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Many Pseudomonas phages recognize lipopolysaccharides (LPS) as the receptor for infection. LPS defective mutants are often recovered from phage treatments, possibly causing the failure of phage applications. In this work, we isolated a lytic Pseudomonas phage, phiZ98, that can specifically lyse LPS defective strains of the genus Pseudomonas. Transmission electron microscopy (TEM) showed that phiZ98 particles were enveloped in a layer of membrane-like structure. Genomic analysis revealed that the phage has a genome of tri-segmented double-stranded RNA molecules of 6627 bp, 3769 bp, and 3075 bp, respectively. The results indicated that phage phiZ98 was the nineth member of the genus Cystovirus. The phiZ98 genome encoded 12 putative proteins with predicted functions and 15 hypothetical proteins. Mass spectrum analysis further identified 11 proteins present in the virions. Antibacterial activity assays showed that phage phiZ98 significantly inhibited cell growth, reduced biofilm formation, and removed mature biofilm. Moreover, phage phiZ98 can significantly control the growth of the host bacterial cells in sterilized milk or canned corned beef. In combination with phage K8 which used LPS as the receptor, phiZ98 can significantly reduce the phage-resistant mutants generated from the K8 treatment in milk. Taken together, the dsRNA phage phiZ98 could be an effective scavenger in removing phage-resistant mutants with defective LPS in cocktail applications.

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.

The survival of viruses in droplets is known to depend on droplets’ chemical composition, which may vary in respiratory fluid between individuals and over the course of disease. This relationship is also important for understanding the persistence of viruses in droplets generated from wastewater, freshwater, and seawater. We investigated the effects of salt (0, 1, and 35 g/L), protein (0, 100, and 1000 μg/mL), surfactant (0, 1, and 10 μg/mL), and droplet pH (4.0, 7.0, and 10.0) on the viability of viruses in 1-μL droplets pipetted onto polystyrene surfaces and exposed to 20%, 50%, and 80% relative humidity (RH) using a culture-based approach. Results showed that viability of MS2, a non-enveloped virus, was generally higher than that of Φ6, an enveloped virus, in droplets after 1 hour. The chemical composition of droplets greatly influenced virus viability. Specifically, the survival of MS2 was similar in droplets at different pH values, but the viability of Φ6 was significantly reduced in acidic and basic droplets compared to neutral ones. The presence of bovine serum albumin protected both MS2 and Φ6 from inactivation in droplets. The effects of sodium chloride and the surfactant sodium dodecyl sulfate varied by virus type and RH. Meanwhile, RH affected the viability of viruses as shown previously: viability was lowest at intermediate to high RH. The results demonstrate that the viability of viruses is determined by the chemical composition of carrier droplets, especially pH and protein content, and environmental factors. These findings emphasize the importance of understanding the chemical composition of carrier droplets in order to predict the persistence of viruses contained in them.

Half a century of research on membrane-containing phages has had a major impact on virology, providing new insights into virus diversity, evolution and ecological importance. The recent revolutionary technical advances in imaging, sequencing and lipid analysis have significantly boosted the depth and volume of knowledge on these viruses. This has resulted in new concepts of virus assembly, understanding of virion stability and dynamics, and the description of novel processes for viral genome packaging and membrane-driven genome delivery to the host. The detailed analyses of such processes have given novel insights into DNA transport across the protein-rich lipid bilayer and the transformation of spherical membrane structures into tubular nanotubes, resulting in the description of unexpectedly dynamic functions of the membrane structures. Membrane-containing phages have provided a framework for understanding virus evolution. The original observation on membrane-containing bacteriophage PRD1 and human pathogenic adenovirus has been fundamental in delineating the concept of viral lineages, postulating that the fold of the major capsid protein can be used as an evolutionary fingerprint to trace long-distance evolutionary relationships that are unrecognizable from the primary sequences. This has brought the early evolutionary paths of certain eukaryotic, bacterial, and archaeal viruses together, and potentially enables the reorganization of the nearly immeasurable virus population (similar to 1 x 10(31)) on Earth into a reasonably low number of groups representing different architectural principles. In addition, the research on membrane-containing phages can support the development of novel tools and strategies for human therapy and crop protection.

Half a century of research on membrane-containing phages has had a major impact on virology, providing new insights into virus diversity, evolution and ecological importance. The recent revolutionary technical advances in imaging, sequencing and lipid analysis have significantly boosted the depth and volume of knowledge on these viruses. This has resulted in new concepts of virus assembly, understanding of virion stability and dynamics, and the description of novel processes for viral genome packaging and membrane-driven genome delivery to the host. The detailed analyses of such processes have given novel insights into DNA transport across the protein-rich lipid bilayer and the transformation of spherical membrane structures into tubular nanotubes, resulting in the description of unexpectedly dynamic functions of the membrane structures. Membrane-containing phages have provided a framework for understanding virus evolution. The original observation on membrane-containing bacteriophage PRD1 and human pathogenic adenovirus has been fundamental in delineating the concept of “viral lineages”, postulating that the fold of the major capsid protein can be used as an evolutionary fingerprint to trace long-distance evolutionary relationships that are unrecognizable from the primary sequences. This has brought the early evolutionary paths of certain eukaryotic, bacterial, and archaeal viruses together, and potentially enables the reorganization of the nearly immeasurable virus population (~1 × 1031) on Earth into a reasonably low number of groups representing different architectural principles. In addition, the research on membrane-containing phages can support the development of novel tools and strategies for human therapy and crop protection.

