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Species of genus Shewanella are among the most frequently identified psychrotrophic bacteria. Here, we have studied the cellular properties, growth dynamics, and stress conditions of cold-active Shewanella strain #4, which was previously isolated from Baltic Sea ice. The cells are rod-shaped of ~2μm in length and 0.5μm in diameter, and they grow between 0 and 25°C, with an optimum at 15°C. The bacterium grows at a wide range of conditions, including 0.5–5.5% w/v NaCl (optimum 0.5–2% w/v NaCl), pH 5.5–10 (optimum pH 7.0), and up to 1mM hydrogen peroxide. In keeping with its adaptation to cold habitats, some polyunsaturated fatty acids, such as stearidonic acid (18:4n-3), eicosatetraenoic acid (20:4n-3), and eicosapentaenoic acid (20:5n-3), are produced at a higher level at low temperature. The genome is 4,456kb in size and has a GC content of 41.12%. Uniquely, strain #4 possesses genes for sialic acid metabolism and utilizes N-acetyl neuraminic acid as a carbon source. Interestingly, it also encodes for cytochrome c3 genes, which are known to facilitate environmental adaptation, including elevated temperatures and exposure to UV radiation. Phylogenetic analysis based on a consensus sequence of the seven 16S rRNA genes indicated that strain #4 belongs to genus Shewanella, closely associated with Shewanella aestuarii with a ~97% similarity, but with a low DNA–DNA hybridization (DDH) level of ~21%. However, average nucleotide identity (ANI) analysis defines strain #4 as a separate Shewanella species (ANI score=76). Further phylogenetic analysis based on the 92 most conserved genes places Shewanella strain #4 into a distinct phylogenetic clade with other cold-active marine Shewanella species. Considering the phylogenetic, phenotypic, and molecular characterization, we conclude that Shewanella strain #4 is a novel species and name it Shewanella glacialimarina sp. nov. TZS-4T, where glacialimarina means sea ice. Consequently, S. glacialimarina TZS-4T constitutes a promising model for studying transcriptional and translational regulation of cold-active metabolism.

The protection of the viral genome during extracellular transport is an absolute requirement for virus survival and replication. In addition to the almost universal proteinaceous capsids, certain viruses add a membrane layer that encloses their double-stranded (ds) DNA genome within the protein shell. Using the membrane-containing enterobacterial virus PRD1 as a prototype, and a combination of nanoindentation assays by atomic force microscopy and finite element modelling, we show that PRD1 provides a greater stability against mechanical stress than that achieved by the majority of dsDNA icosahedral viruses that lack a membrane. We propose that the combination of a stiff and brittle proteinaceous shell coupled with a soft and compliant membrane vesicle yields a tough composite nanomaterial well-suited to protect the viral DNA during extracellular transport., We thank MINECO for the Severo Ochoa Excellence Accreditation to the CIC bioGUNE (SEV-2016-0644) and the University of Helsinki and Academy of Finland (grant 1306833) for the support to Biomolecular Complex Purification (Biocomplex), part of Instruct-FI, used in this study. This research was supported by Bruker – Spain (to S. A. and N. G. A. A.), by the Academy of Finland (Academy Professor funding grants 283072 and 255342 to D. H. B.), by the European Research Council (starting grant FP7-ERC-2012-StG-306435 to R. P. R.), and by the Spanish Ministry of Economy and Competitiveness (MINECO/FEDER; MAT2014-54867-R to R. P. R.; MAT2015-63704-P to G. A. S.; BFU2015-64541-R to N. G. A. A.).

The vertical double β-barrel major capsid protein (MCP) fold, fingerprint of the PRD1-adeno viral lineage, is widespread in many viruses infecting organisms across the three domains of life. The discovery of PRD1-like viruses with two MCPs challenged the known assembly principles. Here, we present the cryo-electron microscopy (cryo-EM) structures of the archaeal, halophilic, internal membrane-containing Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica icosahedral virus 2 (HHIV-2) at 3.7 and 3.8 Å resolution, respectively. Our structures reveal proteins located beneath the morphologically distinct two- and three-tower capsomers and homopentameric membrane proteins at the vertices that orchestrate the positioning of pre-formed vertical single β-barrel MCP heterodimers. The cryo-EM based structures together with the proteomics data provide insights into the assembly mechanism of this type of viruses and into those with membrane-less double β-barrel MCPs., Here, the authors present the cryo-EM structures of two archaeal, halophilic, internal membrane-containing icosahedral viruses at 3.7 and 3.8 Å resolution, providing insights into the assembly process of these and related PRD1-adeno lineage viruses.

