The structure of pariacoto virus reveals a dodecahedral cage of duplex RNA

The 3.0 A resolution crystal structure of Pariacoto virus (PaV) reveals extensive interactions between portions of the viral RNA genome and the icosahedral capsid. Under the protein shell of the T = 3 quasi equivalent capsid lies a dodecahedral cage composed of RNA duplex that accounts for approximately 35% of the single-stranded RNA genome. The highly basic N-terminal regions (residues 7-54) of the subunits, forming pentamers (A subunits) are clearly visible in the density map and make numerous interactions with the RNA cage. The C-terminal segments (residues 394-401) of the A subunits lie in channels near the quasi three-fold axes. Electron cryo-microscopy and image reconstruction of PaV particles clearly show the dodecahedral RNA cage.     

Turnip crinkle virus-encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection

Most plant viruses encode suppressors of RNA silencing (VSRs) to protect themselves from antiviral RNA silencing in host plants. The capsid protein (CP) of Turnip crinkle virus (TCV) is a well-characterized VSR, whereas SUPPRESSOR OF GENE SILENCING 3 (SGS3) is an important plant-encoded component of the RNA silencing pathways. Whether the VSR activity of TCV CP requires it to engage SGS3 in plant cells has yet to be investigated. Here, we report that TCV CP interacts with SGS3 of Arabidopsis in both yeast and plant cells. The interaction was identified with the yeast two-hybrid system, and corroborated with bimolecular fluorescence complementation and intracellular co-localization assays in Nicotiana benthamiana cells. While multiple partial TCV CP fragments could independently interact with SGS3, its hinge domain connecting the surface and protruding domains appears to be essential for this interaction. Conversely, SGS3 enlists its N-terminal domain and the XS rice gene X and SGS3 (XS) domain as the primary CP-interacting sites. Interestingly, SGS3 appears to stimulate TCV accumulation because viral RNA levels of a TCV mutant with low VSR activities decreased in the sgs3 knockout mutants, but increased in the SGS3-overexpressing transgenic plants. Transgenic Arabidopsis plants overexpressing TCV CP exhibited developmental abnormalities that resembled sgs3 knockout mutants and caused similar defects in the biogenesis of trans-acting small interfering RNAs. Our data suggest that TCV CP interacts with multiple RNA silencing pathway components that include SGS3, as well as previously reported DRB4 (dsRNA-binding protein 4) and AGO2 (ARGONAUTE protein 2), to achieve efficient suppression of RNA silencing-mediated antiviral defence.     

Structural analysis of human rhinovirus complexed with ICAM-1 reveals the dynamics of receptor-mediated virus uncoating

Intercellular adhesion molecule 1 (ICAM-1) functions as the cellular receptor for the major group of human rhinoviruses, being not only the target of viral attachment but also the mediator of viral uncoating. The configurations of HRV3-ICAM-1 complexes prepared both at 4 degrees C and physiological temperature (37 degrees C) were analyzed by cryoelectron microscopy and image reconstruction. The particle diameters of two complexes (with and without RNA) representing uncoating intermediates generated at 37 degrees C were each 4% larger than that of those prepared at 4 degrees C. The larger virus particle arose by an expansive movement of the capsid pentamers along the fivefold axis, which loosens interprotomer contacts, particularly at the canyon region where the ICAM-1 receptor bound. Particle expansion required receptor binding and preceded the egress of the viral RNA. These observations suggest that receptor-mediated uncoating could be a consequence of restrained capsid motion, where the bound receptors maintain the viral capsid in an expanded open state for subsequent genome release.     

Specific Encapsidation of Nodavirus RNAs Is Mediated through the C Terminus of Capsid Precursor Protein Alpha

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.     

