Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein (original) (raw)
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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 Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter
Cell, 2015
Segmented negative-strand RNA virus (sNSV) polymerases transcribe and replicate the viral RNA (vRNA) within a ribonucleoprotein particle (RNP). We present cryo-EM and X-ray structures of, respectively, apo- and vRNA bound La Crosse orthobunyavirus (LACV) polymerase that give atomic-resolution insight into how such RNPs perform RNA synthesis. The complementary 3' and 5' vRNA extremities are sequence specifically bound in separate sites on the polymerase. The 5' end binds as a stem-loop, allosterically structuring functionally important polymerase active site loops. Identification of distinct template and product exit tunnels allows proposal of a detailed model for template-directed replication with minimal disruption to the circularised RNP. The similar overall architecture and vRNA binding of monomeric LACV to heterotrimeric influenza polymerase, despite high sequence divergence, suggests that all sNSV polymerases have a common evolutionary origin and mechanism of RNA sy...
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...
Bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation
Proceedings of the National Academy of Sciences, 2013
Bunyamwera virus (BUNV), which belongs to the genus Orthobunyavirus , is the prototypical virus of the Bunyaviridae family. Similar to other negative-sense single-stranded RNA viruses, bunyaviruses possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation and virus replication. The structures of two NPs of members of different genera within the Bunyaviridae family have been reported. However, their structures, RNA-binding features, and functions beyond RNA binding significantly differ from one another. Here, we report the crystal structure of the BUNV NP–RNA complex. The polypeptide of the BUNV NP was found to possess a distinct fold among viral NPs. An N-terminal arm and a C-terminal tail were found to interact with neighboring NP protomers to form a tetrameric ring-shaped organization. Each protomer bound a 10-nt RNA molecule, which was acquired from the expression host, in the positively charged crevice between the N and C lobes. Inhomogeneous oligomerization wa...
Phleboviruses encapsidate their genomes by sequestering RNA bases
Proceedings of the National Academy of Sciences, 2012
Rift Valley fever and Toscana viruses are human pathogens for which no effective therapeutics exist. These and other phleboviruses have segmented negative-sense RNA genomes that are sequestered by a nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes of irregular, asymmetric structure, previously uncharacterized at high resolution. N binds nonspecifically to single-stranded RNA with nanomolar affinity. Crystal structures of Rift Valley fever virus N-RNA complexes reconstituted with defined RNAs of different length capture tetrameric, pentameric and hexameric N-RNA multimers. All N-N subunit contacts are mediated by a highly flexible α-helical arm. Arm movement gives rise to the three multimers in the crystal structures and also explains the asymmetric architecture of the RNP. Despite the flexible association of subunits, the crystal structures reveal an invariant, monomeric RNP building block, consisting of the core of one N subunit, the arm of a neighboring N, and four RNA nucleotides with the flanking phosphates. Up to three additional RNA nucleotides bind between subunits. The monomeric building block is matched in size to the repeating unit in viral RNP, as visualized by electron microscopy. N sequesters four RNA bases in a narrow hydrophobic binding slot and has polar contacts only with the sugar-phosphate backbone, which faces the solvent. All RNA bases, whether in the binding slot or in the subunit interface, face the protein in a manner that is incompatible with base pairing or with "reading" by the viral polymerase.
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.
An encapsidated viral protein and its role in RNA packaging by a non-enveloped animal RNA virus
Virology, 2015
Alphatetraviruses are small (+) ssRNA viruses with non-enveloped, icosahedral, T=4 particles that assemble from 240 copies of a single capsid protein precursor. This study is focused on the mechanisms underlying selection and packaging of genomic vRNAs by Helicoverpa armigera stunt virus. We demonstrate that the viral protein, p17, is packaged at low levels (between 4 and 8 copies per capsid) raising the possibility of icosahedral asymmetry in wild-type particles. p17 promotes packaging of vRNA2 by virus-like particles (VLPs) generated from plasmid-expressed vRNA2. The 5' and 3' UTRs of RNA2 are not required for encapsidation. VLPs produced by recombinant baculoviruses package vRNA2 at detectable levels even in the absence of p17 and apparently excluding baculoviral transcripts. This suggests a role for p17 in vRNA selectivity. This is one of few examples of the packaging of a minor non-structural protein by (+) ssRNA animal viruses.
Journal of Virology, 2000
The genome of Bunyamwera virus (BUN) (family Bunyaviridae, genus Bunyavirus) comprises three negativesense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5 terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5-terminal 32 and 3-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 g of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5 terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.
Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein
Nucleic Acids Research, 2014
Mediterranean Europe where it accounts for 80% of pediatric meningitis cases during the summer. The negativestrand ribonucleic acid (RNA) genome of the virus is wrapped around the virally encoded nucleoprotein N to form the ribonucleoprotein complex (RNP). We determined crystal structures of hexameric N alone (apo) and in complex with a nonameric singlestranded RNA. RNA is sequestered in a sequenceindependent fashion in a deep groove inside the hexamer. At the junction between two adjacent copies of Ns, RNA binding induced an inter-subunit rotation, which opened the RNA-binding tunnel and created a new assembly interface at the outside of the hexamer. Based on these findings, we suggest a structural model for how binding of RNA to N promotes the formation of helical RNPs, which are a characteristic hallmark of many negative-strand RNA viruses.