Basic and applied virus research requires specimens that are purified to high homogeneity. Thus, there is much interest in the efficient production and purification of viruses and their subassemblies. Advances in the production steps have shifted the bottle neck of the process to the purification. Nonetheless, the development of purification techniques for different viruses is challenging due to the complex biological nature of the infected cell cultures as well as the biophysical and -chemical differences in the virus particles. We used bacteriophage phi 6 as a model virus in our attempts to provide a new purification method for enveloped viruses. We compared asymmetrical flow field-flow fractionation (AF4)-based virus purification method to the well-established ultracentrifugation-based purification of phi 6. In addition, binding of phi 6 virions to monolithic anion exchange columns was tested to evaluate their applicability in concentrating the AF4 purified specimens. Our results show that AF4 enables one-hour purification of infectious enveloped viruses with specific infectivity of similar to 1 x 10(13) PFU/mg of protein and similar to 65-95% yields. Obtained purity was comparable with that obtained using ultracentrifugation, but the yields from AF4 purification were 2-3-fold higher. Importantly, high quality virus preparations could be obtained directly from crude cell lysates. Furthermore, when used in combination with inline light scattering detectors, AF4 purification could be coupled to simultaneous quality control of obtained virus specimen.

Phage Cr39582 was induced by mitomycin C from Pseudoalteromonas sp. strain Cr6751, isolated from a marine invertebrate gut. Pseudoalteromonas phage Cr39582 has 85% pairwise nucleotide identity with phage PM2 but lacks sequence homology in the spike protein. This report supports previous bioinformatic identification of corticoviral sequences within aquatic bacterial genomes.

Extremely halophilic Archaea are the only known hosts for pleolipoviruses which are pleomorphic non-lytic viruses resembling cellular membrane vesicles. Recently, pleolipoviruses have been acknowledged by the International Committee on Taxonomy of Viruses (ICTV) as the first virus family that contains related viruses with different DNA genomes. Genomic diversity of pleolipoviruses includes single-stranded and double-stranded DNA molecules and their combinations as linear or circular molecules. To date, only eight viruses belong to the family Pleolipoviridae. In order to obtain more information about the diversity of pleolipoviruses, further isolates are needed. Here we describe the characterization of a new halophilic virus isolate, Haloarcula hispanica pleomorphic virus 4 (HHPV4). All pleolipoviruses and related proviruses contain a conserved core of approximately five genes designating this virus family, but the sequence similarity among different isolates is low. We demonstrate that over half of HHPV4 genome is identical to the genome of pleomorphic virus HHPV3. The genomic regions encoding known virion components are identical between the two viruses, but HHPV4 includes unique genetic elements, e.g., a putative integrase gene. The co-evolution of these two viruses demonstrates the presence of high recombination frequency in halophilic microbiota and can provide new insights considering links between viruses, membrane vesicles, and plasmids.

SummaryRNA viruses have specific mutation rates that balance the conflicting needs of an evolutionary response to host antiviral defenses and avoidance of the error catastrophe. While most mutations are known to originate in replication errors, difficulties of capturing the underlying dynamics have left the mechanochemical basis of viral mutagenesis unresolved. Here, we use multiplexed magnetic tweezers to investigate error incorporation by the bacteriophage Φ6 RNA-dependent RNA polymerase. We extract large datasets fingerprinting real-time polymerase dynamics over four magnitudes in time, in the presence of nucleotide analogs, and under varying NTP and divalent cation concentrations and fork stability. Quantitative analysis reveals a new pause state that modulates polymerase fidelity and so ties viral polymerase pausing to the biological function of optimizing virulence. Adjusting the frequency of such pauses offers a target for therapeutics and may also reflect an evolutionary strategy for virus populations to track the gradual evolution of their hosts.

Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses is to find efficient purification conditions where viruses maintain their infectivity. Asymmetrical flow field-flow fractionation (AF4) is a gentle native chromatography-like technique for size-based separation. It does not have solid stationary phase and the mobile phase composition is readily adjustable according to the sample needs. Due to the high separation power of specimens up to 50 A mu m, AF4 is suitable for virus purification. Here, we applied AF4 for extremophilic viruses representing four morphotypes: lemon-shaped, tailed and tailless icosahedral, as well as pleomorphic enveloped. AF4 was applied to input samples of different purity: crude supernatants of infected cultures, polyethylene glycol-precipitated viruses and viruses purified by ultracentrifugation. All four virus morphotypes were successfully purified by AF4. AF4 purification of culture supernatants or polyethylene glycol-precipitated viruses yielded high recoveries, and the purities were comparable to those obtained by the multistep ultracentrifugation purification methods. In addition, we also demonstrate that AF4 is a rapid monitoring tool for virus production in slowly growing host cells living in extreme conditions.

