Mutually-induced Conformational Switching of RNA and Coat Protein Underpins Efficient Assembly of a Viral Capsid

Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T = 3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T = 3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.     

X-ray Crystal Structure of the Rotavirus Inner Capsid Particle at 3.8 Å Resolution

The rotavirus inner capsid particle, known as the “double-layered particle” (DLP), is the “payload” delivered into a cell in the process of viral infection. Its inner and outer protein layers, composed of viral protein (VP) 2 and VP6, respectively, package the 11 segments of the double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter ∼ 700 Å) in the asymmetric unit of the P2₁2₁2₁ unit cell of dimensions a = 740 Å, b = 1198 Å, and c = 1345 Å. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular replacement model for initial phase determination to about 18.5 Å resolution, and the 60-fold redundancy of icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 Å). The structure of a VP6 trimer (determined previously by others) fits the outer layer density with very little adjustment. The T = 13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus 2 copies per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101-880 fold into a relatively thin principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two “subdomain” boundaries) allow VP2A and VP2B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the 5-fold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: λ1 in orthoreoviruses and VP3 in orbiviruses. Residues 1-80 of VP2A and VP2B fold together with four other such pairs into a “5-fold hub” that projects into the DLP interior along the 5-fold axis; residues 81-100 link the 10 polypeptide chains emerging from a 5-fold hub to the N-termini of their corresponding principal domains, clustered into a decameric assembly unit. The 5-fold hub appears to have several distinct functions. One function is to recruit a copy of VP1 (or of a VP1-VP3 complex), potentially along with a segment of plus-strand RNA, as a decamer of VP2 assembles. The second function is to serve as a shaft around which can coil a segment of dsRNA. The third function is to guide nascent mRNA, synthesized in the DLP interior by VP1 and 5′-capped by the action of VP3, out through a 5-fold exit channel. We propose a model for rotavirus particle assembly, based on known requirements for virion formation, together with the structure of the DLP and that of VP1, determined earlier.     

Identification of the Active Sites in the Methyltransferases of a Transcribing dsRNA Virus

Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2′-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~ 3.8 Å by cryo-electron microscopy. The binding sites for both S-adenosyl-l-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.     

An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5′ untranslated region that contains all residues necessary for efficient RNA packaging (Ψ^WT; residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Ψ^WT]₂) binding 12 ± 2 NC molecules with high affinity (K_d = 17 ± 7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Ψ^M), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (∼ 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.     

Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization

All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.     

Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR

Activation of the latent kinase PKR is a potent innate defense reaction of vertebrate cells towards viral infections, which is triggered by recognition of viral double-stranded (ds) RNA and results in a translational shutdown. A major gap in our understanding of PKR''s antiviral properties concerns the nature of the kinase activating molecules expressed by influenza and other viruses with a negative strand RNA genome, as these pathogens produce little or no detectable amounts of dsRNA. Here we systematically investigated PKR activation by influenza B virus and its impact on viral pathogenicity. Biochemical analysis revealed that PKR is activated by viral ribonucleoprotein (vRNP) complexes known to contain single-stranded RNA with a 5′-triphosphate group. Cell biological examination of recombinant viruses showed that the nucleo-cytoplasmic transport of vRNP late in infection is a strong trigger for PKR activation. In addition, our analysis provides a mechanistic explanation for the previously observed suppression of PKR activation by the influenza B virus NS1 protein, which we show here to rely on complex formation between PKR and NS1''s dsRNA binding domain. The high significance of this interaction for pathogenicity was revealed by the finding that attenuated influenza viruses expressing dsRNA binding-deficient NS1 proteins were rescued for high replication and virulence in PKR-deficient cells and mice, respectively. Collectively, our study provides new insights into an important antiviral defense mechanism of vertebrates and leads us to suggest a new model of PKR activation by cytosolic vRNP complexes, a model that may also be applicable to other negative strand RNA viruses.     

The three-dimensional structure of genomic RNA in bacteriophage MS2: implications for assembly

Using cryo-electron microscopy, single particle image processing and three-dimensional reconstruction with icosahedral averaging, we have determined the three-dimensional solution structure of bacteriophage MS2 capsids reassembled from recombinant protein in the presence of short oligonucleotides. We have also significantly extended the resolution of the previously reported structure of the wild-type MS2 virion. The structures of recombinant MS2 capsids reveal clear density for bound RNA beneath the coat protein binding sites on the inner surface of the T = 3 MS2 capsid, and show that a short extension of the minimal assembly initiation sequence that promotes an increase in the efficiency of assembly, interacts with the protein capsid forming a network of bound RNA. The structure of the wild-type MS2 virion at ∼9 Å resolution reveals icosahedrally ordered density encompassing ∼90% of the single-stranded RNA genome. The genome in the wild-type virion is arranged as two concentric shells of density, connected along the 5-fold symmetry axes of the particle. This novel RNA fold provides new constraints for models of viral assembly.     

