Activation of viral transcription by stepwise largescale folding of an RNA virus genome.

The genomes of RNA viruses contain regulatory elements of varying complexity. Many plus-strand RNA viruses employ largescale intra-genomic RNA-RNA interactions as a means to control viral processes. Here, we describe an elaborate RNA structure formed by multiple distant regions in a tombusvirus genome that activates transcription of a viral subgenomic mRNA. The initial step in assembly of this intramolecular RNA complex involves the folding of a large viral RNA domain, which generates a discontinuous binding pocket. Next, a distally-located protracted stem-loop RNA structure docks, via base-pairing, into the binding site and acts as a linchpin that stabilizes the RNA complex and activates transcription. A multi-step RNA folding pathway is proposed in which rate-limiting steps contribute to a delay in transcription of the capsid protein-encoding viral subgenomic mRNA. This study provides an exceptional example of the complexity of genome-scale viral regulation and offers new insights into the assembly schemes utilized by large intra-genomic RNA structures.

Stability of Cucumber Necrosis Virus at the Quasi-6-Fold Axis Affects Zoospore Transmission.

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus As with other members of the Tombusvirus genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). P73 lies immediately adjacent to a putative zinc binding site (M. Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) that is formed by three icosahedrally related His residues in the N termini of the C subunit at the quasi-6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild-type CNV in the native and swollen state and of the transmission-defective mutant, P73G, under native conditions. With the wild-type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi-6-fold at the ß-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the ß-annulus had been disrupted and that this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the ß-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission.IMPORTANCECucumber necrosis virus (CNV), a member of the genus Tombusvirus, is transmitted in nature via zoospores of the fungus Olpidium bornovanus While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). Here, we show that the P73G mutant is less stable than the wild type, and this appears to be correlated with destabilization of the ß-annulus at the icosahedral 3-fold axes. Therefore, the ß-annulus appears not to be essential for particle assembly but is necessary for interactions with the transmission vector.

Three-dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex.

Positive-strand RNA viruses, which can be devastating pathogens in humans, animals and plants, replicate their genomes on intracellular membranes. Here, we describe the three-dimensional ultrastructural organization of a tombusvirus replicase in yeast, a valuable model for exploring virus-host interactions. We visualized the intracellular distribution of a viral replicase protein using metal-tagging transmission electron microscopy, a highly sensitive nanotechnology whose full potential remains to be developed. These three-dimensional images show how viral replicase molecules are organized when they are incorporated into the active domains of the intracellular replication compartment. Our approach provides a means to study protein activation mechanisms in cells and to identify targets for new antiviral compounds.

Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly.

Uncoating of a virus particle to expose its nucleic acid is a critical aspect of the viral multiplication cycle, as it is essential for the establishment of infection. In the present study, we investigated the role of plant HSP70 homologs in the uncoating process of Cucumber necrosis virus (CNV), a nonenveloped positive-sense single-stranded RNA [(+)ssRNA] virus having a T=3 icosahedral capsid. We have found through Western blot analysis and mass spectrometry that the HSP70 homolog Hsc70-2 copurifies with CNV particles. Virus overlay and immunogold labeling assays suggest that Hsc70-2 is physically bound to virions. Furthermore, trypsin digestion profiles suggest that the bound Hsc70-2 is partially protected by the virus, indicating an intimate association with particles. In investigating a possible role of Hsc70-2 in particle disassembly, we showed that particles incubated with Hsp70/Hsc70 antibody produce fewer local lesions than those incubated with prebleed control antibody on Chenopodium quinoa In conjunction, CNV virions purified using CsCl and having undetectable amounts of Hsc70-2 produce fewer local lesions. We also have found that plants with elevated levels of HSP70/Hsc70 produce higher numbers of local lesions following CNV inoculation. Finally, incubation of recombinant Nicotiana benthamiana Hsc70-2 with virus particles in vitro leads to conformational changes or partial disassembly of capsids as determined by transmission electron microscopy, and particles are more sensitive to chymotrypsin digestion. This is the first report suggesting that a cellular Hsc70 chaperone is involved in disassembly of a plant virus. IMPORTANCE: Virus particles must disassemble and release their nucleic acid in order to establish infection in a cell. Despite the importance of disassembly in the ability of a virus to infect its host, little is known about this process, especially in the case of nonenveloped spherical RNA viruses. Previous work has shown that host HSP70 homologs play multiple roles in the CNV infection cycle. We therefore examined the potential role of these cellular components in the CNV disassembly process. We show that the HSP70 family member Hsc70-2 is physically associated with CNV virions and that HSP70 antibody reduces the ability of CNV to establish infection. Statistically significantly fewer lesions are produced when virions having undetectable HSc70-2 are used as an inoculum. Finally incubation of Hsc70-2 with CNV particles results in conformational changes in particles. Taken together, our data point to an important role of the host factor Hsc70-2 in CNV disassembly.

