dsRNAs.

Cystoviridae is a family of bacterial viruses (bacteriophages) with a tri-segmented dsRNA genome. It includes a single genus Cystovirus, which has presently only one recognised virus species, Pseudomonas virus phi6. However, a large number of additional dsRNA phages have been isolated from various environmental samples, indicating that such viruses are more widespread and abundant than previously recognised. Six of the additional dsRNA phage isolates (Pseudomonas phages phi8, phi12, phi13, phi2954, phiNN and phiYY) have been fully sequenced. They all infect Pseudomonas species, primarily plant pathogenic Pseudomonas syringae strains. Due to the notable genetic and structural similarities with Pseudomonas phage phi6, we propose that these viruses should be included into the Cystovirus genus (and consequently into the Cystoviridae family). Here, we present an updated taxonomy of the family Cystoviridae and give a short overview of the properties of the type member phi6 as well as the putative new members of the family.

Genome replication and transcription of riboviruses are catalyzed by an RNA-dependent RNA polymerase (RdRP). RdRPs are normally associated with other virus- or/and host-encoded proteins that modulate RNA polymerization activity and template specificity. The polymerase complex of double-stranded dsRNA viruses is a large icosahedral particle (inner core) containing RdRP as a minor constituent. In phi6 and other dsRNA bacteriophages from the Cystoviridae family, the inner core is composed of four virus-specific proteins. Of these, protein P2, or Pol subunit, has been tentatively identified as RdRP by sequence comparisons, but the role of this protein in viral RNA synthesis has not been studied until recently. Here, we overview the work on the Pol subunits of phi6 and related viruses from the standpoints of function, structure and evolution.

The family Cystoviridae includes enveloped viruses with a tri-segmented dsRNA genome and a double-layered protein capsid. The innermost protein shell is a polymerase complex responsible for genome packaging, replication and transcription. Cystoviruses infect Gram-negative bacteria, primarily plant-pathogenic Pseudomonas syringae strains. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Cystoviridae, which is available at http://www.ictv.global/report/cystoviridae.

Packaging of the Cystovirus varphi8 genome into the polymerase complex is catalysed by the hexameric P4 packaging motor. The motor is located at the fivefold vertices of the icosahedrally symmetric polymerase complex, and the symmetry mismatch between them may be critical for function. We have developed a novel image-processing approach for the analysis of symmetry-mismatched structures and applied it to cryo-electron microscopy images of P4 bound to the polymerase complex. This approach allowed us to solve the three-dimensional structure of the P4 in situ to 15-A resolution. The C-terminal face of P4 was observed to interact with the polymerase complex, supporting the current view on RNA translocation. We suggest that the symmetry mismatch between the two components may facilitate the ring opening required for RNA loading prior to its translocation.

The enveloped dsRNA bacteriophages phi6 and phi8 are the two most distantly related members of the Cystoviridae family. Their structure and function are similar to that of the Reoviridae but their assembly can be conveniently studied in vitro. Electron cryomicroscopy and three-dimensional icosahedral reconstruction were used to determine the structures of the phi6 virion (14 A resolution), phi8 virion (18 A resolution), and phi8 core (8.5 A resolution). Spikes protrude 2 nm from the membrane bilayer in phi6 and 7 nm in phi8. In the phi6 nucleocapsid, 600 copies of P8 and 72 copies of P4 interact with the membrane, whereas in phi8 it is only P4 and 60 copies of a minor protein. The major polymerase complex protein P1 forms a dodecahedral shell from 60 asymmetric dimers in both viruses, but the alpha-helical fold has apparently diverged. These structural differences reflect the different host ranges and entry and assembly mechanisms of the two viruses.

The isolation of additional bacteriophages besides phi6 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 phi12 was copied as cDNA and its nucleotide sequence determined. This genome's organization is similar to that of the large segment of bacteriophages phi6, phi8, and phi13. In the amino acid sequence of the viral RNA-dependent RNA polymerase (P2), similarity was found to the comparable proteins of phi6, phi8, and phi13. Amino acid sequence similarity was also noted in the nucleotide triphosphate phosphorylase (P4) to the comparable proteins of phi8 and phi13.

