The 3′ End of Hepatitis E Virus (HEV) Genome Binds Specifically to the Viral RNA-Dependent RNA Polymerase (RdRp (original) (raw)
Journal of Biological Chemistry, 2007
Studies of the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV), 3Dpol, have shown that Asn-297 permits this enzyme to distinguish ribose from 2-deoxyribose. All animal RNA viruses have Asn at the structurally homologous position of their polymerases, suggesting a conserved function for this residue. However, all prokaryotic RNA viruses have Glu at this position. In the presence of Mg 2؉ , the apparent affinity of Glu-297 3Dpol for 2-deoxyribonucleotides was decreased by 6-fold relative to wild type without a substantial difference in the fidelity of 2-dNMP incorporation. The fidelity of ribonucleotide misincorporation for Glu-297 3Dpol was reduced by 14-fold relative to wild type. A 4-to 11-fold reduction in the rate of ribonucleotide incorporation was observed. Glu-297 PV was unable to grow in HeLa cells due to a replication defect equivalent to that observed for a mutant PV encoding an inactive polymerase. Evaluation of the protein-(VPg)-primed initiation reaction showed that only half of the Glu-297 3Dpol initiation complexes were capable of producing VPg-pUpU product and that the overall yield of uridylylated VPg products was reduced by 20-fold relative to wild-type enzyme, a circumstance attributable to a reduced affinity for UTP. These studies identify the first RdRp derivative with a mutator phenotype and provide a mechanistic basis for the elevated mutation frequency of RNA phage relative to animal RNA viruses observed in culture. Although protein-primed initiation and RNA-primed elongation complexes employ the same polymerase active site, the functional differences reported here imply significant structural differences between these complexes.
Journal of General Virology, 2016
Hepatitis E virus (HEV) is a positive-sense RNA virus and member of the genus Orthohepevirus in the family Hepeviridae. Although HEV RNA-dependent RNA polymerase (HEV-RdRp) plays an important role in the HEV life cycle, its template specificities are not completely understood. We expressed HEV-RdRp protein with His-tag in a bacterial system and analysed template specificities using different putative cis-regulatory elements in the HEV genome. The enzyme showed highest affinity for the 3¢ non-coding region (NCR), then for the 5¢NCR and least for the putative subgenomic promoter (SgP). The enzyme could co-bind to 3¢NCR and putative SgP templates together, as evident from the supershift in binding assay, indicating presence of different binding sites for these elements. Proteomic analysis revealed that the RNA elements share two common peptides for binding, while a third peptide, which is highly conserved across different HEV genotypes, is specific for 3¢NCR. We propose that, during the early phases of replication, as negative sense antigenome copies accumulate at the replication site, they probably initiate promoter swapping from 3¢NCR to SgP, to favour synthesis of subgenomic RNA and to prevent synthesis of genomic RNA. The conserved site for 3¢NCR binding could be potential antiviral target and needs further evaluation. One supplementary table is available with the online Supplementary Material.
Secondary Structure of the 3' Terminus of Hepatitis C Virus Minus-Strand RNA
Journal of Virology, 2002
The 3-terminal ends of both the positive and negative strands of the hepatitis C virus (HCV) RNA, the latter being the replicative intermediate, are most likely the initiation sites for replication by the viral RNAdependent RNA polymerase, NS5B. The structural features of the very conserved 3 plus [(؉)] strand untranslated region [3 (؉) UTR] are well established (K. J. Blight and C. M. Rice, J. Virol. 71:7345-7352, 1997). However, little information is available concerning the 3 end of the minus [(؊)] strand RNA. In the present work, we used chemical and enzymatic probing to investigate the conformation of that region, which is complementary to the 5 (؉) UTR and the first 74 nucleotides of the HCV polyprotein coding sequence. By combining our experimental data with computer predictions, we have derived a secondary-structure model of this region. In our model, the last 220 nucleotides, where initiation of the (؉) strand RNA synthesis presumably takes place, fold into five stable stem-loops, forming domain I. Domain I is linked to an overall less stable structure, named domain II, containing the sequences complementary to the pseudoknot of the internal ribosomal entry site in the 5 (؉) UTR. Our results show that, even though the (؊) strand 3-terminal region has the antisense sequence of the 5 (؉) UTR, it does not fold into its mirror image. Interestingly, comparison of the replication initiation sites on both strands reveals common structural features that may play key functions in the replication process.
