Removal of Pseudomonas type IV pili by a small RNA virus.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

Vital for Viruses: Intrinsically Disordered Proteins.

Viruses infect all kingdoms of life; their genomes vary from DNA to RNA and in size from 2kB to 1 MB or more. Viruses frequently employ disordered proteins, that is, protein products of virus genes that do not themselves fold into independent three-dimensional structures, but rather, constitute a versatile molecular toolkit to accomplish a range of functions necessary for viral infection, assembly, and proliferation. Interestingly, disordered proteins have been discovered in almost all viruses so far studied, whether the viral genome consists of DNA or RNA, and whatever the configuration of the viral capsid or other outer covering. In this review, I present a wide-ranging set of stories illustrating the range of functions of IDPs in viruses. The field is rapidly expanding, and I have not tried to include everything. What is included is meant to be a survey of the variety of tasks that viruses accomplish using disordered proteins.

Liquid-liquid Phase Separation in Viral Function.

An emerging set of results suggests that liquid-liquid phase separation (LLPS) is the basis for the formation of membrane-less compartments in cells. Evidence is now mounting that various types of virus-induced membrane-less compartments and organelles are also assembled via LLPS. Specifically, viruses appear to use intracellular phase transitions to form subcellular microenvironments known as viral factories, inclusion bodies, or viroplasms. These compartments - collectively referred to as viral biomolecular condensates - can be used to concentrate replicase proteins, viral genomes, and host proteins that are required for virus replication. They can also be used to subvert or avoid the intracellular immune response. This review examines how certain DNA or RNA viruses drive the formation of viral condensates, the possible biological functions of those condensates, and the biophysical and biochemical basis for their assembly.

A Transfectable Fusagravirus from a Japanese Strain of Cryphonectria carpinicola with Spherical Particles.

A novel dsRNA virus (Cryphonectria carpinicola fusagravirus 1, CcFGV1), isolated from a Japanese strain (JS13) of Cryphonectria carpinicola, was thoroughly characterized. The biological comparison of a set of isogenic CcFGV1-infected and -free (JS13VF) strains indicated asymptomatic infection by CcFGV1. The sequence analysis showed that the virus has a two open reading frame (ORF) genome of 9.6 kbp with the RNA-directed RNA polymerase domain encoded by ORF2. The N-terminal sequencing and peptide mass fingerprinting showed an N-terminally processed or degraded product (150 kDa) of the 5'-proximal ORF1-encoded protein (1462 amino acids) to make up the CcFGV1 spherical particles of ~40 nm in diameter. Interestingly, a portion of CcFGV1 dsRNA co-fractionated with a host protein of 70 kDa. The purified CcFGV1 particles were used to transfect protoplasts of JS13VF as well as the standard strain of an experimental model filamentous fungal host Cryphonectria parasitica. CcFGV1 was confirmed to be associated with asymptomatic infection of both fungi. RNA silencing was shown to target the virus in C. parasitica, resulting in reduced CcFGV1 accumulation by comparing the CcFGV1 content between RNA silencing-competent and -deficient strains. These results indicate the transfectability of spherical particles of a fusagravirus associated with asymptomatic infection.

A Capsidless Virus Is trans-Encapsidated by a Bisegmented Botybirnavirus.