Cystoviridae is a family of bacteriophages with a tri-segmented dsRNA genome enclosed in a tri-layered virion structure. Here, we present a new putative member of the Cystoviridae family, bacteriophage ϕNN. ϕNN was isolated from a Finnish lake in contrast to the previously identified cystoviruses, which originate from various legume samples collected in the USA. The nucleotide sequence of the virus reveals a strong genetic similarity (~80 % for the L-segments, ~55 % for the M-segments and ~84 % for the S-segments) to Pseudomonas phage ϕ6, the type member of the virus family. However, the relationship between ϕNN and other cystoviruses is more distant. In general, proteins located in the internal parts of the virion were more conserved than those exposed on the virion surface, a phenomenon previously reported among eukaryotic dsRNA viruses. Structural models of several putative ϕNN proteins propose that cystoviral structures are highly conserved.

In bacteriophages of the cystovirus family, the polymerase complex (PX) encodes a 75-kDa RNA-directed RNA polymerase (P2) that transcribes the double-stranded RNA genome. Also a constituent of the PX is the essential protein P7 that, in addition to accelerating PX assembly and facilitating genome packaging, plays a regulatory role in transcription. Deletion of P7 from the PX leads to aberrant plus-strand synthesis suggesting its influence on the transcriptase activity of P2. Here, using solution NMR techniques and the P2 and P7 proteins from cystovirus ϕ12, we demonstrate their largely electrostatic interaction in vitro. Chemical shift perturbations on P7 in the presence of P2 suggest that this interaction involves the dynamic C-terminal tail of P7, more specifically an acidic cluster therein. Patterns of chemical shift changes induced on P2 by the P7 C-terminus resemble those seen in the presence of single-stranded RNA suggesting similarities in binding. This association between P2 and P7 reduces the affinity of the former towards template RNA and results in its decreased activity both in de novo RNA synthesis and in extending a short primer. Given the presence of C-terminal acidic tracts on all cystoviral P7 proteins, the electrostatic nature of the P2/P7 interaction is likely conserved within the family and could constitute a mechanism through which P7 regulates transcription in cystoviruses.

Double-stranded RNA (dsRNA) viruses are a diverse group of viruses infecting hosts from bacteria to higher eukaryotes. Among the hosts are humans, domestic animals, and economically important plant species. Fine details of high-resolution virion structures have revealed common structural characteristics unique to these viruses including an internal icosahedral capsid built from 60 asymmetric dimers (120 monomers!) of the major coat protein. Here we focus mainly on the structures and assembly principles of large icosahedral dsRNA viruses belonging to the families of Cystoviridae and Reoviridae. It is obvious that there are a variety of assembly pathways utilized by different viruses starting from similar building blocks and reaching in all cases a similar capsid architecture. This is true even with closely related viruses indicating that the assembly pathway per se is not an indicator of relatedness and is achieved with minor changes in the interacting components.

P4 proteins are hexameric RNA packaging ATPases of dsRNA bacteriophages of the Cystoviridae family. P4 hexamers are integral part of the inner polymerase core and play several essential roles in the virus replication cycle. P4 proteins are structurally related to the hexameric helicases and translocases of superfamily 4 (SF4) and other RecA-like ATPases. Recombinant P4 proteins retain their 5' to 3' helicase and translocase activity in vitro and thus serve as a model system for studying the mechanism of action of hexameric ring helicases and RNA translocation. This review summarizes the different roles that P4 proteins play during virus assembly, genome packaging, and transcription. Structural and mechanistic details of P4 action are laid out to and subsequently compared with those of the related hexameric helicases and other packaging motors.

The RNA-directed RNA polymerase P2 from cystovirus ϕ6 catalyzes the de novo synthesis of positive and negative strands of the viral double-stranded RNA genome. P2 is mobile on the slow, microsecond to millisecond time scale with various motional modes, putatively assisting in RNA translocation and catalysis. Here we investigate the influence of the extreme 3′-end sequence of the single-stranded RNA templates and the nature of the substrate nucleotide triphosphates on these motional modes using multiple-quantum NMR spectroscopy. We find that P2, in the presence of templates bearing the proper genomic 3′-ends or the preferred initiation nucleotide, displays unique dynamic signatures that are different from those in the presence of nonphysiological templates or substrates. This suggests that dynamics may play a role in the fidelity of recognition of the correct substrates and template sequences to initiate RNA polymerization.