Distinct families of cis-acting RNA replication elements epsilon from hepatitis B viruses
RNA biology, 2012
The hepadnavirus encapsidation signal, epsilon (ε), is an RNA structure located at the 5' end of the viral pregenomic RNA. It is essential for viral replication and functions in polymerase protein binding and priming. This structure could also have potential regulatory roles in controlling the expression of viral replicative proteins. In addition to its structure, the primary sequence of this RNA element has crucial functional roles in the viral lifecycle. Although the ε elements in hepadnaviruses share common critical functions, there are some significant differences in mammalian and avian hepadnaviruses, which include both sequence and structural variations. Here we present several covariance models for ε elements from the Hepadnaviridae. The model building included experimentally determined data from previous studies using chemical probing and NMR analysis. These models have sufficient similarity to comprise a clan. The clan has in common a highly conserved overall structur...
PLOS Pathogens
There are approximately 20 million events of hepatitis E virus (HEV) infection worldwide annually. The genome of HEV is a single-strand, positive-sense RNA containing 5' and 3' untranslated regions and three open reading frames (ORF). HEV genome has 5' cap and 3' poly(A) tail to mimic host mRNA to escape the host innate immune surveillance and utilize host translational machineries for viral protein translation. The replication mechanism of HEV is poorly understood, especially how the viral polymerase distinguishes viral RNA from host mRNA to synthesize new viral genomes. We hypothesize that the HEV genome contains cis-acting elements that can be recognized by the virally encoded polymerase as "self" for replication. To identify functional cis-acting elements systematically across the HEV genome, we utilized an ORF1 transcomplementation system. Ultimately, we found two highly conserved cis-acting RNA elements within the ORF1 and ORF2 coding regions that are required for viral genome replication in a diverse panel of HEV genotypes. Synonymous mutations in the cis-acting RNA elements, not altering the ORF1 and ORF2 protein sequences, significantly impaired production of infectious viral particles. Mechanistic studies revealed that the cis-acting elements form secondary structures needed to interact with the HEV ORF1 protein to promote HEV replication. Thus, these cis-acting elements function as a scaffold, providing a specific "signal" that recruits viral and host factors to assemble the viral replication complex. Altogether, this work not only facilitates our understanding of the HEV life cycle and provides novel, RNA-directed targets for potential HEV treatments, but also sheds light on the development of HEV as a therapeutic delivery vector.
Kissing-Loop Interaction in the 3' End of the Hepatitis C Virus Genome Essential for RNA Replication
Journal of Virology, 2005
The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3 NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3 NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3 end of the HCV genome that is essential for replication.
Journal of Biological Chemistry, 2016
Dengue virus, an ∼10.7-kb positive-sense RNA virus, is the most common arthropod-communicated pathogen in the world. Despite dengue's clear epidemiological importance, mechanisms for its replication remain elusive. Here, we probed the entire dengue genome for interactions with viral RNA-dependent RNA polymerase (RdRp), and we identified the dominant interaction as a loop-forming ACAG motif in the 3′ positive-stranded terminus, complicating the prevailing model of replication. A subset of interactions coincides with known flaviviral recombination sites inside the viral protein-coding region. Specific recognition of the RNA element occurs via an arginine patch in the C-terminal thumb domain of RdRp. We also show that the highly conserved nature of the consensus RNA motif may relate to its tolerance to various mutations in the interacting region of RdRp. Disruption of the interaction resulted in loss of viral replication ability in cells. This unique RdRp-RNA interface is found thr...
Structural Domains of the 3′-Terminal Sequence of the Hepatitis C Virus Replicative Strand †
Biochemistry, 2008
Here we present the results of a structural analysis of the 3′-terminal region of the replicative strand of hepatitis C virus (HCV), IRES(-), by the Pb 2+-induced cleavage approach and partial digestion with T1 ribonuclease. Oligoribonucleotides that represent selected domains of the earlier proposed in the literature secondary structure models of this region were also synthesized, their structures were analyzed in solution, and the results were compared to those obtained with the full-length molecule. Such "structural fingerprinting" gave better insight into the structure of the IRES(-) region. We showed that in the case of the IRES(-) fragment, which consists of 374 nucleotides, its three domains, D3 (nucleotides 1-104), DM (nucleotides 105-222), and D5 (nucleotides 223-374), independently fold on one another. However, when the IRES(-) molecule is extended by 25 nucleotides of the upstream viral sequence, domains D3 and DM fold autonomously, but a part of domain D5 interacts with that additional RNA stretch. Analysis in silico suggests that similar interactions involving the IRES(-) region and upstream sequences are also possible in other fragments of viral RNA, several hundreds of nucleotides in length. The results of experimental probing are supported by secondary structure predictions in silico and phylogenetic analysis.
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;...