Membrane-containing virus particles exhibit the mechanics of a composite material for genome protection.

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.

Newly isolated Nodularia phage influences cyanobacterial community dynamics.

Cyanophages, that is, viruses infecting cyanobacteria, are a key component driving cyanobacterial community dynamics both ecologically and evolutionarily. In addition to reducing biomass and influencing the genetic diversity of their host populations, they can also have a wider community-level impact due to the release of nutrients by phage-induced cell lysis. In this study, we isolated and characterized a new cyanophage, a siphophage designated as vB_NpeS-2AV2, capable of infecting the filamentous nitrogen fixing cyanobacterium Nodularia sp. AV2 with a lytic cycle between 12 and 18 hours. The role of the phage in the ecology of its host Nodularia and competitor Synechococcus was investigated in a set of microcosm experiments. Initially, phage-induced cell lysis decreased the number of Nodularia cells in the cultures. However, around 18%-27% of the population was resistant against the phage infection. Nitrogen was released from the Nodularia cells as a consequence of phage activity, resulting in a seven-fold increase in Synechococcus cell density. In conclusion, the presence of the cyanophage vB_NpeS-2AV2 altered the ecological dynamics in the cyanobacterial community and induced evolutionary changes in the Nodularia population, causing the evolution from a population dominated by susceptible cells to a population dominated by resistant ones.

Temperature and pH dependence of DNA ejection from archaeal lemon-shaped virus His1.

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.

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015).

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

CO2 laser-based dispersion interferometer utilizing orientation-patterned gallium arsenide for plasma density measurements.

A dispersion interferometer based on the second-harmonic generation of a carbon dioxide laser in orientation-patterned gallium arsenide has been developed for measuring electron density in plasmas. The interferometer includes two nonlinear optical crystals placed on opposite sides of the plasma. This instrument has been used to measure electron line densities in a pulsed radio-frequency generated argon plasma. A simple phase-extraction technique based on combining measurements from two successive pulses of the plasma has been used. The noise-equivalent line density was measured to be 1.7 × 10(17) m(-2) in a detection bandwidth of 950 kHz. One of the orientation-patterned crystals produced 13 mW of peak power at the second-harmonic wavelength from a carbon dioxide laser with 13 W of peak power. Two crystals arranged sequentially produced 58 mW of peak power at the second-harmonic wavelength from a carbon dioxide laser with 37 W of peak power.

DNA ejection from an archaeal virus--a single-molecule approach.

The translocation of genetic material from the viral capsid to the cell is an essential part of the viral infection process. Whether the energetics of this process is driven by the energy stored within the confined nucleic acid or cellular processes pull the genome into the cell has been the subject of discussion. However, in vitro studies of genome ejection have been limited to a few head-tailed bacteriophages with a double-stranded DNA genome. Here we describe a DNA release system that operates in an archaeal virus. This virus infects an archaeon Haloarcula hispanica that was isolated from a hypersaline environment. The DNA-ejection velocity of His1, determined by single-molecule experiments, is comparable to that of bacterial viruses. We found that the ejection process is modulated by the external osmotic pressure (polyethylene glycol (PEG)) and by increased ion (Mg(2+) and Na(+)) concentration. The observed ejection was unidirectional, randomly paused, and incomplete, which suggests that cellular processes are required to complete the DNA transfer.

Lipid fingerprints of intact viruses by MALDI-TOF/mass spectrometry.

A number of viruses contain lipid membranes, which are in close contact with capsid proteins and/or nucleic acids and have an important role in the viral infection process. In this study membrane lipids of intact viruses have been analysed by MALDI-TOF/MS with a novel methodology avoiding lipid extraction and separation steps. To validate the novel method, a wide screening of viral lipids has been performed analysing highly purified intact bacterial and archaeal viruses displaying different virion architectures. Lipid profiles reported here contain all lipids previously detected by mass spectrometry analyses of virus lipid extracts. Novel details on the membrane lipid composition of selected viruses have also been obtained. In addition we show that this technique allows the study of lipid distribution easily in subviral particles during virus fractionation. The possibility to reliably analyse minute amounts of intact viruses by mass spectrometry opens new perspectives in analytical and functional lipid studies on a wider range of viruses including pathogenic human ones, which are difficult to purify in large amounts.