Bacteriophage Φ2954 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.

Background Bacteriophage Φ12 is a member of the Cystoviridae and is distinct from Φ6, the first member of that family. We have recently isolated a number of related phages and five showed high similarity to Φ12 in the amino acid sequences of several proteins. Bacteriophage Φ2954 is a member of this group. Results Φ2954 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 Φ12. However, the host specificity was for the type IV pili of Pseudomonas syringae HB10Y rather than for the rough LPS to which Φ12 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 Φ2954 show no similarity to those of Φ12, segment M of Φ2954 could be acquired by Φ12 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.

Author(s): Wei, Hui; Cheng, R Holland; Berriman, John; Rice, William J; Stokes, David L; Katz, A; Morgan, David Gene; Gottlieb, Paul | Abstract: BackgroundBacteriophage phi12 is a member of the Cystoviridae, a unique group of lipid containing membrane enveloped bacteriophages that infect the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola. The genomes of the virus species contain three double-stranded (dsRNA) segments, and the virus capsid itself is organized in multiple protein shells. The segmented dsRNA genome, the multi-layered arrangement of the capsid and the overall viral replication scheme make the Cystoviridae similar to the Reoviridae.Methodology/principal findingsWe present structural studies of cystovirus phi12 obtained using cryo-electron microscopy and image processing techniques. We have collected images of isolated phi12 virions and generated reconstructions of both the entire particles and the polymerase complex (PC). We find that in the nucleocapsid (NC), the phi12 P8 protein is organized on an incomplete T = 13 icosahedral lattice where the symmetry axes of the T = 13 layer and the enclosed T = 1 layer of the PC superpose. This is the same general protein-component organization found in phi6 NC's but the detailed structure of the entire phi12 P8 layer is distinct from that found in the best classified cystovirus species phi6. In the reconstruction of the NC, the P8 layer includes protein density surrounding the hexamers of P4 that sit at the 5-fold vertices of the icosahedral lattice. We believe these novel features correspond to dimers of protein P7.Conclusions/significanceIn conclusion, we have determined that the phi12 NC surface is composed of an incomplete T = 13 P8 layer forming a net-like configuration. The significance of this finding in regard to cystovirus assembly is that vacancies in the lattice could have the potential to accommodate additional viral proteins that are required for RNA packaging and synthesis.

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 Φ8 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 Φ8 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.

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.

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.

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.

Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Φ12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0·8 to 6·8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5′ GUU and UAC 3′) and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22–30 cycles compared to 30–35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.

To continue the molecular characterization of RNA-dependent RNA polymerases of dsRNA bacteriophages ( Cystoviridae ), we purified and biochemically characterized the wild-type (wt) and a temperature-sensitive ( ts ) point mutant of the polymerase subunit (Pol) from bacteriophage φ12. Interestingly, initiation by both wt and the ts φ12 Pol was notably more sensitive to increased temperatures than the elongation step, the absolute value of the nonpermissive temperature being lower for the ts enzyme. Experiments with the Pol subunit of related cystovirus φ6 revealed a similar differential sensitivity of the initiation and elongation steps. This is consistent with the previous result showing that de novo initiation by RdRp from dengue virus is inhibited at elevated temperatures, whereas the elongation phase is relatively thermostable. Overall, these data suggest that de novo RNA-dependent RNA synthesis in many viral systems includes a specialized thermolabile state of the RdRp initiation complex.

We present the assembly of the polymerase complex (procapsid) of a dsRNA virus from purified recombinant proteins. This molecular machine packages and replicates viral ssRNA genomic precursors in vitro. After addition of an external protein shell, these in vitro self-assembled viral core particles can penetrate the host plasma membrane and initiate a productive infection. Thus, a viral procapsid has been assembled and rendered infectious using purified components. Using this system, we have studied the mechanism of assembly of the common dsRNA virus shell and the incorporation of a symmetry mismatch within an icosahedral capsid. Our work demonstrates that this molecular machine, self-assembled under defined conditions in vitro, can function in its natural environment, the cell cytoplasm.