Towards a structural understanding of RNA synthesis by negative strand RNA viral polymerases (original) (raw)
Cryo-EM structure of the respiratory syncytial virus RNA polymerase
Nature Communications, 2020
The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activities — nucleotide polymerization, cap addition, and cap methylation. How RSV L and P coordinate these activities is poorly understood. Here, we present a 3.67 Å cryo-EM structure of the RSV polymerase (L:P) complex. The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (POD) and C-terminal domain (PCTD) of a tetramer of P. The density of the methyltransferase (MT) domain of L and the N-terminal domain of P (PNTD) is missing. Further analysis and comparison with other RNA polymerases at different stages suggest the structure we obtained is likely to be at an elongation-compatible stage. Together, these data provide enriched insights into the interrelationship, the inhibitors, and the evolutionary implications of the RSV polymerase.
2020
ABSTRACTBunyavirales is an order of segmented negative stranded RNA viruses comprising several life-threatening pathogens such as Lassa fever virus (Arenaviridae), Rift Valley Fever virus (Phenuiviridae) and La Crosse virus (LACV, Peribunyaviridae) against which neither specific treatment nor licenced vaccine is available. Replication and transcription of Bunyavirales genome constitute essential reactions of their viral cycle that are catalysed by the virally encoded RNA-dependent RNA polymerase or L protein. Here we describe the complete high-resolution cryo-EM structure of the full-length (FL) LACV-L protein. It reveals the presence of key C-terminal domains, notably the cap-binding domain that 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 structure of LACV-L FL in two functionally relevant states, pre-initiation and elongation, that reveal large conformational change...
Structural insight into cap-snatching and RNA synthesis by influenza polymerase
Nature, 2014
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.
Molecular architecture of the vesicular stomatitis virus RNA polymerase
Proceedings of the National Academy of Sciences, 2010
Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein–RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which ...
PLOS ONE, 2013
RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2-2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully prepositioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle intermediates.
The Crystal Structure of the RNA-Dependent RNA Polymerase from Human Rhinovirus
Structure, 2004
both healthy and high-risk individuals, antiviral treatment or prophylaxis would be desirable. However, no antiviral agents are currently approved for the prevention/treatment of HRV infection, although several have shown potent in vitro activity against HRV in cell culture, Laboratories such as viral capsid binders (Gwaltney et al., 2002) and protease inhibitors (Patick et al., 1999). San Diego, California 92121 After the binding of a picornavirus to its receptor and insertion of viral RNA into the cytoplasm, the parental RNA serves as mRNA to produce an initial polypeptide Summary which then self cleaves to give enzymes and structural proteins (Racaniello, 2001). Among the best-studied pi-Human rhinoviruses (HRV), the predominant members cornaviral enzymes are two nonstructural proteins deof the Picornaviridae family of positive-strand RNA noted 3C pro and 3D pol . 3C pro is a cysteine protease responviruses, are the major causative agents of the common sible for most of the polyprotein cleavage and has been cold. Given the lack of effective treatments for rhinovia focus of recent drug design efforts (Johnson et al., ral infections, virally encoded proteins have become 2002). 3D pol , encoded by the C-terminal portion of the attractive therapeutic targets. The HRV genome enviral polyprotein, is an RNA-dependent RNA polymerase codes an RNA-dependent RNA polymerase (RdRp) de-(RdRp) which copies the viral genome through an internoted 3D pol , which is responsible for replicating the mediate negative strand prior to encapsidation into inviral genome and for synthesizing a protein primer fectious progeny virions (Cameron et al., 2002). This used in the replication. Here the crystal structures for replication occurs in a primer-dependent manner on three viral serotypes (1B, 14, and 16) of HRV 3D pol have membranous vesicles in the cytoplasm of the infected been determined. The three structures are very similar cell. 3D pol also catalyzes the covalent linkage of UMP to to one another, and to the closely related poliovirus a tyrosine on a short peptide encoded by 3B (denoted (PV) 3D pol enzyme. Because the reported PV crystal VPg), with uridylylated VPg then serving as a protein structure shows significant disorder, HRV 3D pol proprimer for the initiation of RNA replication (Paul et al., vides the first complete view of a picornaviral RdRp. 1998). Therefore, HRV 3D pol possesses two distinct enzy-The folding topology of HRV 3D pol also resembles that matic functions which could be targeted for inhibition. of RdRps from hepatitis C virus (HCV) and rabbit hem-Crystal structures have been determined for several orrhagic disease virus (RHDV) despite very low seviral RdRps, such as poliovirus 3D pol (Hansen et al., quence homology. 