Genome packaging and delivery are fundamental steps in the replication cycle of all viruses. Icosahedral viruses with linear double-stranded DNA (dsDNA) usually pacicage their genome into a preformed, rigid procapsid using the power generated by a virus-encoded packaging ATPase. The pressure and stored energy due to this confinement of DNA at a high density is assumed to drive the initial stages of genome ejection. Membrane-containing icosahedral viruses, such as bacteriophage PRD1, present an additional architectural complexity by enclosing their genome within an internal membrane vesicle. Upon adsorption to a host cell, the PRD1 membrane remodels into a proteo-lipidic tube that provides a conduit for passage of the ejected linear dsDNA through the cell envelope. Based on volume analyses of PRD1 membrane vesicles captured by cryo-electron tomography and modeling of the elastic properties of the vesicle, we propose that the internal membrane makes a crucial and active contribution during infection by maintaining the driving force for DNA ejection and countering the internal turgor pressure of the host These novel functions extend the role of the PRD1 viral membrane beyond tube formation or the mere physical confinement of the genome. The presence and assistance of an internal membrane might constitute a biological advantage that extends also to other viruses that package their linear dsDNA to high density within an internal vesicle. (C) 2016 Elsevier B.V. All rights reserved.

The simplified artificial environments in which highly complex biological systems are studied do not represent the crowded, dense, salty, and dynamic environment inside the living cell. Consequently, it is important to investigate the effect of crowding agents on DNA. We used a dual-trap optical tweezers instrument to perform force spectroscopy experiments at pull speeds ranging from 0.3 to 270 μm/s on single dsDNA molecules in the presence of poly(ethylene glycol) (PEG) and monovalent salt. PEG of sizes 1,500 and 4,000 Da condensed DNA, and force-extension data contained a force plateau at approximately 1 pN. The level of the force plateau increased with increasing pull speed. During slow pulling the dissipated work increased linearly with pull speed. The calculated friction coefficient did not depend on amount of DNA incorporated in the condensate, indicating internal friction is independent of the condensate size. PEG300 had no effect on the dsDNA force-extension curve. The force plateau implies that condensation induced by crowding agents resembles condensation induced by multivalent cations.

Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae , infects young salmon, with a severe impact on the commercial sea farming industry. Of the five mature proteins encoded by the IPNV genome, the multifunctional VP3 has an essential role in morphogenesis; interacting with the capsid protein VP2, the viral double-stranded RNA (dsRNA) genome and the RNA-dependent RNA polymerase VP1. Here we investigate one of these VP3 functions and present the crystal structure of the C-terminal 12 residues of VP3 bound to the VP1 polymerase. This interaction, visualized for the first time, reveals the precise molecular determinants used by VP3 to bind the polymerase. Competition binding studies confirm that this region of VP3 is necessary and sufficient for VP1 binding, while biochemical experiments show that VP3 attachment has no effect on polymerase activity. These results indicate how VP3 recruits the polymerase into birnavirus capsids during morphogenesis.

A temperate haloarchaeal virus, SNJ1, was induced from the lysogenic host, Natrinema sp. J7-1, with mitomycin C, and the virus produced plaques on lawns of Natrinema sp. J7-2. Optimization of the induction conditions allowed us to increase the titer from ∼10 4 PFU/ml to ∼10 11 PFU/ml. Single-step growth curves exhibited a burst size of ∼100 PFU/cell. The genome of SNJ1 was observed to be a circular, double-stranded DNA (dsDNA) molecule (16,341 bp). Surprisingly, the sequence of SNJ1 was identical to that of a previously described plasmid, pHH205, indicating that this plasmid is the provirus of SNJ1. Several structural protein-encoding genes were identified in the viral genome. In addition, the comparison of putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses, as well as the presence of lipid constituents from the host phospholipid pool, strongly suggest that SNJ1 belongs to the PRD1-type lineage of dsDNA viruses, which have an internal membrane.

The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV) is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its apo form and at 2.2 Å resolution bound to the catalytically-activating metal magnesium. We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxy)nucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase) and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA∶polymerase complexes, providing a mechanism for the genesis of viral genome∶polymerase complexes observed in vivo., Author Summary Infectious pancreatic necrosis virus (IPNV) is highly contagious and causes severe disease in fish. As a result of intensive rearing conditions it has become a serious problem for the salmon and trout farming industries. IPNV, like many other viruses, replicates its genome using a protein (a ‘polymerase’) that is itself encoded by the viral genome. Unusually, in infectious IPNV particles the polymerase is found chemically linked to the viral genome. We have determined the atomic structure of IPNV polymerase using X-ray crystallography, revealing some significant differences in the fold of the protein chain compared to other well-characterized viral polymerases. By mutating an amino acid residue at the beginning of the protein we show how the chemical linkage to the viral genome can be disrupted. This provides an elegant mechanism for the attachment of the viral genome to the polymerase observed in vivo.

Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 A resolution by cryo-electron microscopy and image reconstruction1,2, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 A resolution, allows the first detailed analysis of a membrane-containing virus3. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper3. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.

In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail. Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.

Bacteriophage PRD1 shares many structural and functional similarities with adenovirus. A major difference is the PRD1 internal membrane, which acts in concert with vertex proteins to translocate the phage genome into the host. Multiresolution models of the PRD1 capsid, together with genetic analyses, provide fine details of the molecular interactions associated with particle stability and membrane dynamics. The N- and C-termini of the major coat protein (P3), which are required for capsid assembly, act as conformational switches bridging capsid to membrane and linking P3 trimers. Electrostatic P3-membrane interactions increase virion stability upon DNA packaging. Newly revealed proteins suggest how the metastable vertex works and how the capsid edges are stabilized.

