Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter.

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 synthesis. These results will aid development of replication inhibitors of diverse, serious human pathogenic viruses.

The mechanism of genome replication and transcription in bunyaviruses.

Bunyaviruses are negative sense, single-strand RNA viruses that infect a wide range of vertebrate, invertebrate and plant hosts. WHO lists three bunyavirus diseases as priority diseases requiring urgent development of medical countermeasures highlighting their high epidemic potential. While the viral large (L) protein containing the RNA-dependent RNA polymerase is a key enzyme in the viral replication cycle and therefore a suitable drug target, our knowledge on the structure and activities of this multifunctional protein has, until recently, been very limited. However, in the last few years, facilitated by the technical advances in the field of cryogenic electron microscopy, many structures of bunyavirus L proteins have been solved. These structures significantly enhance our mechanistic understanding of bunyavirus genome replication and transcription processes and highlight differences and commonalities between the L proteins of different bunyavirus families. Here, we provide a review of our current understanding of genome replication and transcription in bunyaviruses with a focus on the viral L protein. Further, we compare within bunyaviruses and with the related influenza virus polymerase complex and highlight open questions.

Structural and functional characterization of the Sin Nombre virus L protein.

The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus (SNV) of the Hantaviridae. Despite the high epidemic potential of bunyaviruses, specific medical countermeasures such as vaccines or antivirals are missing. The multifunctional ~250 kDa L protein of hantaviruses, amongst other functional domains, harbors the RNA-dependent RNA polymerase (RdRp) and an endonuclease and catalyzes transcription as well as replication of the viral RNA genome, making it a promising therapeutic target. The development of inhibitors targeting these key processes requires a profound understanding of the catalytic mechanisms. Here, we established expression and purification protocols of the full-length SNV L protein bearing the endonuclease mutation K124A. We applied different biochemical in vitro assays to provide an extensive characterization of the different enzymatic functions as well as the capacity of the hantavirus L protein to interact with the viral RNA. By using single-particle cryo-EM, we obtained a 3D model including the L protein core region containing the RdRp, in complex with the 5' promoter RNA. This first high-resolution model of a New World hantavirus L protein shows striking similarity to related bunyavirus L proteins. The interaction of the L protein with the 5' RNA observed in the structural model confirms our hypothesis of protein-RNA binding based on our biochemical data. Taken together, this study provides an excellent basis for future structural and functional studies on the hantavirus L protein and for the development of antiviral compounds.

Structural Analysis of dsRNA Binding to Anti-viral Pattern Recognition Receptors LGP2 and MDA5.

RIG-I and MDA5 sense virus-derived short 5'ppp blunt-ended or long dsRNA, respectively, causing interferon production. Non-signaling LGP2 appears to positively and negatively regulate MDA5 and RIG-I signaling, respectively. Co-crystal structures of chicken (ch) LGP2 with dsRNA display a fully or semi-closed conformation depending on the presence or absence of nucleotide. LGP2 caps blunt, 3' or 5' overhang dsRNA ends with 1 bp longer overall footprint than RIG-I. Structures of 1:1 and 2:1 complexes of chMDA5 with short dsRNA reveal head-to-head packing rather than the polar head-to-tail orientation described for long filaments. chLGP2 and chMDA5 make filaments with a similar axial repeat, although less co-operatively for chLGP2. Overall, LGP2 resembles a chimera combining a MDA5-like helicase domain and RIG-I like CTD supporting both stem and end binding. Functionally, RNA binding is required for LGP2-mediated enhancement of MDA5 activation. We propose that LGP2 end-binding may promote nucleation of MDA5 oligomerization on dsRNA.

Structural insight into cap-snatching and RNA synthesis by influenza polymerase.

Influenza virus polymerase uses a capped primer, derived by 'cap-snatching' from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves in situ rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.

Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein.

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 N- and 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.

Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers.

Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms. Apo polymerase protomers can adopt two drastically different conformations, which assemble into two distinct symmetric homodimers, that can themselves gather to form hexamers. Polymerase dimerization induces the stabilization of most polymerase domains, including the C-terminal domain that contributes the most to dimer's interface, along with a lariat region that participates to the polymerase steadying. Binding to viral RNA induces significant conformational changes resulting in symmetric oligomer disruption and polymerase activation, suggesting the possible involvement of apo multimers as protecting systems that would stabilize the otherwise flexible C-terminal domains. Overall, these results provide insights into the multimerization capability of Hantavirus polymerase and may help to define antiviral compounds to counteract these life-threatening viruses.

The low-resolution structure of nHDL reconstituted with DMPC with and without cholesterol reveals a mechanism for particle expansion.

Small-angle neutron scattering (SANS) with contrast variation was used to obtain the low-resolution structure of nascent HDL (nHDL) reconstituted with dimyristoyl phosphatidylcholine (DMPC) in the absence and presence of cholesterol, [apoA1:DMPC (1:80, mol:mol) and apoA1:DMPC:cholesterol (1:86:9, mol:mol:mol)]. The overall shape of both particles is discoidal with the low-resolution structure of apoA1 visualized as an open, contorted, and out of plane conformation with three arms in nascent HDL/dimyristoyl phosphatidylcholine without cholesterol (nHDL(DMPC)) and two arms in nascent HDL/dimyristoyl phosphatidylcholine with cholesterol (nHDL(DMPC+Chol)). The low-resolution shape of the lipid phase in both nHDL(DMPC) and nHDL(DMPC+Chol) were oblate ellipsoids, and fit well within their respective protein shapes. Modeling studies indicate that apoA1 is folded onto itself in nHDL(DMPC), making a large hairpin, which was also confirmed independently by both cross-linking mass spectrometry and hydrogen-deuterium exchange (HDX) mass spectrometry analyses. In nHDL(DMPC+Chol), the lipid was expanded and no hairpin was visible. Importantly, despite the overall discoidal shape of the whole particle in both nHDL(DMPC) and nHDL(DMPC+Chol), an open conformation (i.e., not a closed belt) of apoA1 is observed. Collectively, these data show that full length apoA1 retains an open architecture that is dictated by its lipid cargo. The lipid is likely predominantly organized as a bilayer with a micelle domain between the open apoA1 arms. The apoA1 configuration observed suggests a mechanism for accommodating changing lipid cargo by quantized expansion of hairpin structures.

Structures of active Hantaan virus polymerase uncover the mechanisms of Hantaviridae genome replication.

Hantaviruses are causing life-threatening zoonotic infections in humans. Their tripartite negative-stranded RNA genome is replicated by the multi-functional viral RNA-dependent RNA-polymerase. Here we describe the structure of the Hantaan virus polymerase core and establish conditions for in vitro replication activity. The apo structure adopts an inactive conformation that involves substantial folding rearrangement of polymerase motifs. Binding of the 5' viral RNA promoter triggers Hantaan virus polymerase reorganization and activation. It induces the recruitment of the 3' viral RNA towards the polymerase active site for prime-and-realign initiation. The elongation structure reveals the formation of a template/product duplex in the active site cavity concomitant with polymerase core widening and the opening of a 3' viral RNA secondary binding site. Altogether, these elements reveal the molecular specificities of Hantaviridae polymerase structure and uncover the mechanisms underlying replication. They provide a solid framework for future development of antivirals against this group of emerging pathogens.

Molecular adaptation of the DegQ protease to exert protein quality control in the bacterial cell envelope.

To react to distinct stress situations and to prevent the accumulation of misfolded proteins, all cells employ a number of proteases and chaperones, which together set up an efficient protein quality control system. The functionality of proteins in the cell envelope of Escherichia coli is monitored by the HtrA proteases DegS, DegP, and DegQ. In contrast with DegP and DegS, the structure and function of DegQ has not been addressed in detail. Here, we show that substrate binding triggers the conversion of the resting DegQ hexamer into catalytically active 12- and 24-mers. Interestingly, substrate-induced oligomer reassembly and protease activation depends on the first PDZ domain but not on the second. Therefore, the regulatory mechanism originally identified in DegP should be a common feature of HtrA proteases, most of which encompass only a single PDZ domain. Using a DegQ mutant lacking the second PDZ domain, we determined the high resolution crystal structure of a dodecameric HtrA complex. The nearly identical domain orientation of protease and PDZ domains within 12- and 24-meric HtrA complexes reveals a conserved PDZ1 → L3 → LD/L1/L2 signaling cascade, in which loop L3 senses the repositioned PDZ1 domain of higher order, substrate-engaged particles and activates protease function. Furthermore, our in vitro and in vivo data imply a pH-related function of DegQ in the bacterial cell envelope.

