The modeled structure of the RNA dependent RNA polymerase of GBV-C Virus suggests a role for motif E in Flaviviridae RNA polymerases
© Ferron et al; licensee BioMed Central Ltd. 2005
Received: 18 January 2005
Accepted: 14 October 2005
Published: 14 October 2005
The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp) plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV) is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV) is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C). Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet.
We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites.
Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA polymerases.
The Flaviviridae virus family comprises three genera pestivirus, hepacivirus, and the large group of flavivirus. HCV causes acute and chronic hepatitis that may lead to cirrhosis and/or liver cancer. HCV is a major human pathogen, with 170 million people infected worldwide and 3 to 4 million of newly infected people each year . Despite its large socio-economic impact, there is neither a vaccine nor an efficient, side-effect free therapy against this virus. Thus, the identification of potent drugs would be a major public health achievement. However, convenient small-animal models or productively infected cell systems to study HCV are still lacking. Consequently, compounds are often directly validated in HCV infected chimpanzees, or in cultured cells infected with related, surrogate viruses such as pestiviruses. The latter are animal pathogens showing similarity to hepaciviruses and flaviviruses  in genome structure, replication strategy, and individual gene products.
The RNA-dependent RNA polymerase (RdRp) is an enzyme playing a key role in the RNA replication process. Despite the increasing number of studies on the characterization of RdRp activity and structure, the precise molecular mechanism remains unclear. The postulated RNA replication process is a two-step mechanism. First, the initiation step of RNA synthesis begins at or near the 3' end of the (+) RNA template by means of a primer-independent (de novo) mechanism . The de novo initiation consists in the addition of a nucleotide tri-phosphate (NTP) to the 3'-OH of the first initiating NTP. During the following so-called elongation phase, this nucleotidyl transfer reaction is repeated with subsequent NTPs to generate the complementary RNA product [3–6].
Based on the structure of HCV RdRp solved in complex with NTPs , several GTP and NTP binding sites have been proposed. One is located behind the thumb, in a pocket on the surface of the structure, and has been called the allosteric (or surface) GTP binding site. The second one is in the catalytic cavity, where NTP can bind at various sites called P (priming), C (catalytic), and I (interrogating). Recently, the crystal structure of the RdRp of Bovine viral diarrhea virus (BVDV) has been published . Another GTP binding site was found in the catalytic site, distinct from the P, C, and I sites of HCV NS5B. In the latter structure, this site corresponds to a cavity filled with water.
BVDV and HCV polymerases share a similar fold (Figure 1), but exhibit differences in the fingers and thumb subdomains due to differences in the number of secondary structure elements. As for the HCV polymerase, the shape of the BVDV polymerase is a semi-closed right hand made of fingers, palm, and thumb. Fingers are made of eleven β-strands, and twelve α-helices. The palm domain shows great conservation with the HCV palm domain. It consists of four strands forming a central β-sheet surrounded by three α-helices. The thumb contains height α-helices and five β-strands. The flap is lacking in BVDV RNA polymerase although Choi & et al  proposed that two β-strands with their connecting loops play the same role.
A number of structural differences in the flap and other subdomains raise the question of the relevance of BVDV as a surrogate model to discover HCV RNA polymerase inhibitors. Few years ago, "GB" viruses were identified and characterized as Flaviviridae agents leading to hepatitis  but not belonging to hepacivirus. Previous phylogenetic studies of GBV viruses were based on NS3 sequence comparisons . Out of the three GB viruses identified so far, namely GBV-A, -B, and -C, two of them (GBV-A and GBV-B) are most likely monkey viruses while GBV-C can infect humans. HCV and GB virus genomes are organized in a similar way [13, 14]. This similarity has been extended to the functional level with the characterization of the polymerase activity carried out by NS5B [15, 16]. GBV-C virus allows a productive infection of cultured cells, that makes it a relevant alternate virus to be used as a model for HCV antiviral drug screening. In this study, we show using a NS5B-based phylogenetic analysis that GB viruses indeed carry the closest known RdRp to HCV in Flaviviridae. We have built a structural model for the GBV-C polymerase, which allows comparative analysis with HCV, and BVDV polymerase. Results presented in this paper suggest a novel model for the initiation of RNA synthesis in Flaviviridae. Due to its phylogenetic closeness to HCV, GBV-C might be an alternate and more relevant surrogate viral system than BVDV to HCV. Finally, the GBV-C polymerase model proposed in this study might help drug discovery and guide the characterization of the RNA polymerization mechanism.