Influenza A viruses (IAVs) impact the public health and global economy by causing yearly epidemics and occasional pandemics. Several anti-IAV drugs are available and many are in development. However, the question remains which of these antiviral agents may allow activation of immune responses and protect patients against co- and re-infections. To answer to this question, we analysed immuno-modulating properties of the antivirals saliphenylhalamide (SaliPhe), SNS-032, obatoclax, and gemcitabine, and found that only gemcitabine did not impair immune responses in infected cells. It also allowed activation of innate immune responses in lipopolysaccharide (LPS)- and interferon alpha (IFN alpha)-stimulated macrophages. Moreover, immuno-mediators produced by gemcitabine-treated IAV-infected macrophages were able to prime immune responses in non-infected cells. Thus, we identified an antiviral agent which might be beneficial for treatment of patients with severe viral infections. (C) 2015 The Authors. Published by Elsevier B.V.

Detailed biochemical and biophysical characterization of viruses requires viral preparations of high quantity and purity. The optimization of virus production and purification is an essential, but laborious and time-consuming process. Asymmetric flow field flow fractionation (AF4) is an attractive alternative method for virus purification because it is a rapid and gentle separation method that should preserve viral infectivity. Here we optimized the AF4 conditions to be used for purification of a model virus, bacteriophage PRD1, from various types of starting materials. Our results show that AF4 is well suited for PRD1 purification as monitored by virus recovery and specific infectivity. Short analysis time and high sample loads enabled us to use AF4 for preparative scale purification of PRD1. Furthermore, we show that AF4 enables the rapid real-time analysis of progeny virus production in infected cells. (C) 2016 Elsevier B.V. All rights reserved.

Our understanding of the third domain of life, Archaea , has greatly increased since its establishment some 20 years ago. The increasing information on archaea has also brought their viruses into the limelight. Today, about 100 archaeal viruses are known, which is a low number compared to the numbers of characterized bacterial or eukaryotic viruses. Here, we have performed a comparative biological and structural study of seven pleomorphic viruses infecting extremely halophilic archaea. The pleomorphic nature of this novel virion type was established by sedimentation analysis and cryo-electron microscopy. These nonlytic viruses form virions characterized by a lipid vesicle enclosing the genome, without any nucleoproteins. The viral lipids are unselectively acquired from host cell membranes. The virions contain two to three major structural proteins, which either are embedded in the membrane or form spikes distributed randomly on the external membrane surface. Thus, the most important step during virion assembly is most likely the interaction of the membrane proteins with the genome. The interaction can be driven by single-stranded or double-stranded DNA, resulting in the virions having similar architectures but different genome types. Based on our comparative study, these viruses probably form a novel group, which we define as pleolipoviruses.

Summary VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV-1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by β-elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy. Such analyses showed that the major VP4-derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5-N-formyl-legionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid-like sugar, in an archaeal-derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with N-acetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC-ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4-derived peptides bearing the N-glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N-glycosylation apparatus, as determined by LC-ESI/MS of heterologously expressed VP4. Here, however, the N-linked pentasaccharide was the same as shown to decorate the S-layer glycoprotein in this species. Hence, N-glycosylation of the haloarchaeal viral protein, VP4, is host-specific. These results thus present additional examples of archaeal N-glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.

RNA-dependent RNA polymerases (RdRps) are key to the replication of RNA viruses. A common divalent cation binding site, distinct from the positions of catalytic ions, has been identified in many viral RdRps. We have applied biochemical, biophysical, and structural approaches to show how the RdRp from bacteriophage ϕ6 uses the bound noncatalytic Mn 2+ to facilitate the displacement of the C-terminal domain during the transition from initiation to elongation. We find that this displacement releases the noncatalytic Mn 2+ , which must be replaced for elongation to occur. By inserting a dysfunctional Mg 2+ at this site, we captured two nucleoside triphosphates within the active site in the absence of Watson-Crick base pairing with template and mapped movements of divalent cations during preinitiation. These structures refine the pathway from preinitiation through initiation to elongation for the RNA-dependent RNA polymerization reaction, explain the role of the noncatalytic divalent cation in ϕ6 RdRp, and pinpoint the previously unresolved Mn 2+ -dependent step in replication.

International audience; The number of viral particles in the biosphere is enormous. Virus classification helps to comprehend the virosphere and to understand the relationship between different virus groups. However, the evolutionary reach of the currently employed sequence-based approaches in virus taxonomy is rather limited, producing a fragmented view of the virosphere. As a result, viruses are currently classified into 87 different families. However, studies on virion architectures have unexpectedly revealed that their structural diversity is far more limited. Here we describe structures of the major capsid proteins of double-stranded DNA viruses infecting hosts residing in different domains of life. We note that viruses belonging to 20 different families fall into only five distinct structural groups, suggesting that optimal virus classification approach should equally rely on both sequence and structural information.