RNA channelling by the eukaryotic exosome.

The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states. In the RNA-bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate-threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome-like complexes from all domains of life, and might have been present in the most recent common ancestor.

Structural snapshots of La Crosse virus polymerase reveal the mechanisms underlying Peribunyaviridae replication and transcription.

Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse virus polymerase and visualize its conformational dynamics by cryo-electron microscopy, unveiling the precise molecular mechanics underlying its essential activities. We find that replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that prompt primer cleavage prior to primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The uncovered detailed mechanics should be helpful for the future design of antivirals counteracting bunyaviral life threatening pathogens.

Mechanisms of RNA recruitment by the exosome.

Exosome-like protein complexes are essential 3'−>5' ribonucleases involved in processing and degradation of many RNAs. They are conserved in the three domains of life and share a common architecture comprised of a ring-like core structure organized around a central channel. RNA degradation by bacterial and archaeal exosome-like complexes requires threading through this single-stranded RNA specific channel to reach the phosphorolytic active sites buried deep within the barrel-shaped complex. In contrast most eukaryotic exosomes appear to have lost phosphorolytic activity and instead rely on hydrolytic RNases for catalytic activity raising the question of the degree of conservation of RNA recruitment mechanisms between prokaryotic and eukaryotic complexes. Recent single particle electron microscopy reconstructions of apo and RNA bound yeast exosomes provide the first direct structural evidence for a channeling mechanism by a eukaryotic exosome suggesting that this mechanism is conserved between all exosome-like complexes.

The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket.

Macro domains (also called "X domains") constitute a protein module family present in all kingdoms of life, including viruses of the Coronaviridae and Togaviridae families. Crystal structures of the macro domain from the Chikungunya virus (an "Old World" alphavirus) and the Venezuelan equine encephalitis virus (a "New World" alphavirus) were determined at resolutions of 1.65 and 2.30 A, respectively. These domains are active as adenosine di-phosphoribose 1''-phosphate phosphatases. Both the Chikungunya and the Venezuelan equine encephalitis virus macro domains are ADP-ribose binding modules, as revealed by structural and functional analysis. A single aspartic acid conserved through all macro domains is responsible for the specific binding of the adenine base. Sequence-unspecific binding to long, negatively charged polymers such as poly(ADP-ribose), DNA, and RNA is observed and attributed to positively charged patches outside of the active site pocket, as judged by mutagenesis and binding studies. The crystal structure of the Chikungunya virus macro domain with an RNA trimer shows a binding mode utilizing the same adenine-binding pocket as ADP-ribose, but avoiding the ADP-ribose 1''-phosphate phosphatase active site. This leaves the AMP binding site as the sole common feature in all macro domains.

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes.

Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors.

Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex.

The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that inside the LdcI-RavA cage, RavA hexamers adopt an asymmetric spiral conformation in which the nucleotide-free seam is constrained to two opposite orientations. Cryo-EM reconstructions of free RavA reveal two co-existing structural states: an asymmetric spiral, and a flat C2-symmetric closed ring characterised by two nucleotide-free seams. The closed ring RavA state bears close structural similarity to the pseudo two-fold symmetric crystal structure of the AAA+ unfoldase ClpX, suggesting a common ATPase mechanism. Based on these structures, and in light of the current knowledge regarding AAA+ ATPases, we propose different scenarios for the ATP hydrolysis cycle of free RavA and the LdcI-RavA cage-like complex, and extend the comparison to other AAA+ ATPases of clade 7.

High resolution cryo-EM structure of the helical RNA-bound Hantaan virus nucleocapsid reveals its assembly mechanisms.