RNA viruses usually have linear genomes and are encapsidated by their own capsids. Here, we newly identified four mycoviruses and two previously reported mycoviruses (a fungal reovirus and a botybirnavirus) in the hypovirulent strain SCH941 of Sclerotinia sclerotiorum. One of the newly discovered mycoviruses, Sclerotinia sclerotiorum yadokarivirus 1 (SsYkV1), with a nonsegmented positive-sense single-stranded RNA (+ssRNA) genome, was molecularly characterized. SsYkV1 is 5,256 nucleotides (nt) in length, excluding the poly(A) structure, and has a large open reading frame that putatively encodes a polyprotein with the RNA-dependent RNA polymerase (RdRp) domain and a 2A-like motif. SsYkV1 was phylogenetically positioned into the family Yadokariviridae and was most closely related to Rosellinia necatrix yadokarivirus 2 (RnYkV2), with 40.55% identity (78% coverage). Although SsYkV1 does not encode its own capsid protein, the RNA and RdRp of SsYkV1 are trans-encapsidated in virions of Sclerotinia sclerotiorum botybirnavirus 3 (SsBV3), a bisegmented double-stranded RNA (dsRNA) mycovirus within the genus Botybirnavirus. In this way, SsYkV1 likely replicates inside the heterocapsid comprised of the SsBV3 capsid protein, like a dsRNA virus. SsYkV1 has a limited impact on the biological features of S. sclerotiorum. This study represents an example of a yadokarivirus trans-encapsidated by an unrelated dsRNA virus, which greatly deepens our knowledge and understanding of the unique life cycles of RNA viruses. IMPORTANCE RNA viruses typically encase their linear genomes in their own capsids. However, a capsidless +ssRNA virus (RnYkV1) highjacks the capsid of a nonsegmented dsRNA virus for the trans-encapsidation of its own RNA and RdRp. RnYkV1 belongs to the family Yadokariviridae, which already contains more than a dozen mycoviruses. However, it is unknown whether other yadokariviruses except RnYkV1 are also hosted by a heterocapsid, although dsRNA viruses with capsid proteins were detected in fungi harboring yadokarivirus. It is noteworthy that almost all presumed partner dsRNA viruses of yadokariviruses belong to the order Ghabrivirales (most probably a totivirus or toti-like virus). Here, we found a capsidless +ssRNA mycovirus, SsYkV1, from hypovirulent strain SCH941 of S. sclerotiorum, and the RNA and RdRp of this mycovirus are trans-encapsidated in virions of a bisegmented dsRNA virus within the free-floating genus Botybirnavirus. Our results greatly expand our knowledge of the unique life cycles of RNA viruses.

Detailed Analyses of Molecular Interactions between Favipiravir and RNA Viruses In Silico.

There are currently no antiviral agents for human metapneumovirus (HMPV), respiratory syncytial virus (RSV), mumps virus (MuV), or measles virus (MeV). Favipiravir has been developed as an anti-influenza agent, and this agent may be effective against these viruses in vitro. However, the molecular mechanisms through which the agent affects virus replication remain to be fully elucidated. Thus, to clarify the detailed molecular interactions between favipiravir and the RNA-dependent RNA polymerase (RdRp) of HMPV, RSV, MuV, MeV, and influenza virus, we performed in silico studies using authentic bioinformatics technologies. As a result, we found that the active form of favipiravir (favipiravir ribofuranosyl-5'-triphosphate [F-RTP]) can bind to the RdRp active sites of HMPV, RSV, MuV, and MeV. The aspartic acid residue of RdRp active sites was involved in the interaction. Moreover, F-RTP was incorporated into the growing viral RNA chain in the presence of nucleotide triphosphate and magnesium ions. The results suggested that favipiravir shows two distinct mechanisms in various viruses: RdRp active site inhibition and/or genome replication inhibition.

Mechanisms for destabilisation of RNA viruses at air-water and liquid-liquid interfaces.

Understanding the interactions between viruses and surfaces or interfaces is important, as they provide the principles underpinning the cleaning and disinfection of contaminated surfaces. Yet, the physics of such interactions is currently poorly understood. For instance, there are longstanding experimental observations suggesting that the presence of air-water interfaces can generically inactivate and kill viruses, yet the mechanism underlying this phenomenon remains unknown. Here we use theory and simulations to show that electrostatics may provide one such mechanism, and that this is very general. Thus, we predict that the electrostatic free energy of an RNA virus should increase by several thousands of kBT as the virion breaches an air-water interface. We also show that the fate of a virus approaching a generic liquid-liquid interface depends strongly on the detailed balance between interfacial and electrostatic forces, which can be tuned, for instance, by choosing different media to contact a virus-laden respiratory droplet. Tunability arises because both the electrostatic and interfacial forces scale similarly with viral size. We propose that these results can be used to design effective strategies for surface disinfection.

Evaluating RNA Structural Flexibility: Viruses Lead the Way.

Our understanding of RNA structure has lagged behind that of proteins and most other biological polymers, largely because of its ability to adopt multiple, and often very different, functional conformations within a single molecule. Flexibility and multifunctionality appear to be its hallmarks. Conventional biochemical and biophysical techniques all have limitations in solving RNA structure and to address this in recent years we have seen the emergence of a wide diversity of techniques applied to RNA structural analysis and an accompanying appreciation of its ubiquity and versatility. Viral RNA is a particularly productive area to study in that this economy of function within a single molecule admirably suits the minimalist lifestyle of viruses. Here, we review the major techniques that are being used to elucidate RNA conformational flexibility and exemplify how the structure and function are, as in all biology, tightly linked.