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.

The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little or no homology to existing databases (referred to as the “viral dark matter”). Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies.

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.

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. Non

P2, an RNA-directed RNA polymerase (RdRP), is encoded on the largest of the three segments of the double-stranded RNA genome of cystoviruses. P2 performs the dual tasks of replication and transcription de novo on single-stranded RNA templates, and plays a critical role in the viral life-cycle. Work over the last few decades has yielded a wealth of biochemical and structural information on the functional regulation of P2, on its role in the spatiotemporal regulation of RNA synthesis and its variability across the Cystoviridae family. These range from atomic resolution snapshots of P2 trapped in functionally significant states, in complex with catalytic/structural metal ions, polynucleotide templates and substrate nucleoside triphosphates, to P2 in the context of viral capsids providing structural insight into the assembly of supramolecular complexes and regulatory interactions therein. They include in vitro biochemical studies using P2 purified to homogeneity and in vivo studies utilizing infectious core particles. Recent advances in experimental techniques have also allowed access to the temporal dimension and enabled the characterization of dynamics of P2 on the sub-nanosecond to millisecond timescale through measurements of nuclear spin relaxation in solution and single molecule studies of transcription from seconds to minutes. Below we summarize the most significant results that provide critical insight into the role of P2 in regulating RNA synthesis in cystoviruses.

Prior studies of clay–virus interactions have focused on the stability and infectivity of nonenveloped viruses, yielding contradictory results. We hypothesize that the surface charge distribution of the clay and virus envelope dictates how the components react and affect aggregation, viral stability, and infectivity. The bacteriophage Cystoviridae species φ6 used in this study is a good model for enveloped pathogens. The interaction between φ6 and montmorillonite (MMT) clay (the primary component of bentonite) is explored by transmission electron microscopy. The analyses show that MMT–φ6 mixtures undergo heteroaggregation, forming structures in which virtually all the virions are either sequestered between MMT platelet layers or attached to platelet edges. The virions swell and undergo disassembly resulting in partial or total envelope loss. Edge-attached viral envelopes distort to increase contact area with the positively charged platelet edges indicating that the virion surface is negatively charged. The nucleocapsid (NCs) remaining after envelope removal also exhibit distortion, in contrast to detergent-produced NCs which exhibit no distortion. This visually discernible disassembly is a mechanism for loss of infectivity previously unreported by studies of nonenveloped viruses. The MMT-mediated sequestration and disassembly result in reduced infectivity, suggesting that clays may reduce infectivity of enveloped pathogenic viruses in soils and sediments.

Genome packaging into an empty capsid is an essential step in the assembly of many complex viruses. In double-stranded RNA (dsRNA) bacteriophages of the Cystoviridae family this step is performed by a hexameric helicase P4 which is one of the simplest packaging motors found in nature. Biochemical and structural studies of P4 proteins have led to a surprising finding that these proteins bear mechanistic and structural similarities to a variety of the pervasive RecA/F1-ATPase-like motors that are involved in diverse biological functions. This review describes the role of P4 proteins in assembly, transcription and replication of dsRNA bacteriophages as it has emerged over the past decade while focusing on the most recent structural studies. The P4 mechanism is compared with the models proposed for the related hexameric motors.

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.

Summary The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity., Graphical Abstract, Highlights • Crystal structure of the major capsid protein P1 of the Pseudomonas phage Φ8 • Φ8 P1 shares a common ancestor with mammalian reoviruses • Φ8 P1’s trapezoidal shape may facilitate capsid expansion during maturation • The pentameric organization of Φ8 P1 suggests a revised assembly pathway, El Omari et al. report a structure of the dsRNA bacteriophage ϕ8 capsid protein P1. P1 crystallizes as a pentamer, suggesting a new pathway for procapsid assembly. P1 displays a novel fold and a trapezoidal shape, distinct from that of other dsRNA virus, which may facilitate capsid expansion during maturation.

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.

The 3-dimensional structure of the nucleocapsid (NC) of bacteriophage φ6 is described utilizing component tree analysis, a topological and geometric image descriptor. The component trees are derived from density maps of cryo-electron microscopy single particle reconstructions. Analysis determines position and occupancy of structure elements responsible for RNA packaging and transcription. Occupancy of the hexameric nucleotide triphosphorylase (P4) and RNA polymerase (P2) are found to be essentially complete in the NC. The P8 protein lattice likely fixes P4 and P2 in place during maturation. We propose that the viral procapsid (PC) is a dynamic structural intermediate where the P4 and P2 can attach and detach until held in place in mature NCs. During packaging, the PC expands to accommodate the RNA, and P2 translates from its original site near the inner 3-fold axis (20 sites) to the inner 5-fold axis (12 sites) with excess P2 positioned inside the central region of the NC.