Protein A33 is a type II membrane protein present in the outer envelope of extracellular as well as cell-associated Vaccinia virus particles. A33 has been implicated in mediating cell-to-cell virus spread in an antibody-resistant manner. Here, using state-of-the-art structure prediction methods and structural modeling, we show that A33 has most likely evolved from a C-type lectin-like protein. Comparison of the three-dimensional A33 model to the X-ray structures of distant cellular homologues revealed that A33 retained the key residues required for adopting the C-type lectin-like fold. Our results provide insights into the structure and origin of protein A33.

Coxsackievirus B3 (CBV3) is a member of the human enterovirus B species and a common human pathogen. Even though much is known about the enteroviral life cycle, no specific drugs are available to treat enterovirus infections. RNA interference (RNAi) has evolved to be an important tool for antiviral experimental therapies and gene function studies. We describe here a novel approach for RNAi against CBVs by using a short interfering (siRNA) pool covering 3.5 kb of CBV3 genomic sequence. The RNA-dependent RNA polymerase (RdRP) of bacteriophage φ6 was used to synthesize long double-stranded RNA (dsRNA) from a cloned region (nt 3837–7399) of the CBV3 genome. The dsRNA was cleaved using Dicer, purified and introduced to cells by transfection. The siRNA pool synthesized using the φ6 RdRP (φ6–siRNAs) was considerably more effective than single-site siRNAs. The φ6–siRNA pool also inhibited replication of other enterovirus B species, such as coxsackievirus B4 and coxsackievirus A9.

Icosahedral-tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses translocate viral DNA into a preformed procapsid in an ATP-driven reaction by a packaging complex that operates at a portal vertex. A similar packaging system operates in the tailless dsDNA phage PRD1 (Tectiviridae family), except that there is an internal membrane vesicle in the procapsid. The unit-length linear dsDNA genome with covalently linked 5'-terminal proteins enters the procapsid through a unique vertex. Two small integral membrane proteins, P20 and P22, provide a conduit for DNA translocation. The packaging machinery also contains the packaging ATPase P9 and the packaging efficiency factor P6. Here we describe a method used to obtain purified packaging-competent PRD1 procapsids. The optimized in vitro packaging system allowed efficient packaging of defined DNA substrates. We determined that the genome terminal protein P8 is necessary for packaging and provided an estimation of the packaging rate.

The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced. The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced. The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.

The replication and transcription of double-stranded RNA (dsRNA) viruses occur within a polymerase complex particle in which the viral genome is enclosed throughout the entire life cycle of the virus. A single protein subunit in the polymerase complex is responsible for the template-dependent RNA polymerization activity. The isolated polymerase subunit of the dsRNA bacteriophage φ6 was previously shown to replicate and transcribe given RNA molecules. In this study, we show that this enzyme also catalyzes nontemplated nucleotide additions to single-stranded and double-stranded nucleic acid molecules. This terminal nucleotidyltransferase activity not only is a property of the isolated enzyme but also is detected to take place within the viral nucleocapsid. This is the first time terminal nucleotidyltransferase activity has been reported for a dsRNA virus as well as for a viral particle. The results obtained together with previous high-resolution structural data on the φ6 RNA-dependent RNA polymerase suggest a mechanism for terminal nucleotidyl addition. We propose that the activity is involved in the termination of the template-dependent RNA polymerization reaction on the linear φ6 genome.

Polymyxin B (PMB) is a cationic antibiotic that interacts with the envelopes of gram-negative bacterial cells. The therapeutic use of PMB was abandoned for a long time due to its undesirable side effects; however, the spread of resistance to currently used antibiotics has forced the reevaluation of PMB for clinical use. Previous studies have used enteric bacteria to examine the mode of PMB action, resulting in a somewhat limited understanding of this process. This study examined the effects of PMB on marine pseudoalteromonads and demonstrates that the frequently accepted view that “what is true for Escherichia coli is true for all bacteria” does not hold true. We show here that in contrast to the growth inhibition observed for enteric bacteria, PMB induces lysis of pseudoalteromonads, which is not prevented by high concentrations of divalent cations. Furthermore, we demonstrate that a high membrane voltage is required for the interaction of PMB with the cytoplasmic membranes of pseudoalteromonads, further elucidating the mechanisms by which PMB interacts with the cell envelopes of those gram-negative bacteria.

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A2. We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid–protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.