The structure of a plant-specific partitivirus capsid reveals a unique coat protein domain architecture with an intrinsically disordered protrusion.

Persistent plant viruses may be the most common viruses in wild plants. A growing body of evidence for mutualism between such viruses and their hosts, suggests that they play an important role in ecology and agriculture. Here we present the capsid structure of a plant-specific partitivirus, Pepper cryptic virus 1, at 2.9 Å resolution by Cryo-EM. Structural features, including the T = 1 arrangement of 60 coat protein dimers, are shared with fungal partitiviruses and the picobirnavirus lineage of dsRNA viruses. However, the topology of the capsid is markedly different with protrusions emanating from, and partly comprising, the binding interface of coat protein dimers. We show that a disordered region at the apex of the protrusion is not required for capsid assembly and represents a hypervariable site unique to, and characteristic of, the plant-specific partitiviruses. These results suggest a structural basis for the acquisition of additional functions by partitivirus coat proteins that enables mutualistic relationships with diverse plant hosts.

Structural 3D Domain Reconstruction of the RNA Genome from Viruses with Secondary Structure Models.

Three-dimensional RNA domain reconstruction is important for the assembly, disassembly and delivery functionalities of a packed proteinaceus capsid. However, to date, the self-association of RNA molecules is still an open problem. Recent chemical probing reports provide, with high reliability, the secondary structure of diverse RNA ensembles, such as those of viral genomes. Here, we present a method for reconstructing the complete 3D structure of RNA genomes, which combines a coarse-grained model with a subdomain composition scheme to obtain the entire genome inside proteinaceus capsids based on secondary structures from experimental techniques. Despite the amount of sampling involved in the folded and also unfolded RNA molecules, advanced microscope techniques can provide points of anchoring, which enhance our model to include interactions between capsid pentamers and RNA subdomains. To test our method, we tackle the satellite tobacco mosaic virus (STMV) genome, which has been widely studied by both experimental and computational communities. We provide not only a methodology to structurally analyze the tertiary conformations of the RNA genome inside capsids, but a flexible platform that allows the easy implementation of features/descriptors coming from both theoretical and experimental approaches.

Structure and Expression of Large (+)RNA Genomes of Viruses of Higher Eukaryotes.

Viral positive-sense RNA genomes evolve rapidly due to the high mutation rates during replication and RNA recombination, which allowing the viruses to acquire and modify genes for their adaptation. The size of RNA genome is limited by several factors, including low fidelity of RNA polymerases and packaging constraints. However, the 12-kb size limit is exceeded in the two groups of eukaryotic (+)RNA viruses - animal nidoviruses and plant closteroviruses. These virus groups have several traits in common. Their genomes contain 5'-proximal genes that are expressed via ribosomal frameshifting and encode one or two papain-like protease domains, membrane-binding domain(s), methyltransferase, RNA helicase, and RNA polymerase. In addition, some nidoviruses (i.e., coronaviruses) contain replication-associated domains, such as proofreading exonuclease, putative primase, nucleotidyltransferase, and endonuclease. In both nidoviruses and closteroviruses, the 3'-terminal part of the genome contains genes for structural and accessory proteins expressed via a nested set of coterminal subgenomic RNAs. Coronaviruses and closteroviruses have evolved to form flexuous helically symmetrical nucleocapsids as a mean to resolve packaging constraints. Since phylogenetic reconstructions of the RNA polymerase domains indicate only a marginal relationship between the nidoviruses and closteroviruses, their similar properties likely have evolved convergently, along with the increase in the genome size.

Structure Unveils Relationships between RNA Virus Polymerases.

