Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein (original) (raw)
Related papers
Nucleic Acids Research, 2013
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 RNAbinding 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.
Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein
Proceedings of the National Academy of Sciences, 2013
The nucleoprotein (NP) of segmented negative-strand RNA viruses such as Orthomyxo-, Arena-, and Bunyaviruses coats the genomic viral RNA and together with the polymerase forms ribonucleoprotein particles (RNPs), which are both the template for replication and transcription and are packaged into new virions. Here we describe the crystal structure of La Crosse Orthobunyavirus NP both RNA free and a tetrameric form with single-stranded RNA bound. La Crosse Orthobunyavirus NP is a largely helical protein with a fold distinct from other bunyavirus genera NPs. It binds 11 RNA nucleotides in the positively charged groove between its two lobes, and hinged Nand C-terminal arms mediate oligomerization, allowing variable protein-protein interface geometry. Oligomerization and RNA binding are mediated by residues conserved in the Orthobunyavirus genus. In the twofold symmetric tetramer, 44 nucleotides bind in a closed ring with sharp bends at the NP-NP interfaces. The RNA is largely inaccessible within a continuous internal groove. Electron microscopy of RNPs released from virions shows them capable of forming a hierarchy of more or less compact irregular helical structures. We discuss how the planar, tetrameric NP-RNA structure might relate to a polar filament that upon supercoiling could be packaged into virions. This work gives insight into the RNA encapsidation and protection function of bunyavirus NP, but also highlights the need for dynamic rearrangements of the RNP to give the polymerase access to the template RNA.
mBio, 2012
Influenza A virions contain eight ribonucleoproteins (RNPs), each comprised of a negative-strand viral RNA, the viral polymerase, and multiple nucleoproteins (NPs) that coat the viral RNA. NP oligomerization along the viral RNA is mediated largely by a 28-amino-acid tail loop. Influenza viral RNPs, which serve as the templates for viral RNA synthesis in the nuclei of infected cells, are not linear but rather are organized in hairpin-like double-helical structures. Here we present results that strongly support a coherent model for the assembly of the double-helical influenza virus RNP structure. First, we show that NP self-associates much more weakly in the absence of RNA than in its presence, indicating that oligomerization is very limited in the cytoplasm. We also show that once NP has oligomerized, it can dissociate in the absence of bound RNA, but only at a very slow rate, indicating that the NP scaffold remains intact when viral RNA dissociates from NPs to interact with the polymerase during viral RNA synthesis. In addition, we identify a previously unknown NP-NP interface that is likely responsible for organizing the double-helical viral RNP structure. This identification stemmed from our observation that NP lacking the oligomerization tail loop forms monomers and dimers. We determined the crystal structure of this NP dimer, which reveals this new NP-NP interface. Mutation of residues that disrupt this dimer interface does not affect oligomerization of NPs containing the tail loop but does inactivate the ability of NPs containing the tail loop to support viral RNA synthesis in minigenome assays. Importance Influenza A virus, the causative agent of human pandemics and annual epidemics, contains eight RNA gene segments. Each RNA segment assumes the form of a rod-shaped, double-helical ribonucleoprotein (RNP) that contains multiple copies of a viral protein, the nucleoprotein (NP), which coats the RNA segment along its entire length. Previous studies showed that NP molecules can polymerize via a structural element called the tail loop, but the RNP assembly process is poorly understood. Here we show that influenza virus RNPs are likely assembled from NP monomers, which polymerize through the tail loop only in the presence of viral RNA. Using X-ray crystallography, we identified an additional way that NP molecules interact with each other. We hypothesize that this new interaction is responsible for organizing linear, single-stranded influenza virus RNPs into double-helical structures. Our results thus provide a coherent model for the assembly of the double-helical influenza virus RNP structure.
The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein
PLoS Pathogens, 2013
Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ,12 nts of RNA, shorter than the 24-28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions. Citation: Zheng W, Olson J, Vakharia V, Tao YJ (2013) The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein. PLoS Pathog 9(9): e1003624.
Crystal Structure of a Nucleocapsid-Like Nucleoprotein-RNA Complex of Respiratory Syncytial Virus
Science, 2009
The respiratory syncytial virus (RSV) is an important human pathogen, yet neither a vaccine nor effective therapies are available to treat infection. To help elucidate the replication mechanism of this RNA virus, we determined the three-dimensional (3D) crystal structure at 3.3 A resolution of a decameric, annular ribonucleoprotein complex of the RSV nucleoprotein (N) bound to RNA. This complex mimics one turn of the viral helical nucleocapsid complex, which serves as template for viral RNA synthesis. The RNA wraps around the protein ring, with seven nucleotides contacting each N subunit, alternating rows of four and three stacked bases that are exposed and buried within a protein groove, respectively. Combined with electron microscopy data, this structure provides a detailed model for the RSV nucleocapsid, in which the bases are accessible for readout by the viral polymerase. Furthermore, the nucleoprotein structure highlights possible key sites for drug targeting.
RNA, 2009
Bunyamwera virus (BUNV) is the prototypic member of both the Orthobunyavirus genus and the Bunyaviridae family of negative stranded RNA viruses. In common with all negative stranded RNA viruses, the BUNV genomic and anti-genomic strands are not naked RNAs, but instead are encapsidated along their entire lengths with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex. This association is critical for the negative strand RNA virus life cycle because only RNPs are active for productive RNA synthesis and RNA packaging. We are interested in understanding the molecular details of how N and RNA components associate within the bunyavirus RNP, and what governs the apparently selective encapsidation of viral replication products. Toward this goal, we recently devised a protocol that allowed generation of native BUNV N protein that maintained solubility under physiological conditions and allowed formation of crystals that yielded high-resolution x-ray diffract...
RNA, 2013
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.
The respiratory syncytial virus nucleoprotein-RNA complex forms a left-handed helical nucleocapsid
Journal of General Virology, 2013
Respiratory syncytial virus (RSV) is an important human pathogen. Its nucleocapsid (NC), which comprises the negative sense RNA viral genome coated by the viral nucleoprotein N, is a critical assembly that serves as template for both mRNA synthesis and genome replication. We have previously described the X-ray structure of an NC-like structure: a decameric ring formed of N-RNA that mimics one turn of the helical NC. In the absence of experimental data we had hypothesized that the NC helix would be right-handed, as the N–N contacts in the ring appeared to more easily adapt to that conformation. We now unambiguously show that the RSV NC is a left-handed helix. We further show that the contacts in the ring can be distorted to maintain key N–N-protein interactions in a left-handed helix, and discuss the implications of the resulting atomic model of the helical NC for viral replication and transcription.
Conformational flexibility of nonamer ribonucleoprotein complexes from Influenza A virus
The nucleoprotein (NP) of negative-sense single-strand RNA viruses is the major component of the ribonucleoprotein (vRNP) complex, which is responsible for viral transcription and replication. NP of Influenza A virus consists of two helical head and body domains, arranged in the banana-shaped configuration. We evaluated the conformational peculiarities of wild-type NP nonamers from the influenza A/Hong Kong/1/68 (H3N2) strain and mutant (E292G) NP nonamers from cold-adapted strain using the molecular dynamics method. We found that the E292G mutation in NP may lead to changing of the vRNP nonamer planar structure both at 299 And 312 K. It may lead to change in the complex functionality and shed the light to the explanation of influenza A cold-adaptivity mechanisms. Our molecular dynamics experiments also expands recent the cryoelectronic microscopy structural data. We have proposed that the observed mutation in the position 292 of NP can contribute to the development of the genetical...