The assembly of bacteriophage PRD1 proceeds via formation of empty procapsids containing an internal lipid membrane, into which the linear double-stranded DNA genome is subsequently packaged. The packaging ATPase P9 and other putative packaging proteins have been shown to be located at a unique vertex of the PRD1 capsid. Here, we describe the isolation and characterization of a suppressor-sensitive PRD1 mutant deficient in the unique vertex protein P6. Protein P6 was found to be an essential part of the PRD1 packaging machinery; its absence leads to greatly reduced packaging efficiency. Lack of P6 was not found to affect particle assembly, because in the P6-deficient mutant infection, wild-type (wt) amounts of particles were produced, although most were empty. P6 was determined not to be a specificity factor, as the few filled particles seen in the P6-deficient infection contained only PRD1-specific DNA. The presence of P6 was not necessary for retention of DNA in the capsid once packaging had occurred, and P6-deficient DNA-containing particles were found to be stable and infectious, albeit not as infectious as wt PRD1 virions. A packaging model for bacteriophage PRD1, based on previous results and those obtained in this study, is presented.

The archaeal virus His1 isolated from a hypersaline environment infects an extremely halophilic archaeon Haloarcula hispanica. His1 features a lemon-shaped capsid, which is so far found only in archaeal viruses. This unique capsid can withstand high salt concentrations, and can transform into a helical tube, which in turn is resistant to extremely harsh conditions. Hypersaline environments exhibit a wide range of temperatures and pH conditions, which present an extra challenge to their inhabitants. We investigated the influence of pH and temperature on DNA ejection from His1 virus using single-molecule fluorescence experiments. The observed number of ejecting viruses is constant in pH 5 to 9, while the ejection process is suppressed at pH below 5. Similarly, the number of ejections within 15-42 °C shows only a minor increase around 25-37 °C. The maximum velocity of single ejected DNA increases with temperature, in qualitative agreement with the continuum model of dsDNA ejection.

Viruses infecting archaea show a variety of virion morphotypes, and they are currently classified into more than ten viral families or corresponding groups. A pleomorphic virus morphotype is very common among haloarchaeal viruses, and to date, several such viruses have been isolated. Here, we propose the classification of eight such viruses and formation of a new family, Pleolipoviridae (from the Greek pleo for more or many and lipos for lipid), containing three genera, Alpha-, Beta-, and Gammapleolipovirus. The proposal is currently under review by the International Committee on Taxonomy of Viruses (ICTV). The members of the proposed family Pleolipoviridae infect halophilic archaea and are nonlytic. They share structural and genomic features and differ from any other classified virus. The virion of pleolipoviruses is composed of a pleomorphic membrane vesicle enclosing the genome. All pleolipoviruses have two major structural protein species, internal membrane and spike proteins. Although the genomes of the pleolipoviruses are single- or double-stranded, linear or circular DNA molecules, they share the same genome organization and gene synteny and show significant similarity at the amino acid level. The canonical features common to all members of the proposed family Pleolipoviridae show that they are closely related and thus form a new viral family.

Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae , comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea ; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress. IMPORTANCE Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea , structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and significant insights into the organization and architecture of spindle-shaped virions. The obtained data permit the comparison between spindle-shaped viruses residing in widely different ecological niches, improving our understanding of the adaptation of viruses with unusual morphotypes to extreme environmental conditions.

The temperate double-stranded DNA bacteriophage Bam35 infects gram-positive Bacillus thuringiensis cells. Bam35 has an icosahedral protein coat surrounding the viral membrane that encloses the linear 15-kbp DNA genome. The protein coat of Bam35 uses the same assembly principle as that of PRD1, a lytic bacteriophage infecting gram-negative hosts. In this study, we dissected the process of Bam35 entry into discrete steps: receptor binding, peptidoglycan penetration, and interaction with the plasma membrane (PM). Bam35 very rapidly adsorbs to the cell surface, and N -acetyl-muramic acid is essential for Bam35 binding. Zymogram analysis demonstrated that peptidoglycan-hydrolyzing activity is associated with the Bam35 virion. We showed that the penetration of Bam35 through the PM is a divalent-cation-dependent process, whereas adsorption and peptidoglycan digestion are not.

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.

A diarrhoeal outbreak among adults in China was caused by a new rotavirus, termed ADRV-N, that does not react with antisera directed against group A, B or C rotaviruses [Zhonghua Liu Xing Bing Xue Za Zhi (Chin. Epidemiol.) 19 (1998) 336]. ADRV-N can be propagated in cell cultures [Zhonghua Yi Xue Za Zhi (Natl. Med. J. China) 82 (2002) 14]. We present the complete sequences for ADRV-N genome segments 5 and 6, and a full ORF sequence of genome segment 7. The deduced amino acid sequences suggest that these segments encode NSP1, VP6 and NSP3, respectively. These three ADRV-N genome segments have a unique -ACCCC-3′ terminal sequence. The 5′-GG- terminus of segments 5 and 6 is the same as that of other rotaviruses. The amino acid similarity between VP6 and NSP3 of ADRV-N and the cognate sequences of their closest counterpart, group B IDIR, was 37 and 35%, respectively. The ADRV-N NSP1 has a double-stranded RNA binding motif (DSRM) and a putative autoproteolytic cleavage motif upstream from the DSRM. The putative ADRV-N NSP3 has a truncated C-terminus compared to the cognate protein of group B rotaviruses. All the available data demonstrate that ADRV-N differs significantly from the known rotaviruses and strongly suggest that ADRV-N is the first recognized member of a new group of rotaviruses infecting humans.