Negative-strand RNA viruses condense their genome into helical nucleocapsids that constitute essential templates for viral replication and transcription. The intrinsic flexibility of nucleocapsids usually prevents their full-length structural characterisation at high resolution. Here, we describe purification of full-length recombinant metastable helical nucleocapsid of Hantaan virus (Hantaviridae family, Bunyavirales order) and determine its structure at 3.3 Å resolution by cryo-electron microscopy. The structure reveals the mechanisms of helical multimerisation via sub-domain exchanges between protomers and highlights nucleotide positions in a continuous positively charged groove compatible with viral genome binding. It uncovers key sites for future structure-based design of antivirals that are currently lacking to counteract life-threatening hantavirus infections. The structure also suggests a model of nucleoprotein-polymerase interaction that would enable replication and transcription solely upon local disruption of the nucleocapsid.

Structural and functional analysis of methylation and 5'-RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5.

The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase(DV), is involved in two of four steps required for the formation of the viral mRNA cap (7Me)GpppA(2'OMe), the guanine-N7 and the adenosine-2'O methylation. Its S-adenosyl-l-methionine (AdoMet) dependent 2'O-methyltransferase (MTase) activity has been shown on capped (7Me+/-)GpppAC(n) RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 A to 2.8 A resolution of NS5MTase(DV) in complex with cap analogues and the co-product of methylation S-adenosyl-l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP-binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of (7Me)GpppAC(4) and (7Me)GpppAC(5) RNAs is inhibited in the presence of GTP, (7Me)GTP and (7Me)GpppA but not by ATP. This particular position of the cap is in accordance with the 2'O-methylation step. A model was generated of a ternary 2'O-methylation complex of NS5MTase(DV), (7Me)GpppA and AdoMet. RNA-binding increased when (7Me+/-)GpppAGC(n-1) starting with the consensus sequence GpppAG, was used instead of (7Me+/-)GpppAC(n). In the NS5MTase(DV)-GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5' end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2'O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.

The flavivirus polymerase as a target for drug discovery.

Flaviviruses are emerging pathogens of increasingly important public health concern in the world. For most flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) neither vaccine nor antiviral treatment is available. The viral RNA-dependent RNA polymerase (RdRp) non-structural protein 5 (NS5) has no equivalent in the host cell and is essential for viral replication. Here, we give an overview of the current knowledge regarding Flavivirus RdRp function and structure as it represents an attractive target for drug design. Flavivirus RdRp exhibits primer-independent activity, thus initiating RNA synthesis de novo. Following initiation, a conformational change must occur to allow the elongation process. Structure-function studies of Flavivirus RdRp are now facilitated by the crystal structures of DENV (serotype 3) and WNV RdRp domains. Both adopt a classic viral RdRp fold and present a closed pre-initiation conformation. The so-called priming loop is thought to provide the initiation platform stabilizing the de novo initiation complex. A zinc-ion binding site at the hinge between two subdomains might be involved in opening up the RdRp structure towards a conformation for elongation. Using two different programs we predicted common potential allosteric inhibitor binding sites on both structures. We also review ongoing approaches of in vitro and cell-based screening programs aiming at the discovery of nucleosidic and non-nucleosidic inhibitors targeting Flavivirus RdRps.

RIP2 filament formation is required for NOD2 dependent NF-κB signalling.

Activation of the innate immune pattern recognition receptor NOD2 by the bacterial muramyl-dipeptide peptidoglycan fragment triggers recruitment of the downstream adaptor kinase RIP2, eventually leading to NF-κB activation and proinflammatory cytokine production. Here we show that full-length RIP2 can form long filaments mediated by its caspase recruitment domain (CARD), in common with other innate immune adaptor proteins. We further show that the NOD2 tandem CARDs bind to one end of the RIP2 CARD filament, suggesting a mechanism for polar filament nucleation by activated NOD2. We combine X-ray crystallography, solid-state NMR and high-resolution cryo-electron microscopy to determine the atomic structure of the helical RIP2 CARD filament, which reveals the intermolecular interactions that stabilize the assembly. Using structure-guided mutagenesis, we demonstrate the importance of RIP2 polymerization for the activation of NF-κB signalling by NOD2. Our results could be of use to develop new pharmacological strategies to treat inflammatory diseases characterised by aberrant NOD2 signalling.