1997), hepatitis C virus (HCV) NS5B (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999), rabbit hem-Introduction orrhagic disease virus (RHDV) polymerase (Ng et al., 2002), bovine viral diarrhea virus (BVDV) polymerase Picornaviruses are a large group of nonenveloped, posi-(Choi et al., 2004), and bacteriophage φ6 polymerase tive-strand RNA viruses with a common genetic organi-(Butcher et al., 2001). These structures display a comzation and replication strategy (Racaniello, 2001). This mon overall architecture found in all oligonucleotide family includes the human rhinoviruses (HRV), the major polymerases and described as a right hand with thumb, causative agents of the common cold, and enterovirfingers, and palm domains (Steitz, 1999). In addition, uses, as represented by poliovirus (PV). HRV and PV are RdRps have the unique feature of bridging finger and very similar in genome organization 0027ف( and 7500 thumb domains, giving a relatively closed and spherical nucleotides, respectively), polyprotein structure and appearance. In the structure of PV 3D pol , much of the processing, and viral protein function (Kitamura et al., fingers domain is disordered; thus characterization of 1981; Skern et al., 1985). Over 100 serotypes of rhinovipicornaviral RdRp architecture has remained incomruses have been reported, and the majority recognize plete. Here we report the crystal structures of HRV 3D pol ICAM-1 as a cellular receptor (Greve et al., 1989) while derived from three distinct serotypes: 1B from the minor the rest utilize the LDL receptor (Hofer et al., 1994). The LDL receptor class, and 16 and 14 from the major ICAM-1 complete genome sequence has been determined for receptor binding class. Each structure reveals a fully only a few HRV serotypes: 14, 16, and 89 from the ICAM-1 ordered enzyme with the same tertiary fold and clear class; and 1B and 2 from the LDL class. similarity to other members of the RdRp family. Unlike poliovirus, the development of vaccines against rhinovirus is hindered by the large number of serotypes and weak cross-protection between serotypes. While Results and Discussion most HRV infections are self-limiting, they can cause serious complications in persons with chronic respira-Structure Determination tory disease or immunodeficiencies (Couch, 2001). For The crystal structure of HRV14 3D pol was solved by taking advantage of the strong binding of lanthinides at a magnesium site in the enzyme's catalytic center. Crys-*Correspondence: robert.love@pfizer.com Data from an early apo-HRV14 crystal (without bound samarium) RNA-dependent RNA polymerase of hepatitis C virus. Proc. Natl. Acad. Sci. USA 96, 13034-13039. were obtained in-house at lower resolution 2.3ف( Å ), but these crystals could not be reproduced for synchrotron analysis. Butcher, S.J., Grimes, J.M., Makeyev, E.V., Bamford, D.H., and Stuart, D.I. (2001). A mechanism for initiating RNA-dependent RNA Structure Determination and Refinement polymerization. Nature 410, 235-240. Heavy atom parameter refinement and SAD phase calculations were Cameron, C.E., Gohara, D.W., and Arnold, J.J. (2002). Poliovirus performed with SHARP (De La Fortelle and Bricogne, 1997), using RNA-dependent RNA polymerase (3Dpol): structure, function, and the anomalous signal from a single samarium atom which bound in mechanism. In Molecular Biology of Picornaviruses, B.L. Semler and the HRV14 3D pol active site at a catalytic magnesium position (f″ of E. Wimmer, eds. (Washington, DC: ASM Press), pp. 255-267. 29.8 at peak absorption) The electron density map (Figure 1) was CCP4 (Collaborative Computational Project, Number 4) (1994). The improved with solvent flattening using SOLOMON (Abrahams, 1996) CCP4 suite: programs for protein crystallography. Acta Crystallogr. as implemented in SHARP. SAD phasing statistics to 2.8 Å included D Biol. Crystallogr. 