Cryoelectron microscopy analysis of the structural changes associated with human rhinovirus type 14 uncoating

Release of the human rhinovirus (HRV) genome into the cytoplasm of the cell involves a concerted structural modification of the viral capsid. The intracellular adhesion molecule 1 (ICAM-1) cellular receptor of the major-group HRVs and the low-density lipoprotein (LDL) receptor of the minor-group HRVs have different nonoverlapping binding sites. While ICAM-1 binding catalyzes uncoating, LDL receptor binding does not. Uncoating of minor-group HRVs is initiated by the low pH of late endosomes. We have studied the conformational changes concomitant with uncoating in the major-group HRV14 and compared them with previous results for the minor-group HRV2. The structure of empty HRV14 was determined by cryoelectron microscopy, and the atomic structure of native HRV14 was used to examine the conformational changes of the capsid and its constituent viral proteins. For both HRV2 and HRV14, the transformation from full to empty capsid involves an overall 4% expansion and an iris type of movement of viral protein VP1 to open up a 10-A-diameter channel on the fivefold axis to allow exit of the RNA genome. The beta-cylinders formed by the N termini of the VP3 molecules inside the capsid on the fivefold axis all open up in HRV2, but we propose that only one opens up in HRV14. The release of VP4 is less efficient in HRV14 than in HRV2, and the N termini of VP1 may exit at different points. The N-terminal loop of VP2 is modified in both viruses, probably to detach the RNA, but it bends only inwards in HRV2.     

Insights into minor group rhinovirus uncoating: the X-ray structure of the HRV2 empty capsid

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.     

Removal of divalent cations induces structural transitions in red clover necrotic mosaic virus, revealing a potential mechanism for RNA release

The structure of Red clover necrotic mosaic virus (RCNMV), an icosahedral plant virus, was resolved to 8.5 A by cryoelectron microscopy. The virion capsid has prominent surface protrusions and subunits with a clearly defined shell and protruding domains. The structures of both the individual capsid protein (CP) subunits and the entire virion capsid are consistent with other species in the Tombusviridae family. Within the RCNMV capsid, there is a clearly defined inner cage formed by complexes of genomic RNA and the amino termini of CP subunits. An RCNMV virion has approximately 390 +/- 30 Ca2+ ions bound to the capsid and 420 +/- 25 Mg2+ ions thought to be in the interior of the capsid. Depletion of both Ca2+ and Mg2+ ions from RCNMV leads to significant structural changes, including (i) formation of 11- to 13-A-diameter channels that extend through the capsid and (ii) significant reorganization within the interior of the capsid. Genomic RNA within native capsids containing both Ca2+ and Mg2+ ions is extremely resistant to nucleases, but depletion of both of these cations results in nuclease sensitivity, as measured by a significant reduction in RCNMV infectivity. These results indicate that divalent cations play a central role in capsid dynamics and suggest a mechanism for the release of viral RNA in low-divalent-cation environments such as those found within the cytoplasm of a cell.     

Encapsidation of turnip crinkle virus is defined by a specific packaging signal and RNA size.

A protoplast infection assay has been used to reliably examine the viral RNA encapsidation of turnip crinkle virus (TCV). Analysis of the encapsidation of various mutant viral RNAs revealed that a 186-nucleotide (nt) region at the 3' end of the coat protein (CP) gene, with a bulged hairpin loop of 28 nt as its most essential element, was indispensable for TCV RNA encapsidation. When RNA fragments containing the 186-nt region were used to replace the CP gene of a different virus, tomato bushy stunt virus, the resulting chimeric viral RNAs were encapsidated into TCV virions. Furthermore, analysis of the encapsidated chimeric RNA species established that the RNA size was an important determinant of the TCV assembly process.     

The Assembly Pathway of an Icosahedral Single-Stranded RNA Virus Depends on the Strength of Inter-Subunit Attractions

The strength of attraction between capsid proteins (CPs) of cowpea chlorotic mottle virus (CCMV) is controlled by the solution pH. Additionally, the strength of attraction between CP and the single-stranded RNA viral genome is controlled by ionic strength. By exploiting these properties, we are able to control and monitor the in vitro co-assembly of CCMV CP and single-stranded RNA as a function of the strength of CP–CP and CP–RNA attractions. Using the techniques of velocity sedimentation and electron microscopy, we find that the successful assembly of nuclease-resistant virus-like particles (VLPs) depends delicately on the strength of CP–CP attraction relative to CP–RNA attraction. If the attractions are too weak, the capsid cannot form; if they are too strong, the assembly suffers from kinetic traps. Separating the process into two steps—by first turning on CP–RNA attraction and then turning on CP–CP attraction—allows for the assembly of well-formed VLPs under a wide range of attraction strengths. These observations establish a protocol for the efficient in vitro assembly of CCMV VLPs and suggest potential strategies that the virus may employ in vivo.     