The phospholipid class and molecular species compositions of bacteriophage phi6 and its host Pseudomonas syringae were determined quantitatively using TLC and liquid-chromatography/electrospray ionization mass-spectrometry. In addition, the fatty acid compositions of the phospholipids were analyzed by gas-chromatography/mass-spectrometry. The phage contained significantly more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host cytoplasmic (CM) and outer (OM) membranes. In addition, the phospholipid molecular species composition of the viral membrane differed from those of the host membranes, but resembled that of CM more than OM as shown by principal component analysis (PCA). The membrane of phi6 contained more 34:1 and 34:2, and less 32:1 PE and PG molecular species than the host CM or OM. Also, phi6 contained negligible amounts of saturated phospholipid molecular species. These data provide the first biochemical evidence suggesting that phi6 obtains its lipids from the CM. This process is not unselective, but certain phospholipid species are preferentially incorporated in the phage membrane. Common factors leading to similar enrichment of PG in every membrane-containing bacterial virus system studied so far (phi6, PM2, PRD1, PR4, Bam35) are discussed.

This review summarizes the combined insights from recent structural and functional studies of viral RNA-dependent RNA polymerases (RdRPs) with the primary focus on the mechanisms of initiation of RNA synthesis. Replication of RNA viruses has traditionally been approached using a combination of biochemical and genetic methods. Recently, high-resolution structures of six viral RdRPs have been determined. For three RdRPs, enzyme complexes with metal ions, single-stranded RNA and/or nucleoside triphosphates have also been solved. These advances have expanded our understanding of the molecular mechanisms of viral RNA synthesis and facilitated further RdRP studies by informed site-directed mutagenesis. What transpires is that the basic polymerase right hand shape provides the correct geometrical arrangement of substrate molecules and metal ions at the active site for the nucleotidyl transfer catalysis, while distinct structural elements have evolved in the different systems to ensure efficient initiation of RNA synthesis. These elements feed the template, NTPs and ions into the catalytic cavity, correctly position the template 3′ terminus, transfer the products out of the catalytic site and orchestrate the transition from initiation to elongation.

The double-stranded DNA (dsDNA) virus PRD1 carries its genome in a membrane surrounded by an icosahedral protein shell. The shell contains 240 copies of the trimeric P3 protein arranged with a pseudo T = 25 triangulation that is reminiscent of the mammalian adenovirus. DNA packaging and infection are believed to occur through the vertices of the particle. We have used immunolabeling to define the distribution of proteins on the virion surface. Antibodies to protein P3 labeled the entire surface of the virus. Most of the 12 vertices labeled with antibodies directed against proteins P5, P2, and P31. These proteins are known to function in virus binding to the cell surface. Proteins P6, P11, and P20 were found on a single vertex per virion. The P6 and P20 proteins are believed to function in DNA packaging. Protein P11 is a pilot protein that is involved in a complex that mediates the early stages of DNA entry to the host cell. Labeling with antibodies to P5 or P2 did not affect the labeling of P6, the unique vertex protein. Labeling with antibodies to the unique vertex protein P6 interfered with the labeling by antibodies to the unique vertex protein P20. We conclude that PRD1 utilizes 11 of its vertices for initial receptor binding. It utilizes a single, unique vertex for both DNA packing during assembly and DNA delivery during infection.

Holin proteins are phage-induced integral membrane proteins which regulate the access of lytic enzymes to host cell peptidoglycan at the time of release of progeny viruses by host cell lysis. We describe the identification of the membrane-containing phage PRD1 holin gene (gene XXXV ). The PRD1 holin protein (P35, 12.8 kDa) acts similarly to its functional counterpart from phage lambda (gene S ), and the defect in PRD1 gene XXXV can be corrected by the presence of gene S of lambda. Several nonsense, missense, and insertion mutations in PRD1 gene XXXV were analyzed. These studies support the overall conclusion that the charged amino acids at the protein C terminus are involved in the timing of host cell lysis.

Summary DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mech- anisms offer an opportunity to obtain useful informa- tion in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double- stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recogni- tion. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) pen- etration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA transloca- tion. We present a model for PRD1 DNA entry propos- ing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.

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

The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae , infecting two gram-negative Pseudoalteromonas species. The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins. The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer. The C-terminal part of P1 is distal and is responsible for receptor binding activity. The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome. This internal particle is designated the lipid core. The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family Tectiviridae .