Biochemical and structural characterisation of membrane-containing icosahedral dsDNA bacteriophages infecting thermophilic Thermus thermophilus.

Icosahedral dsDNA viruses isolated from hot springs and proposed to belong to the Tectiviridae family infect the gram-negative thermophilic Thermus thermophilus bacterium. Seven such viruses were obtained from the Promega Corporation collection. The structural protein patterns of three of these viruses, growing to a high titer, appeared very similar but not identical. The most stable virus, P23-77, was chosen for more detailed studies. Analysis of highly purified P23-77 by thin layer chromatography for neutral lipids showed lipid association with the virion. Cryo-EM based three-dimensional image reconstruction of P23-77 to 1.4 nm resolution revealed an icosahedrally-ordered protein coat, with spikes on the vertices, and an internal membrane. The capsid architecture of P23-77 is most similar to that of the archaeal virus SH1. These findings further complicate the grouping of icosahedrally-symmetric viruses containing an inner membrane. We propose a single superfamily or order with members in several viral families.

Total hip replacement after intra-articular injection of local anaesthetic and steroid.

Intra-articular injections of steroid into the hip are used for a variety of reasons in current orthopaedic practice. Recently their safety prior to ipsilateral total hip replacement has been called into question owing to concerns about deep joint infection. We undertook a retrospective analysis of all patients who had undergone local anaesthetic and steroid injections followed by ipsilateral total hip replacement over a five-year period. Members of the surgical team, using a lateral approach to the hip, performed all the injections in the operating theatre using a strict aseptic technique. The mean time between injection and total hip replacement was 18 months (4 to 50). The mean follow-up after hip replacement was 25.8 months (9 to 78), during which time no case of deep joint sepsis was found. In our series, ipsilateral local anaesthetic and steroid injections have not conferred an increased risk of infection in total hip replacement. We believe that the practice of intra-articular local anaesthetic and steroid injections to the hip followed by total hip replacement is safer than previously reported.

Cloning and sequence analysis of dsRNA segments 5, 6 and 7 of a novel non-group A, B, C adult rotavirus that caused an outbreak of gastroenteritis in China.

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.

Crystallization of the membrane-containing bacteriophage PRD1 in quartz capillaries by vapour diffusion.

Crystals of bacteriophage PRD1, a virus containing an internal lipid bilayer, have been grown in thin-walled quartz capillary tubes by vapour diffusion as a means of eliminating mechanical handling of the crystals during data collection. It has been found that the addition of polyethylene glycol 20 000 (PEG 20K) to the mother liquor that bathes the crystals allows far higher resolution diffraction intensities to be observed. Growing and treating the crystals in this way has produced a small number of crystals which are particularly amenable to X-ray diffraction analysis.

Safety and efficacy of intra-articular injection of the hip.

The aim of the present study was twofold: a) to establish the therapeutic efficacy of the hip injection with marcaine and steroid; (b) to establish the safety of the procedure with respect to possible bacterial contamination at the time of passage of needle into the hip joint. Thirty-five patients with osteoarthritis of the hip were injected. All patients were admitted as day cases and the procedure was performed in a laminar flow theatre under full aseptic conditions. The hip was aspirated before injection. Aspirate and injecting needles were sent for microbiological examination. Both aerobic and anaerobic cultures were performed. All patients were followed up for three months after hip injection. Microbiology results were not revealed to reviewing clinicians. All patients were reviewed at two weeks and at twelve weeks. Thirty-three patients (94.3%) had no growth in the samples. Two cases out of thirty-five hips had a positive culture but none of the patients went on to develop clinical sepsis. Patient response for pain in the hip was graded using a 10 point visual analogue scale (VAS) with 10 point as maximum pain and 0 points as no pain. Pre-injection the patient VAS for pain was a mean value of 6.4 0.77 (median, 6). The mean value of VAS score at 2 weeks dropped to 2.6 2.7 with median of 2. This difference was highly significant (p=0.003). At 12 weeks mean VAS score was 2.7 2.5 with median value of 2 (p=001). Three patients were worse after the injection. We found the hip injection to be safe. The risk of bacterial contamination is low provided a strict aseptic protocol is observed. In this study, one third of the patients had excellent pain relief for 3 months after the injection but in two thirds of cases it failed to provide complete, long-term pain relief (> 3 months). (Hip International 2002; 4: 378-82).