Transmission electron microscopy and the molecular structure of icosahedral viruses.

The field of structural virology developed in parallel with methodological advances in X-ray crystallography and cryo-electron microscopy. At the end of the 1970s, crystallography yielded the first high resolution structure of an icosahedral virus, the T=3 tomato bushy stunt virus at 2.9Å. It took longer to reach near-atomic resolution in three-dimensional virus maps derived from electron microscopy data, but this was finally achieved, with the solution of complex icosahedral capsids such as the T=25 human adenovirus at ∼3.5Å. Both techniques now work hand-in-hand to determine those aspects of virus assembly and biology that remain unclear. This review examines the trajectory followed by EM imaging techniques in showing the molecular structure of icosahedral viruses, from the first two-dimensional negative staining images of capsids to the latest sophisticated techniques that provide high resolution three-dimensional data, or snapshots of the conformational changes necessary to complete the infectious cycle.

Noncanonical role for the host Vps4 AAA+ ATPase ESCRT protein in the formation of Tomato bushy stunt virus replicase.

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation.

From lows to highs: using low-resolution models to phase X-ray data.

The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ~13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.

A synthetic nanomaterial for virus recognition produced by surface imprinting.

Major stumbling blocks in the production of fully synthetic materials designed to feature virus recognition properties are that the target is large and its self-assembled architecture is fragile. Here we describe a synthetic strategy to produce organic/inorganic nanoparticulate hybrids that recognize non-enveloped icosahedral viruses in water at concentrations down to the picomolar range. We demonstrate that these systems bind a virus that, in turn, acts as a template during the nanomaterial synthesis. These virus imprinted particles then display remarkable selectivity and affinity. The reported method, which is based on surface imprinting using silica nanoparticles that act as a carrier material and organosilanes serving as biomimetic building blocks, goes beyond simple shape imprinting. We demonstrate the formation of a chemical imprint, comparable to the formation of biosilica, due to the template effect of the virion surface on the synthesis of the recognition material.

Tomato bushy stunt viruses (TBSV) in nanotechnology investigated by scanning force and scanning electron microscopy.

Spherical plant viruses like the tomato bushy stunt virus (TBSV) allow for multiple applications in nanotechnology due to their shape. In this article, different types of the virus were created by extending coat protein (CP) at carboxylic termini with 2 different charged amino acids by point mutation. The obtained CPs carried 6 aspartic acid (negative charge) and 4 histamine (positive charge) residues. The ability of TBSV to form self assembled monolayers with large ordered areas on native and chemically modified mica will be presented. The structural differences between layers formed by the wild type and by the genetically modified types will be discussed in detail.

A highly basic KGKKGK sequence in the RNA-binding domain of the Cucumber necrosis virus coat protein is associated with encapsidation of full-length CNV RNA during infection.

The Cucumber necrosis virus particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. The N-terminal 58 aa residue segment of the CP R domain is believed to bind viral RNA within virions and during assembly. We report results of in vivo experiments that examine the role of the R domain in assembly. Deletion analyses identified 3 conserved 5-10 aa regions as playing critical roles. A highly basic KGKKGK sequence was found to be both necessary and sufficient for encapsidation of the full-length genome and polymorphic virions were produced in mutants lacking the KGKKGK sequence. The amount of full-length RNA present in virions was substantially reduced in R domain mutants where 2 of the 4 lysine residues were substituted with alanine, whereas substitution of 4 lysines by arginine had only a modest effect. The potential role of the R domain in formation of a scaffold for particle assembly is discussed.

Induction of particle polymorphism by cucumber necrosis virus coat protein mutants in vivo.

The Cucumber necrosis virus (CNV) particle is a T=3 icosahedron consisting of 180 identical coat protein (CP) subunits. Plants infected with wild-type CNV accumulate a high number of T=3 particles, but other particle forms have not been observed. Particle polymorphism in several T=3 icosahedral viruses has been observed in vitro following the removal of an extended N-terminal region of the CP subunit. In the case of CNV, we have recently described the structure of T=1 particles that accumulate in planta during infection by a CNV mutant (R1+2) in which a large portion of the N-terminal RNA binding domain (R-domain) has been deleted. In this report we further describe properties of this mutant and other CP mutants that produce polymorphic particles. The T=1 particles produced by R1+2 mutants were found to encapsidate a 1.9-kb RNA species as well as smaller RNA species that are similar to previously described CNV defective interfering RNAs. Other R-domain mutants were found to encapsidate a range of specifically sized less-than-full-length CNV RNAs. Mutation of a conserved proline residue in the arm domain near its junction with the shell domain also influenced T=1 particle formation. The proportion of polymorphic particles increased when the mutation was incorporated into R-domain deletion mutants. Our results suggest that both the R-domain and the arm play important roles in the formation of T=3 particles. In addition, the encapsidation of specific CNV RNA species by individual mutants indicates that the R-domain plays a role in the nature of CNV RNA encapsidated in particles.