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.

Molecular dynamics (MD) simulations combined with biochemical studies have suggested the presence of long-range networks of functionally relevant conformational flexibility on the nanosecond time scale in single-subunit RNA polymerases in many RNA viruses. However, experimental verification of these dynamics at a sufficient level of detail has been lacking. Here we describe the fast, picosecond to nanosecond dynamics of an archetypal viral RNA-directed RNA polymerase (RdRp), the 75 kDa P2 protein from cystovirus ϕ12, using analyses of (1)H-(1)H dipole-dipole cross-correlated relaxation at the methyl positions of Ile (δ1), Leu, Val, and Met residues. Our results, which represent the most detailed experimental characterization of fast dynamics in a viral RdRp until date, reveal a highly connected dynamic network as predicted by MD simulations of related systems. Our results suggest that the entry portals for template RNA and substrate NTPs are relatively disordered, while conserved motifs involved in metal binding, nucleotide selection, and catalysis display greater rigidity. Perturbations at the active site through metal binding or functional mutation affect dynamics not only in the immediate vicinity but also at remote regions. Comparison with the limited experimental and extensive functional and in silico results available for homologous systems suggests conservation of the overall pattern of dynamics in viral RdRps.

Bacteriophages are the most numerous entities in the biosphere. Despite this numerical dominance, the genetic structure of bacteriophage populations is poorly understood. Here, we present a biogeography study involving 25 previously undescribed bacteriophages from the Cystoviridae clade, a group characterized by a dsRNA genome divided into three segments. Previous laboratory manipulation has shown that, when multiple Cystoviruses infect a single host cell, they undergo ( i ) rare intrasegment recombination events and ( ii ) frequent genetic reassortment between segments. Analyzing linkage disequilibrium (LD) within segments, we find no significant evidence of intrasegment recombination in wild populations, consistent with ( i ). An extensive analysis of LD between segments supports frequent reassortment, on a time scale similar to the genomic mutation rate. The absence of LD within this group of phages is consistent with expectations for a completely sexual population, despite the fact that some segments have >50% nucleotide divergence at 4-fold degenerate sites. This extraordinary rate of genetic exchange between highly unrelated individuals is unprecedented in any taxa. We discuss our results in light of the biological species concept applied to viruses.

We have identified two new lysozyme-like protein families by using a combination of sequence similarity searches, domain architecture analysis, and structural predictions. First, the P5 protein from bacteriophage phi8, which belongs to COG3926 and Pfam family DUF847, is predicted to have a new lysozyme-like domain. This assignment is consistent with the lytic function of P5 proteins observed in several related double-stranded RNA bacteriophages. Domain architecture analysis reveals two lysozyme-associated transmembrane modules (LATM1 and LATM2) in a few COG3926/DUF847 members. LATM2 is also present in two proteins containing a peptidoglycan binding domain (PGB) and an N-terminal region that corresponds to COG5526 with uncharacterized function. Second, structure prediction and sequence analysis suggest that COG5526 represents another new lysozyme-like family. Our analysis offers fold and active-site assignments for COG3926/DUF847 and COG5526. The predicted enzymatic activity is consistent with an experimental study on the zliS gene product from Zymomonas mobilis, suggesting that bacterial COG3926/DUF847 members might be activators of macromolecular secretion.

The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity.

Double-stranded RNA viruses sequester their genomes within a protein shell, called the polymerase complex. Translocation of ssRNA into (packaging) and out (transcription) of the polymerase complex are essential steps in the life cycle of the dsRNA bacteriophages of the Cystoviridae family (phi6-phi14). Both processes require a viral molecular motor P4, an NTPase, which bears structural and functional similarities to hexameric helicases. In effect, switching between the packaging and the transcription mode requires the translocation direction of the P4 motor to reverse. However, the mechanism of the reversal remains elusive. Here we characterize the P4 protein from bacteriophage phi12 and exploit its purine nucleotide specificity to delineate P4 role in transcription. The results indicate that while P4 actively translocates RNA during packaging it acts as a passive conduit for RNA export. The directionality switching is accomplished via the regulation of P4 NTPase activity within the polymerase core.

Bacteriophages are classified into one order and 13 families. Over 5100 phages have been examined in the electron microscope since 1959. At least 4950 phages (96%) are tailed. They constitute the order Caudovirales and three families. Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages). Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses. Bacteriophages occur in over 140 bacterial or archaeal genera. Their distribution reflects their origin and bacterial phylogeny. Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent. Tailed phages appear as monophyletic and as the oldest known virus group.

The inner core of bacteriophage φ8 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 φ8 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 φ8 does package RNA in the order s / m / l. The inner core of bacteriophage φ8 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.

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.