A minor capsid protein P30 is essential for bacteriophage PRD1 capsid assembly.

Bacteriophage PRD1 is a double-stranded DNA virus infecting Gram-negative hosts. It has a membrane component located in the interior of the isometric capsid. In addition to the major capsid protein P3, the capsid contains a 9 kDa protein P30. Protein P30 is proposed to be located between the adjacent facets of the icosahedral capsid and is required for stable capsid assembly. In its absence, an empty phage-specific membrane vesicle is formed. The major protein component of this vesicle is a phage-encoded assembly factor, protein P10, that is not present in the final structure.

Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions.

BACKGROUND: The dsDNA bacteriophage PRD1 has a membrane inside its icosahedral capsid. While its large size (66 MDa) hinders the study of the complete virion at atomic resolution, a 1.65-A crystallographic structure of its major coat protein, P3, is available. Cryo-electron microscopy (cryo-EM) and three-dimensional reconstruction have shown the capsid at 20-28 A resolution. Striking architectural similarities between PRD1 and the mammalian adenovirus indicate a common ancestor. RESULTS: The P3 atomic structure has been fitted into improved cryo-EM reconstructions for three types of PRD1 particles: the wild-type virion, a packaging mutant without DNA, and a P3-shell lacking the membrane and the vertices. Establishing the absolute EM scale was crucial for an accurate match. The resulting "quasi-atomic" models of the capsid define the residues involved in the major P3 interactions, within the quasi-equivalent interfaces and with the membrane, and show how these are altered upon DNA packaging. CONCLUSIONS: The new cryo-EM reconstructions reveal the structure of the PRD1 vertex and the concentric packing of DNA. The capsid is essentially unchanged upon DNA packaging, with alterations limited to those P3 residues involved in membrane contacts. These are restricted to a few of the N termini along the icosahedral edges in the empty particle; DNA packaging leads to a 4-fold increase in the number of contacts, including almost all copies of the N terminus and the loop between the two beta barrels. Analysis of the P3 residues in each quasi-equivalent interface suggests two sites for minor proteins in the capsid edges, analogous to those in adenovirus.

Comparison of polymerase subunits from double-stranded RNA bacteriophages.

The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage phi6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3' termini from the phi6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from phi6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the phi6-related bacteriophages phi8 and phi13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from phi6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3'-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit.

Solution structure of bacteriophage PRD1 vertex complex.

Bacteriophage PRD1 is a prototype of viruses with an internal membrane. The icosahedral capsid and major coat protein share structural similarity with the corresponding structures of adenovirus. The present study further explores similarities between these viruses, considering the 5-fold vertex assemblies. The vertex structure of bacteriophage PRD1 consists of proteins P2, P5, and P31. The vertex complex mediates host cell binding and controls double-stranded DNA delivery. Quaternary structures and interactions of purified spike proteins were studied by synchrotron radiation x-ray solution scattering. Low resolution models of the vertex proteins P5, P2, and P31 were reconstructed ab initio from the scattering data. Protein P5 is a long trimer that resembles the adenovirus spike protein pIV. The receptor-binding protein P2 is a 15.5-nm long, thin monomer and does not have an adenovirus counterpart. P31 forms a pentameric base with a maximum diameter of 8.5 nm, which is thinner than the adenovirus penton pIII. P5 further polymerize into a nonameric form ((P5(3))(3)). In the presence of P31, P5 associates into a P5(6):P31 complex. The constructed models of these assemblies provided support for a model of vertex assembly onto the virion. Although similar in overall architecture, clear differences between PRD1 and adenovirus spike assemblies have been revealed.

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

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

Self-assembly of a viral molecular machine from purified protein and RNA constituents.

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

A mechanism for initiating RNA-dependent RNA polymerization.

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.

Macromolecular assemblies: greater than their parts.

Increasingly powerful methods of analysis have opened up complex macromolecular assemblies to scrutiny at atomic detail. They reveal not only examples of assembly from preformed and prefolded components, but also examples in which the act of assembly drives changes to the components. In the most extreme of these examples, some of the components only achieve a folded state when the complex is formed. Striking results have appeared for systems ranging from the already mature field of virus structure and assembly, where notable progress has been made for rather complex capsids, to descriptions of ribosome structures in atomic detail, where recent results have emerged at breathtaking speed.