Formation of two-dimensional crystals of icosahedral RNA viruses.

UNLABELLED: The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A novel form of macroscopic 3D crystals of turnip yellow mosaic virus has been grown in the negative staining solution. On the basis of the experimental results, the hypothesis is advanced that microscopic arrays might be planar crystallisation nuclei. The formation of 2D crystals and the enhancing effect of polyethylene glycol on the self-organisation of virions at the air/water interface are discussed. SYNOPSIS: The formation of 2D arrays of icosahedral viruses was investigated by spectroscopic and transmission electron microscopic methods.

Quantitative analysis of efficient endogenous gene silencing in Nicotiana benthamiana plants using tomato bushy stunt virus vectors that retain the capsid protein gene.

Tomato bushy stunt virus (TBSV) coat protein (CP) replacement vectors have been used previously to silence transgenes (e.g., the green fluorescent protein gene) but have not been effective for silencing endogenous plant genes. New TBSV vectors which retained the CP gene were developed by engineering an XhoI restriction site in three positions (3f, CEB, and CEA) of the pTBSV-100 infectious clone. Magnesium chelatase (ChlH) and phytoene desaturase (PDS) were chosen as targets for endogenous gene silencing. Initial experiments using CP replacement vectors with a 230-bp sense or antisense ChlH insert gave a silencing phenotype prominent only in the first new leaves above those inoculated. No silencing phenotype was apparent beyond these leaves whereas, for PDS, no silencing phenotype was observed. When plants were inoculated with the XhoI insert vectors containing ChlH and PDS sequences, plants showed a silencing phenotype extensively throughout the challenged plant, indicating an improved ability for virus movement and silencing in Nicotiana benthamiana host plants. Silencing efficiencies were quantified using realtime reverse-transcription polymerase chain reaction, indicating specific silencing effects of each individual silencing vector. Only one recombinant vector (pPD-3f5), where the XhoI insert was at the 3' end of the CP gene, failed to give effective silencing. Here, we show that our new CP-retaining TBSV vectors (CEA-CEB) form typical TBSV virions, retain silencing inserts of variable lengths (110 to 260 nucleotides), and can systemically silence endogenous genes in N. benthamiana.

Characterization of polymorphism displayed by the coat protein mutants of tomato bushy stunt virus.

Expression of full-length and N-terminal deletion mutants of the coat protein (CP) of tomato bushy stunt virus (TBSV) using the recombinant baculovirus system resulted in spontaneously assembled virus-like particles (VLPs). Deletion of the majority of the R-domain sequence of the CP, residues 1-52 (CP-NDelta52) and 1-62 (CP-NDelta62), produced capsids similar to wild-type VLPs. Interestingly, the CP-NDelta62 mutant that retains the last 3 residues of R-domain is capable of forming both the T = 1 and T = 3 particles. However, between the two types of VLPs, formation of the T = 1 capsids appears to be preferred. Another mutant, CP-NDelta72, in which R-domain (residues 1-65) was completely removed but contains most of the beta-annulus and extended arm (betaA) regions exclusively formed T = 1 particles. These results suggest that as few as 3 residues (63-65) of the R-domain, which includes 2 basic amino acids together with the arm (betaA) and beta-annulus regions, may be sufficient for the formation of T = 3 particles. However, anywhere between 4 to 13 residues of the R-domain may be required for proper positioning of betaA and beta-annulus structural elements of the C-type subunits to facilitate an error free assembly of T = 3 capsids.

Divalent ion-dependent swelling of tomato bushy stunt virus: a multi-approach study.

Time-resolved small-angle X-ray and neutron scattering (SAXS and SANS) in solution were used to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. SAXS intensities comprise contribution from the protein capsid and the RNA moiety, while neutron scattering, recorded in 72% D2O, is essentially due to the protein capsid. Cryo-electron micrographs of compact and swollen virus were used to produce 3D reconstructions of the initial and final conformations of the virus at a resolution of 13 A and 19 A, respectively. While compact particles appear to be very homogeneous in size, solutions of swollen particles exhibit some size heterogeneity. A procedure has been developed to compute the SAXS pattern from the 3D reconstruction for comparison with experimental data. Cryo-electron microscopy thereby provides an invaluable starting (and ending) point for the analysis of the time-resolved swelling process using the scattering data.