Viral Genomic Single-Stranded RNA Directs the Pathway Toward a T = 3 Capsid

The molecular mechanisms controlling genome packaging by single-stranded RNA viruses are still largely unknown. It is necessary in most cases for the protein to adopt different conformations at different positions on the capsid lattice in order to form a viral capsid from multiple copies of a single protein. We showed previously that such quasi-equivalent conformers of RNA bacteriophage MS2 coat protein dimers (CP₂) can be switched by sequence-specific interaction with a short RNA stem-loop (TR) that occurs only once in the wild-type phage genome. In principle, multiple switching events are required to generate the phage T = 3 capsid. We have therefore investigated the sequence dependency of this event using two RNA aptamer sequences selected to bind the phage coat protein and an analogous packaging signal from phage Qβ known to be discriminated against by MS2 coat protein both in vivo and in vitro. All three non-cognate stem-loops support T = 3 shell formation, but none shows the kinetic-trapping effect seen when TR is mixed with equimolar CP₂. We show that this reflects the fact that they are poor ligands compared with TR, failing to saturate the coat protein under the assay conditions, ensuring that sufficient amounts of both types of dimer required for efficient assembly are present in these reactions. Increasing the non-cognate RNA concentration restores the kinetic trap, confirming this interpretation. We have also assessed the effects of extending the TR stem-loop at the 5′ or 3′ end with short genomic sequences. These longer RNAs all show evidence of the kinetic trap, reflecting the fact that they all contain the TR sequence and are more efficient at promoting capsid formation than TR. Mass spectrometry has shown that at least two pathways toward the T = 3 shell occur in TR-induced assembly reactions: one via formation of a 3-fold axis and another that creates an extended 5-fold complex. The longer genomic RNAs suppress the 5-fold pathway, presumably as a consequence of steric clashes between multiply bound RNAs. Reversing the orientation of the extension sequences with respect to the TR stem-loop produces RNAs that are poor assembly initiators. The data support the idea that RNA-induced protein conformer switching occurs throughout assembly of the T = 3 shell and show that both positional and sequence-specific effects outside the TR stem-loop can have significant impacts on the precise assembly pathway followed.     

Insight into the Ebola virus nucleocapsid assembly mechanism: crystal structure of Ebola virus nucleoprotein core domain at 1.8 ? resolution

Ebola virus (EBOV) is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA (-ssRNA) viruses, EBOV possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation to form viral ribonucleoprotein complex (RNP) together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. However, the mechanism of EBOV RNP formation remains unclear. In this work, we solved the high resolution structure of core domain of EBOV NP. The polypeptide of EBOV NP core domain (NP_core) possesses an N-lobe and C-lobe to clamp a RNA binding groove, presenting similarities with the structures of the other reported viral NPs encoded by the members from Mononegavirales order. Most strikingly, a hydrophobic pocket at the surface of the C-lobe is occupied by an α-helix of EBOV NP_core itself, which is highly conserved among filoviridae family. Combined with other biochemical and biophysical evidences, our results provides great potential for understanding the mechanism underlying EBOV RNP formation via the mobility of EBOV NP element and enables the development of antiviral therapies targeting EBOV RNP formation.     

Insight into DNA and protein transport in double-stranded DNA viruses: the structure of bacteriophage N4

Bacteriophage N4 encapsidates a 3500-aa-long DNA-dependent RNA polymerase (vRNAP), which is injected into the host along with the N4 genome upon infection. The three-dimensional structures of wild-type and mutant N4 viruses lacking gp17, gp50, or gp65 were determined by cryoelectron microscopy. The virion has an icosahedral capsid with T = 9 quasi-symmetry that encapsidates well-organized double-stranded DNA and vRNAP. The tail, attached at a unique pentameric vertex of the head, consists of a neck, 12 appendages, and six ribbons that constitute a non-contractile sheath around a central tail tube. Comparison of wild-type and mutant virus structures in conjunction with bioinformatics established the identity and virion locations of the major capsid protein (gp56), a decorating protein (gp17), the vRNAP (gp50), the tail sheath (gp65), the appendages (gp66), and the portal protein (gp59). The N4 virion organization provides insight into its assembly and suggests a mechanism for genome and vRNAP transport strategies utilized by this unique system.     