Interactions of the RNA polymerase with the viral genome at the 5'- and 3'-ends contribute to 20S RNA narnavirus persistence in yeast

20S RNA narnavirus is a positive strand RNA virus found in the yeast Saccharomyces cerevisiae. The viral genome (2514 nucleotides) only encodes a single protein (p91), the RNA-dependent RNA polymerase and does not have capsid proteins to form intracellular virions. The genomic RNA has no 3' poly(A) tail and perhaps no cap structure at the 5'-end; thus resembling an intermediate of mRNA degradation. The virus, however, escapes the host surveillance and replicates in the yeast cytoplasm persistently. The viral genome is not naked but exists in the form of a ribonucleoprotein complex with p91 in a 1:1 stoichiometry. Here we investigated interactions between p91 and the viral genome. Our results indicate that p91 directly or indirectly interacts with the RNA at the 5'- and 3'-end regions and to a lesser extent at a central part. The 3'-end site is identical to or overlaps with the 3' cis signal for replication identified previously. The 5'-site is at the second stem loop structure from the 5'-end (nucleotides 72-104), and this structure also contains a cis signal for replication. Analysis of mutants in the structure revealed a tight correlation between replication and formation of complexes. These results highlight the importance of ribonucleoprotein complexes for the viral life cycle. We will discuss implications of these findings especially on how the virus escapes from mRNA degradation pathways and resides in the cytoplasm persistently despite the lack of a protective capsid.     

Influence of viral genes on the cell-to-cell spread of RNA silencing

The turnip crinkle virus-based vector TCV-GFP Delta CP had been devised previously to study cell-to-cell and long-distance spread of virus-induced RNA silencing. TCV-GFP Delta CP, which had been constructed by replacing the coat protein (CP) gene with a green fluorescent protein (GFP) coding sequence, was able to induce RNA silencing in single epidermal cells, from which RNA silencing spread from cell-to-cell. Using this unique local silencing assay together with mutagenesis analysis, two TCV genes, p8 and p9, which were involved in the intercellular spread of virus-induced RNA silencing, were identified. TCV-GFP Delta CP and its p8- or p9-mutated derivatives, TCVmp8-GFP Delta CP and TCVmp9-GFP Delta CP, replicated efficiently but were restricted to single Nicotiana benthamiana epidermal cells. TCV-GFP Delta CP, TCVmp8-GFP Delta CP, or TCVmp9-GFP Delta CP was able to initiate RNA silencing that targeted and degraded recombinant viral RNAs in inoculated leaves of the GFP-expressing N. benthamiana line 16c. However, cell-to-cell spread of silencing to form silencing foci was triggered only by TCV-GFP Delta CP. Non-replicating TCVmp88-GFP Delta CP and TCVmp28mp88-GFP Delta CP with dysfunctional replicase genes, and single-stranded gfp RNA did not induce RNA silencing. Transient expression of the TCV p9 protein could effectively complement TCVmp9-GFP Delta CP to facilitate intercellular spread of silencing. These data suggest that the plant cellular trafficking machinery could hijack functional viral proteins to permit cell-to-cell movement of RNA silencing.     

Interaction with capsid protein alters RNA structure and the pathway for in vitro assembly of cowpea chlorotic mottle virus

Viruses use sophisticated mechanisms to allow the specific packaging of their genome over that of host nucleic acids. We examined the in vitro assembly of the Cowpea chlorotic mottle virus (CCMV) and observed that assembly with viral RNA follows two different mechanisms. Initially, CCMV capsid protein (CP) dimers bind RNA with low cooperativity and form virus-like particles of 90 CP dimers and one copy of RNA. Longer incubation reveals a different assembly path. At a stoichiometry of about ten CP dimers per RNA, the CP slowly folds the RNA into a compact structure that can be bound with high cooperativity by additional CP dimers. This folding process is exclusively a function of CP quaternary structure and is independent of RNA sequence. CP-induced folding is distinct from RNA folding that depends on base-pairing to stabilize tertiary structure. We hypothesize that specific encapsidation of viral RNA is a three-step process: specific binding by a few copies of CP, RNA folding, and then cooperative binding of CP to the "labeled" nucleoprotein complex. This mechanism, observed in a plant virus, may be applicable to other viruses that do not halt synthesis of host nucleic acid, including HIV.     