50, 760-763. a phasing power of 2.0 (0.6 in the highest shell) and an overall figure Choi, K.H., Groarke, J.M., Young, D.C., Kuhn, R.J., Smith, J.L., Pevof merit before and after density modification of 0.41 and 0.78. A ear, D.C., and Rossmann, M.G. (2004). The structure of the RNAmodel was constructed for HRV14 3D pol using XFIT (McRee, 1992), dependent RNA polymerase from bovine viral diarrhea virus estaband was refined initially with CNX (Accelrys, Inc). This model permitlishes the role of GTP in de novo initiation. Proc. Natl. Acad. Sci. ted the HRV1B and HRV16 3D pol structures to be determined with USA 101, 4425-4430. molecular replacement (CCP4, 1994). Replacement of amino acid side chains to transform HRV14 into other serotypes was performed Couch, R.B. (2001). Rhinoviruses. In Fields Virology, D.M. Knipe and with MODELLER (Sali and Blundell, 1993). After several rounds of P.M. Howley, eds. (Philadelphia: Lippincott Williams & Wilkins), pp. XFIT and CNX for all models, REFMAC5 (CCP4, 1994) was used in 777-797. final stages of refinement (maximum likelihood target). Refinement De La Fortelle, E., and Bricogne, G. (1997). Macromolecular crystalstatistics are shown in Table 1. The HRV14/Sm 3ϩ 3D pol model with lography. Methods Enzymol. 276, 472-494. samarium removed was also refined against in-house 3.2 Å diffrac-Doublie, S., Sawaya, M.R., and Ellenberger, T. (1999). An open and tion data obtained from a small crystal of apo-HRV14 in order to closed case for all polymerases. Struct. Fold. Des. 7, R31-R35. study conformational changes induced by the metal; these refine-Gerber, K., Wimmer, E., and Paul, A.V. (2001). Biochemical and ment statistics were R/R free (20-3.2 Å ) ϭ 25.6/27.2, rmsd bond genetic studies of the initiation of human rhinovirus 2 RNA replicalengths and angles of 0.012 Å and 1.41Њ. Stereochemical quality of tion: purification and enzymatic analysis of the RNA-dependent RNA all models was checked using PROCHECK (Laskowski et al., 1993). polymerase 3D(pol). J. Virol. 75, 10969-10978. Only Lys275 (second residue of type-IIЈ -turn 274-277) was consistently an outlier in the Ramachandran plot, probably a result of salt bridges to Asp55 and Glu45 along with several main chain-main Cameron, C.E. (2000). Poliovirus RNA-dependent RNA polymerase chain hydrogen bonds. (3Dpol): structural, biochemical, and biological analysis of con-During solvent incorporation for the HRV1B 3D pol structure, a water served structural motifs A and B. J. Biol. Chem. 275, 25523-25532. molecule placed into the single strongest positive peak (10 ) of Goodfellow, I., Chaudhry, Y., Richardson, A., Meredith, J., Almond, (F o Ϫ F c ) maps refined to a B factor 5ف( Å 2 ) much lower than the J.W., Barclay, W., and Evans, D.J. (2000). Identification of a cisaverage for surrounding atoms (28 Å 2 ). Since this site involved interacting replication element within the poliovirus coding region. J. actions with four main chain carbonyl oxygens (average distance Virol. 74, 4590-4600. 2.64 Å ) and a serine hydryoxyl (2.73 Å ) without any regular coordina-Greasley, S.E., Horton, P., Ramcharan, J., Beardsley, G.P., Benkovic, tion geometry, monovalent cation binding was suspected. HRV1B S.J., and Wilson, I.A. (2001). Crystal structure of a bifunctional transcrystallized using 1.0 M Na-K tartrate;...
Crystal Structure of Complete Rhinovirus RNA Polymerase Suggests Front Loading of Protein Primer
Journal of Virology, 2005
Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3D pol ) at a 2.4-Å resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3D pol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3D po1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.
Journal of Virology, 2014
Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3D pol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8-and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3D pol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cationinteraction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3D pol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3D pol , we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.
The structure of the RNA-dependent RNA polymerase (RdRP) from the rabbit hemorrhagic disease virus has been determined by x-ray crystallography to a 2.5-Å resolution. The overall structure resembles a " right hand, " as seen before in other polymerases, including the RdRPs of polio virus and hepatitis C virus. Two copies of the polymerase are present in the asymmetric unit of the crystal, revealing active and inactive conformations within the same crystal form. The fingers and palm domains form a relatively rigid unit, but the thumb domain can adopt either " closed " or " open " conformations differing by a rigid body rotation of 8 degrees. Metal ions bind at different positions in the two conformations and suggest how structural changes may be important to enzymatic function in RdRPs. Comparisons between the structures of the alternate conformational states of rabbit hemorrhagic disease virus RdRP and the structures of RdRPs from hepatitis C virus and polio virus suggest novel structure-function relationships in this medically important class of enzymes.