The Impact of Viral RNA on Assembly Pathway Selection

Many single-stranded RNA viruses self-assemble their protein containers around their genomes. The roles that the RNA plays in this assembly process have mostly been ignored, resulting in a protein-centric view of assembly that is unable to explain adequately the fidelity and speed of assembly in such viruses. Using bacteriophage MS2, we demonstrate here via a combination of mass spectrometry and kinetic modelling how viral RNA can bias assembly towards only a small number of the many possible assembly pathways, thus increasing assembly efficiency. Assembly reactions have been studied in vitro using phage coat protein dimers, the known building block of the T = 3 shell, and short RNA stem-loops based on the translational operator of the replicase cistron, a 19 nt fragment (TR). Mass spectrometry has unambiguously identified two on-pathway intermediates in such reactions that have stoichiometry consistent with formation of either a particle 3-fold or 5-fold axis. These imply that there are at least two sub-pathways to the final capsid. The flux through each pathway is controlled by the length of the RNA stem-loop triggering the assembly reaction and this effect can be understood in structural terms. The kinetics of intermediate formation have been studied and show steady-state concentrations for intermediates between starting materials and the T = 3 shell, consistent with an assembly process in which all the steps are in equilibrium. These data have been used to derive a kinetic model of the assembly reaction that in turn allows us to determine the dominant assembly pathways explicitly, and to estimate the effect of the RNA on the free energy of association between the assembling protein subunits. The results reveal that there are only a small number of dominant assembly pathways, which vary depending on the relative ratios of RNA and protein. These results suggest that the genomic RNA plays significant roles in defining the precise assembly sub-pathway followed to create the final capsid.     

Crystal structure of Schmallenberg orthobunyavirus nucleoprotein–RNA complex reveals a novel RNA sequestration mechanism

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus (family Bunyaviridae) that has caused severe disease in the offspring of farm animals across Europe. Like all orthobunyaviruses, SBV contains a tripartite negative-sense RNA genome that is encapsidated by the viral nucleocapsid (N) protein in the form of a ribonucleoprotein complex (RNP). We recently reported the three-dimensional structure of SBV N that revealed a novel fold. Here we report the crystal structure of the SBV N protein in complex with a 42-nt-long RNA to 2.16 Å resolution. The complex comprises a tetramer of N that encapsidates the RNA as a cross-shape inside the protein ring structure, with each protomer bound to 11 ribonucleotides. Eight bases are bound in the positively charged cleft between the N- and C-terminal domains of N, and three bases are shielded by the extended N-terminal arm. SBV N appears to sequester RNA using a different mechanism compared with the nucleoproteins of other negative-sense RNA viruses. Furthermore, the structure suggests that RNA binding results in conformational changes of some residues in the RNA-binding cleft and the N- and C-terminal arms. Our results provide new insights into the novel mechanism of RNA encapsidation by orthobunyaviruses.     

Size-independent and noncooperative recognition of dsRNA by the Rice stripe virus RNA silencing suppressor NS3

Plant and animal viruses employ diverse suppressor proteins to thwart the host antiviral reaction of RNA silencing. Many suppressors bind dsRNA with different size specificity. Here, we examine the dsRNA recognition mechanism of the Rice stripe virus NS3 suppressor using quantitative biochemical approaches, as well as mutagenesis and suppression activity analyses in plants. We show that dimeric NS3 is a size-independent, rather than small interfering RNA-specific, dsRNA-binding protein that recognizes a minimum of 9 bp and can bind to long dsRNA with two or more copies. Global analysis using a combinatorial approach reveals that NS3 dimer has an occluded site size of ∼ 13 bp on dsRNA, an intrinsic binding constant of 1 × 10⁸ M^− 1, and virtually no binding cooperativity. This lack of cooperativity suggests that NS3 is not geared to target long dsRNA. The larger site size of NS3, compared with its interacting size, indicates that the NS3 structure has a border region that has no direct contact with dsRNA but occludes a ∼ 4-bp region from binding. We also develop a method to correct the border effect of ligand by extending the lattice length. In addition, we find that NS3 recognizes the helical structure and 2′-hydroxyl group of dsRNA with moderate specificity. Analysis of dsRNA-binding mutants suggests that silencing of the suppression activity of NS3 is mechanistically related to its dsRNA binding ability.