Degenerate RNA packaging signals in the genome of Satellite Tobacco Necrosis Virus: implications for the assembly of a T=1 capsid

Using a recombinant, T = 1 Satellite Tobacco Necrosis Virus (STNV)-like particle expressed in Escherichia coli, we have established conditions for in vitro disassembly and reassembly of the viral capsid. In vivo assembly is dependent on the presence of the coat protein (CP) N-terminal region, and in vitro assembly requires RNA. Using immobilised CP monomers under reassembly conditions with “free” CP subunits, we have prepared a range of partially assembled CP species for RNA aptamer selection. SELEX directed against the RNA-binding face of the STNV CP resulted in the isolation of several clones, one of which (B3) matches the STNV-1 genome in 16 out of 25 nucleotide positions, including across a statistically significant 10/10 stretch. This 10-base region folds into a stem-loop displaying the motif ACAA and has been shown to bind to STNV CP. Analysis of the other aptamer sequences reveals that the majority can be folded into stem-loops displaying versions of this motif. Using a sequence and secondary structure search motif to analyse the genomic sequence of STNV-1, we identified 30 stem-loops displaying the sequence motif AxxA. The implication is that there are many stem-loops in the genome carrying essential recognition features for binding STNV CP. Secondary structure predictions of the genomic RNA using Mfold showed that only 8 out of 30 of these stem-loops would be formed in the lowest-energy structure. These results are consistent with an assembly mechanism based on kinetically driven folding of the RNA.     

Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus

Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.     

Assembly of two independent populations of flock house virus particles with distinct RNA packaging characteristics in the same cell

Flock House virus (FHV; Nodaviridae) is a positive-strand RNA virus that encapsidates a bipartite genome consisting of RNA1 and RNA2. We recently showed that specific recognition of these RNAs for packaging into progeny particles requires coat protein translated from replicating viral RNA. In the present study, we investigated whether the entire assembly pathway, i.e., the formation of the initial nucleating complex and the subsequent completion of the capsid, is restricted to the same pool of coat protein subunits. To test this, coat proteins carrying either FLAG or hemagglutinin epitopes were synthesized from replicating or nonreplicating RNA in the same cell, and the resulting particle population and its RNA packaging phenotype were analyzed. Results from immunoprecipitation analysis and ion-exchange chromatography showed that the differentially tagged proteins segregated into two distinct populations of virus particles with distinct RNA packaging phenotypes. Particles assembled from coat protein that was translated from replicating RNA contained the FHV genome, whereas particles assembled from coat protein that was translated from nonreplicating mRNA contained random cellular RNA. These data demonstrate that only coat proteins synthesized from replicating RNA partake in the assembly of virions that package the viral genome and that RNA replication, coat protein translation, and virion assembly are processes that are tightly coupled during the life cycle of FHV.     

The Impact of Viral RNA on the Association Rates of Capsid Protein Assembly: Bacteriophage MS2 as a Case Study

A large number of single-stranded RNA viruses, which form a major class of all viruses, co-assemble their protein container and their genomic material. The multiple roles of the viral genome in this process are presently only partly understood. Recent experimental results indicate that RNA, in addition to its function as a repository for genetic information, could play important functional roles during the assembly of the viral protein containers. An investigation of the impact of genomic RNA on the association of the protein subunits may therefore provide further insights into the mechanism of virus assembly. We study here the impact of viral RNA on the association rates of the capsid proteins during virus assembly. As a case study, we consider the viral capsid of bacteriophage MS2, which is formed from 60 asymmetric (AB) and 30 symmetric (CC) protein dimers. Using Brownian dynamics simulations, we investigate the effect of the binding of an RNA stem-loop (the translational repressor) on the association rates of the capsid protein dimers. Our analysis shows that translational repressor binding results in self-association of AB dimers being inhibited, whilst association of AB with CC dimers is greatly enhanced. This provides an explanation for experimental results in which an alternating assembly pattern of AB and CC dimer addition to the growing assembly intermediate has been observed to be the dominant mode of assembly. The presence of the RNA hence dramatically decreases the number of dominant assembly pathways and thereby reduces the complexity of the self-assembly process of these viruses.