RNA viruses are the fastest evolving known biological entities. Consequently, the sequence similarity between homologous viral proteins disappears quickly, limiting the usability of traditional sequence-based phylogenetic methods in the reconstruction of relationships and evolutionary history among RNA viruses. Protein structures, however, typically evolve more slowly than sequences, and structural similarity can still be evident, when no sequence similarity can be detected. Here, we used an automated structural comparison method, homologous structure finder, for comprehensive comparisons of viral RNA-dependent RNA polymerases (RdRps). We identified a common structural core of 231 residues for all the structurally characterized viral RdRps, covering segmented and non-segmented negative-sense, positive-sense, and double-stranded RNA viruses infecting both prokaryotic and eukaryotic hosts. The grouping and branching of the viral RdRps in the structure-based phylogenetic tree follow their functional differentiation. The RdRps using protein primer, RNA primer, or self-priming mechanisms have evolved independently of each other, and the RdRps cluster into two large branches based on the used transcription mechanism. The structure-based distance tree presented here follows the recently established RdRp-based RNA virus classification at genus, subfamily, family, order, class and subphylum ranks. However, the topology of our phylogenetic tree suggests an alternative phylum level organization.

Identification of a novel host protein interacting with the structural protein VP2 of deformed wing virus by yeast two-hybrid screening.

Deformed wing virus (DWV) interacting with Varroa destructor is a possible cause of honeybee colony mortality. VP2 is the structural protein of DWV but its function remains unknown. To clarify the function of VP2 and screen for novel binding proteins that interact with VP2, we carried out a membrane protein yeast two-hybrid screening using VP2 as bait. Subsequently, the interaction between VP2 and the host interacting protein [heat shock protein 10 (Hsp10)] was further verified using glutathione S-transferase pull-down assay in vitro and co-immunoprecipitation assay in cells. Furthermore, fluorescence confocal microscopy revealed that VP2 and Hsp10 were mainly co-localized in the cytoplasm. Using real-time polymerase chain reaction, we found that Hsp10 expression in DWV-infected worker honey bees were downregulated compared with that in healthy honey bees. Additionally, we showed that overexpression of VP2 protein could reduce the expression of Hsp10. These results suggest that Hsp10 plays a vital role in host immunity and antiviral effects.

A Divergent Articulavirus in an Australian Gecko Identified Using Meta-Transcriptomics and Protein Structure Comparisons.

The discovery of highly divergent RNA viruses is compromised by their limited sequence similarity to known viruses. Evolutionary information obtained from protein structural modelling offers a powerful approach to detect distantly related viruses based on the conservation of tertiary structures in key proteins such as the RNA-dependent RNA polymerase (RdRp). We utilised a template-based approach for protein structure prediction from amino acid sequences to identify distant evolutionary relationships among viruses detected in meta-transcriptomic sequencing data from Australian wildlife. The best predicted protein structural model was compared with the results of similarity searches against protein databases. Using this combination of meta-transcriptomics and protein structure prediction we identified the RdRp (PB1) gene segment of a divergent negative-sense RNA virus, denoted Lauta virus (LTAV), in a native Australian gecko (Gehyra lauta). The presence of this virus was confirmed by PCR and Sanger sequencing. Phylogenetic analysis revealed that Lauta virus likely represents a newly described genus within the family Amnoonviridae, order Articulavirales, that is most closely related to the fish virus Tilapia tilapinevirus (TiLV). These findings provide important insights into the evolution of negative-sense RNA viruses and structural conservation of the viral replicase among members of the order Articulavirales.

Mapping RNA-capsid interactions and RNA secondary structure within virus particles using next-generation sequencing.

To characterize RNA-capsid binding sites genome-wide within mature RNA virus particles, we have developed a Next-Generation Sequencing (NGS) platform: viral Photo-Activatable Ribonucleoside CrossLinking (vPAR-CL). In vPAR-CL, 4-thiouridine is incorporated into the encapsidated genomes of virus particles and subsequently UV-crosslinked to adjacent capsid proteins. We demonstrate that vPAR-CL can readily and reliably identify capsid binding sites in genomic viral RNA by detecting crosslink-specific uridine to cytidine transitions in NGS data. Using Flock House virus (FHV) as a model system, we identified highly consistent and significant vPAR-CL signals across virus RNA genome, indicating a clear tropism of the encapsidated RNA genome. Certain interaction sites coincide with previously identified functional RNA motifs. We additionally performed dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to generate a high-resolution profile of single-stranded genomic RNA inside viral particles. Combining vPAR-CL and DMS-MaPseq reveals that the predominant RNA-capsid interaction sites favored double-stranded RNA regions. We disrupted secondary structures associated with vPAR-CL sites using synonymous mutations, resulting in varied effects to virus replication, propagation and packaging. Certain mutations showed substantial deficiency in virus replication, suggesting these RNA-capsid sites are multifunctional. These provide further evidence to support that FHV packaging and replication are highly coordinated and inter-dependent events.