Double-stranded RNA (dsRNA) is an inducer molecule of the RNA silencing (RNA interference, RNAi) pathway that is present in all higher eukaryotes and controls gene expression at the posttranscriptional level. This mechanism allows the cell to recognize aberrant genetic material in a highly sequence specific manner. This ultimately leads to degradation of the homologous target sequence, rendering the plant cell resistant to subcellular pathogens. Consequently, dsRNA-mediated resistance has been exploited in transgenic plants to convey resistance against viruses. In addition, it has been shown that enzymatically synthesized specific dsRNA molecules can be applied directly onto plant tissue to induce resistance against the cognate virus. This strongly implies that dsRNA molecules are applicable as efficacious agents in crop protection, which will fuel the demand for cost-effective dsRNA production methods. In this chapter, the different methods for dsRNA production-both in vitro and in vivo-are described in detail.

P3 has been imaged with X-ray crystallography to reveal a trimeric molecule with strikingly similar characteristics to hexon, the major coat protein of adenovirus. The structure of native P3 has now been extended to 1.65 A resolution (R(work) = 19.0% and R(free) = 20.8%). The new high-resolution model shows that P3 forms crystals through hydrophobic patches solvated by 2-methyl-2,4-pentanediol molecules. It reveals details of how the molecule's high stability may be achieved through ordered solvent in addition to intra- and intersubunit interactions. Of particular importance is a 'puddle' at the top of the molecule containing a four-layer deep hydration shell that cross-links a complex structural feature formed by 'trimerization loops'. These loops also link subunits by extending over a neighbor to reach the third subunit in the trimer. As each subunit has two eight-stranded viral jelly rolls, the trimer has a pseudo-hexagonal shape to allow close packing in its 240 hexavalent capsid positions. Flexible regions in P3 facilitate these interactions within the capsid and with the underlying membrane. A selenometh-ionine P3 derivative, with which the structure was solved, has been refined to 2.2 A resolution (R(work) = 20.1% and R(free) = 22.8%). The derivatized molecule is essentially unchanged, although synchrotron radiation has the curious effect of causing it to rotate about its threefold axis. P3 is a second example of a trimeric 'double-barrel' protein that forms a stable building block with optimal shape for constructing a large icosahedral viral capsid. A major difference is that hexon has long variable loops that distinguish different adenovirus species. The short loops in P3 and the severe constraints of its various interactions explain why the PRD1 family has highly conserved coat proteins.

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.

Bacteriophagephi;6 has a three-segmented double-stranded (ds) RNA genome, which resides inside a polymerase complex particle throughout the entire life cycle of the virus. The polymerase subunit P2, a minor constituent of the polymerase complex, has previously been reported to replicate bothphi;6-specific and heterologous single-stranded (ss) RNAs, giving rise to dsRNA products. In this study, we show that the enzyme is also able to use dsRNA templates to perform semi-conservative RNA transcription in vitro without the assistance of other proteins. The polymerase synthesizes predominantly plus-sense copies ofphi;6 dsRNA, medium and small segments being more efficient templates than the large one. This distribution of the test-tube reaction products faithfully mimics viral transcription in vivo. Experiments with chimeric ssRNAs and dsRNAs show that short terminal nucleotide sequences can account for the difference in efficiency of RNA synthesis. Taken together, these results suggest a model explaining important aspects of viral RNA metabolism regulation in terms of enzymatic properties of the polymerase subunit.

Phage PRD1 and adenovirus share a number of structural and functional similarities, one of which is the vertex organization at the fivefold-symmetry positions. We developed an in vitro mutagenesis system for the linear PRD1 genome in order to make targeted mutations. The role of protein P5 in the vertex structure was examined by this method. Mutation in gene V revealed that protein P5 is essential. The absence of P5 did not compromise the particle assembly or DNA packaging but led to a deficient vertex structure where the receptor binding protein P2, in addition to protein P5, was missing. P5 − particles also lost their DNA upon purification. Based on this and previously published information we propose a spatial model for the spike structure at the vertices. This resembles to the corresponding structure in adenovirus.

BacteriophagePRD1 has remarkable structural similarities to adenovirus, but is unusual in containing a membrane beneath its icosahedral capsid. Its monomeric receptor-binding protein, P2, is part of a complex at each capsid vertex and so is the functional equivalent of adenovirus fiber. P2 has been crystallized by the “hanging-drop” method of vapor diffusion and two different crystal forms were obtained. Macroseeding, used to increase the size of the initial small needles, gave rod-shaped crystals. These grew to a size of 0.08 × 0.08 × 0.50 mm3 and diffracted to 2.6 A resolution. They have the orthorhombic space group P2221, with unit cell dimensions a = 137.8 A, b = 46.5 A, c = 136.4 A. A few single crystals of a second form were grown without seeding under slightly different conditions. A parallelepiped crystal (0.10 × 0.10 × 0.35 mm3), with space group C2221 and unit cell dimensions a = 182.3 A, b = 204.8 A, c = 133.3 A, diffracted to 3.5 A resolution. A rotation function for the second form revealed that four monomers of P2 are related by a noncrystallographic twofold axis. The structure of P2 will reveal how this arrangement relates to the trimeric adenovirus fiber.