Results and Discussion
Sequence analysis and phylogenic distribution
A listing of Flaviviridae. Viruses used in the study, together with their correspondent VaZyMolO and NCBI accession numbers.
Data Base Accession
NCBI Acc Protein
Dengue virus type 2
Omsk hemorrhagic fever virus [Bogoluvovska]
West Nile virus
Kamiti River virus – isolate SR-82
Yokose virus [Oita 36]
Hepatitis C virus type 1a – isolate H77
Murray Valley encephalitis virus
Japanese encephalitis virus
Pestivirus type 1 [NADL]
Cell fusing agent virus
Yellow fever virus [Flavivirus (mosquito-borne)]
Hepatitis GB virus B
Bovine viral diarrhea virus genotype 2 [C413]
Pestivirus type 3 [X818; Clover Lane]
Tick-borne encephalitis virus
Powassan virus [LB]
Hepatitis GB virus C
Pestivirus type 2 [Eystrup]
Langat virus [TP21]
Louping ill virus [369/T2]
Deer tick virus [ctb30] – isolate CT95
Tamana bat virus
Hepatitis GB virus A
Modoc virus [M544]
Montana myotis leukoencephalitis virus
Rio Bravo virus [RiMAR]
Alkhurma virus 
Apoi virus [ApMAR]
Pestivirus – isolate reindeer-1 V60-Krefeld
Pestivirus – isolate giraffe-1 H138
Homology modeling of the GBV-C Virus RNA Polymerase
Quality of the model. A: Parameters reflecting the quality of the model checked by «WHAT IF» . B: Quality of chain of the model. The model is verified at 2Å resolution. Parameter values in the table represent observed values for the GBV-C polymerase model compared with typical values obtained for well refined structures at the same resolution .
1st generation packing quality
2nd generation packing quality
Ramachandran plot appearance
chi-1/chi-2 rotamer normality
RMS Z-scores, should be close to 1.0:
Omega angle restraints
N° of data
N° of bandwidth
Stereochemistry of main-chain
Percentage residues in A, B, L
Omega angle S.D
Bad contacts 100 residues
Zeta angle S.D.
Hydrogen bond energy S.D.
Stereochemistry of side-chain
Chi-1 gauche minus S.D.
Chi-1 trans S.D.
Chi-1 gauche plus S.D.
Chi-1 pooled S.D.
Chi-2 trans S.D.
As expected with such good scores, the model of the GBV-C polymerase is similar to that of HCV, and displays the essential features of the typical RNA dependent RNA polymerase fold (Figure 4A and 4B). However, we note two small differences between the HCV structure and the GBV-C model. First, Cys 283 and Cys 308 are spatially close enough to model a disulphide bridge (Figure 3 and additional file 3). This bond connects the fingers and the palm, and may stabilize the protein. Second, the superimposition of the GBV-C model and the HCV structure (Figure 4C) shows little but notable differences in the palm and thumb. The secondary structure elements are conserved in place and type, but they are shorter in the model than in the structure. These secondary structure elements should have similar functions, though. For example His 428 overlaps Tyr 448 of the HCV flap (Figure 4D) and replacement of the aromatic ring of the tyrosine by the histidine ring could play the same role during initiation (see discussion below).
In the HCV polymerase, the allosteric site forms a pocket where GTP binds. Such a pocket does exist in GBV-C despite sequence variability (Figure 3), and is located behind the thumb subdomain. The surface analysis shows that the pocket has a hydrophobic nature, except for the side chains of Asp 30 and Lys 473 that may however participate in the binding of a GTP molecule (see below).