Structural Characterization of Open Reading Frame-Encoded Functional Genes from Tilapia Lake Virus (TiLV).

In recent years, large-scale mortalities are observed in tilapia due to infection with a novel orthomyxo-like virus named, tilapia lake virus (TiLV) which is marked to be a severe threat to universal tilapia industry. Currently, there are knowledge gaps relating to the antiviral peptide as well as there are no affordable vaccines or drugs available against TiLV yet. To understand the spreading of infection of TiLV in different organs of Oreochromis niloticus, RT-PCR analysis has been carried out. The gene segments of TiLV were retrieved from the NCBI database for computational biology analysis. The 14 functional genes were predicted from the 10 gene segments of TiLV. Phylogenetic analysis was employed to find out a better understanding for the evolution of tilapia lake virus genes. Out of 14 proteins, only six proteins show transmembrane helix region. Moreover, molecular modeling and molecular dynamics simulations of the predicted proteins revealed structural stability of the protein stabilized after 10-ns simulation. Overall, our study provided a basic bioinformatics on functional proteome of TiLV. Further, this study could be useful for development of novel peptide-based therapeutics to control TiLV infection.

Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome.

Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process.

Anomalous small viral shells and simplest polyhedra with icosahedral symmetry: the rhombic triacontahedron case.

The development of antiviral strategies requires a clear understanding of the principles that control the protein arrangements in viral shells. Considered here are those capsids that violate the paradigmatic Caspar and Klug (CK) model, and it is shown that the important structural features of such anomalous shells from the Picobirnaviridae, Flaviviridae and Leviviridae families can be revealed by models in the form of spherical icosahedral packings of equivalent rhombic structural units (SUs). These SUs are composed of protein dimers forming the investigated capsids which, as shown here, are based on the rhombic triacontahedron (RT) geometry. How to modify the original CK approach in order to make it compatible with the considered rhombic tessellations of a sphere is also discussed. Analogies between capsids self-assembled from dimers and trimers are demonstrated. This analysis reveals the principles controlling the localization of receptor proteins (which recognize the host cell) on the capsid surface.

Virion structure and genome delivery mechanism of sacbrood honeybee virus.

Infection by sacbrood virus (SBV) from the family Iflaviridae is lethal to honey bee larvae but only rarely causes the collapse of honey bee colonies. Despite the negative effect of SBV on honey bees, the structure of its particles and mechanism of its genome delivery are unknown. Here we present the crystal structure of SBV virion and show that it contains 60 copies of a minor capsid protein (MiCP) attached to the virion surface. No similar MiCPs have been previously reported in any of the related viruses from the order Picornavirales. The location of the MiCP coding sequence within the SBV genome indicates that the MiCP evolved from a C-terminal extension of a major capsid protein by the introduction of a cleavage site for a virus protease. The exposure of SBV to acidic pH, which the virus likely encounters during cell entry, induces the formation of pores at threefold and fivefold axes of the capsid that are 7 Å and 12 Å in diameter, respectively. This is in contrast to vertebrate picornaviruses, in which the pores along twofold icosahedral symmetry axes are currently considered the most likely sites for genome release. SBV virions lack VP4 subunits that facilitate the genome delivery of many related dicistroviruses and picornaviruses. MiCP subunits induce liposome disruption in vitro, indicating that they are functional analogs of VP4 subunits and enable the virus genome to escape across the endosome membrane into the cell cytoplasm.

Viral RNA-Dependent RNA Polymerases: A Structural Overview.

Most emerging and re-emerging human and animal viral diseases are associated with RNA viruses. All these pathogens, with the exception of retroviruses, encode a specialized enzyme called RNA-dependent RNA polymerase (RdRP), which catalyze phosphodiester-bond formation between ribonucleotides (NTPs) in an RNA template-dependent manner. These enzymes function either as single polypeptides or in complex with other viral or host components to transcribe and replicate the viral RNA genome. The structures of RdRPs and RdRP catalytic complexes, currently available for several members of (+) ssRNA, (-)ssRNA and dsRNA virus families, have provided high resolution snapshots of the functional steps underlying replication and transcription of viral RNA genomes and their regulatory mechanisms.