Viruses and the development of quantitative biological electron microscopy.

The electron microscope has become an important tool for determining the structure of biological materials of all kinds. Many technical advances in specimen preparation and in sophisticated methods of image analysis, initially based on optical systems but latterly on computer processing, have contributed to the development of the subject. Viruses of various kinds have often provided a convenient and appropriate test specimen. This paper describes the major technical advances and shows how viruses have had an important role in most of the developments.

Pelargonium necrotic spot virus: a new member of the genus Tombusvirus.

A virus isolate from Pelargonium spp., provisionally designated UPEV (unknown pelargonium virus), had isometric particles 31-33 nm in diameter, with a granular surface structure similar to that of viruses in three genera of family Tombusviridae. Immunoelectron microscopy proved that UPEV was serologically distinct from all examined morphologically similar members of the family Tombusviridae. The induced cytopathology was characterized by large cytoplasmic virion aggregates and the formation of multivesicular bodies derived from mitochondria. Analysis of the complete ssRNA genome sequence revealed four open reading frames (ORFs) arranged like those of viruses in the genera Tombusvirus and Aureusvirus. Sequence comparisons indicated that three of the four ORFs had a high identity (52-97% identical amino acids) with the respective ORFs of tombusvirus species, especially with Carnation Italian ringspot virus, but not with those of viruses in other genera in Tombusviridae. On the contrary, UPEV coat protein had a low indentity (36-53% identical amino acids) with that of the aureusvirus Pothos latent virus. The data suggested that UPEV originated in a recombination event between a tombus- and an aureusvirus. According to its original host and symptom expression we proposed the new virus be named Pelargonium necrotic spot virus (PeNSV) and classified it as a distinct and new species in the genus Tombusvirus.

Host-dependent recombination of a Tomato bushy stunt virus coat protein mutant yields truncated capsid subunits that form virus-like complexes which benefit systemic spread.

This study examined the contribution of the Tomato bushy stunt virus (TBSV) coat protein (CP) and its corresponding RNA to systemic infection of plants. Compared to results obtained with a mutant lacking the 5'-half of the CP gene, the presence of those CP-RNA sequences in another mutant benefited TBSV infection on Nicotiana benthamiana even though wild-type CP expression was eliminated by introduction of a small out-of-frame deletion. RT-PCR of viral RNA associated with rapid infections established by this CP frameshift deletion mutant revealed that in planta recombination had provided the progeny with the ability to express a truncated CP (tCP) with a block of N-proximal 30 residues deleted from the 66 amino acid RNA-binding domain. Subsequent biochemical characterizations revealed the presence of large ribonucleoprotein complexes that were shown to contain viral RNA as well as the approximately 38-kDa tCP. Electron microscopic examination of purified complexes showed particle-like structures that were nonuniform in size and shape compared to wild-type TBSV particles. Inoculation of pepper with the tCP-containing ribonucleoprotein complexes resulted in a rapid systemic infection similar to that caused by wild-type TBSV. In contrast, infections established in pepper by the original CP frameshift deletion mutant transcripts were restricted to inoculated leaves and did not yield recombinants capable of systemically infecting this host. In summary, TBSV possesses the flexibility to form alternative virion-like structures even if a substantial portion of the RNA-binding domain is deleted from the CP; mutants producing the tCP-containing particle-like structures are more effective for virus spread than those devoid of CP expression; and recombination events to produce the alternative tCP-RNA complexes are host-dependent.

Detection and characterization of an intermediate conformation during the divalent ion-dependent swelling of tomato bushy stunt virus.

We have used time-resolved small-angle X-ray scattering (SAXS) in solution to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. The reaction was initiated by rapid mixing of a virus solution with the same buffer containing a variable amount of EDTA. The X-ray scattering data sets recorded after mixing were submitted to a singular value decomposition analysis which demonstrated the existence of an intermediate state in addition to the compact and fully swollen forms of the virion. The kinetics of the reaction display an initial lag, and a linear combination of three exponential terms is required for a satisfactory analytical fit. Accordingly, a model is put forward involving three sequential irreversible processes between four species. Beyond the three structural species mentioned above, the fourth one, which is the second species along the time sequence, is proposed to represent those viruses which, although partially deprived of Ca2+ ions, are still in the compact conformation. Using the combination of the kinetic model and the structural data, an estimate of the intermediate scattering pattern can be derived from each time resolved frame. These patterns are all very similar after a slight drift towards the swollen pattern over the first 2 min. The curve presents well-resolved minima and maxima, corresponding to an isometric particle with an outer radius of about 172 A for the intermediate conformation.