In the HCV structure, several NTP molecules can bind to the catalytic site at P, C, and I sites. Indeed, up to 9 phosphate moieties can be seen in the crystal structure. Only the nucleotide bound at the C site is well defined, although its nucleobase is probably incorrectly located in the absence of the RNA template . Clearly, a better definition of nucleotides and template is needed to understand the RNA synthesis process. On the other hand, the BVDV polymerase structure in complex with GTP in the catalytic cavity suggests a role for this nucleotide in the initiation of RNA synthesis, as proposed below.
Docking of GTP in GBV-C
The recently published high-resolution three-dimensional structure of BVDV and HCV polymerase has allowed the structural comparison of the two polymerases. Major differences in fingers and thumb suggest that molecular interactions during the initiation mechanism are different. BVDV has been used as a model in the study of hepaciviruses. However, phylogenic analysis shows that GBV-C is more closely related to HCV than BVDV. We propose here a reliable model of the GBV-C polymerase structure.
The model of the GBV-C polymerase is poorly defined in loopy regions where most of the gaps have been introduced. Despite this imprecision, the very good scores of the structural indicators make us very confident of the reliability of our model. Moreover, the model is consistent with the known three-dimensional structure of RNA dependent RNA polymerases, and show conservation of all structural elements involved in polymerization (catalytic site, RNA positive channel, NTP tunnel). As expected after the alignment and prediction study, the GBV-C model is very close to the HCV structure, even with a conserved allosteric GTP binding site. Based on the BVDV polymerase/GTP complex structure, we generated a model of a corresponding complex of GBV-C. We propose a role for the GTP molecule bound at a site involved in the initiation of RNA synthesis. Our study provides useful information of the location of residues involved in the polymerization process and hence presents a useful resource for future biochemical analysis and drug discovery.
The sequences related to the different kind of polymerase were retrieved with a PSI-BLAST  with standard parameters from the public available protein database Swiss-Prot , Protein Data Bank (PDB)  and VaZyMolO . For this study we have used different structures of HCV (PDB code: [1GX5, 1GX6]), and BVDV (PDB code: [1S48, 1S49]).
Sequence alignment comparison
Alignment of representative sequences from several members of Flaviviridae were performed using CLUSTALW  with the following parameter. Slow Algorithm, Identity matrix for pairwise alignment and BLOSUM series matrix for multiple alignments. The alignment was then carefully analyzed and optimized with SEAVIEW , taking into account the secondary structure prediction and structural elements when existing. This alignment was cross checked using 3DJURY .
The secondary structure predictions were carried out using JPRED2 , PSI-PRED  and PREDICT-PROTEIN Server . We used PREDICT-PROTEIN with a window of 150 amino acids in order to increase the sensitivity of the prediction. 20 amino acids overlap with each common superimposed window. The results presented are consensus. Sequence alignment with structural information (structure or predictions) and the comparison of the structure one dimension of the known viral polymerases was performed using ESPript 2.0  and ENDscript 1.0 .
To visualize conserved region in amino acids composition on the reference structure, we used BOBSCRIPT . The similarity scores were calculated from the CLUSTALW  alignment and they are shown on this structure with a white (low score) to red (identity) color ramp.
The sampling variance of the distance values was estimated from 1000 bootstrap resamplings of the alignment columns. The evolutionary inference was performed according to the Neighbor-joining method. Multiple runs were conducted with randomized sequence input order to avoid the tree being caught in a local statistical minimum. The tree was generated using Phylodendron (©1997 Gilbert).
Model building, refinement and evaluation
The resulting multiple sequence alignment with the consensus secondary structure prediction was used as template to generate the threading alignment. The derived pairwise alignment serves as reference for preparing the file for the model. SWISS-PDB VIEWER  was used to generate a first threading model. The three dimensional model of the GBV-C RdRp was constructed using the crystal structure coordinates of the HCV polymerase [8, 7] (PDB code: 1GX5, 1QUV). Main gaps appear in loops and smaller ones in helices. This alignment and the threading model serve as a template file for SWISS-MODEL . The non-modeled loops were manually built after scanning the loop database. The model was then minimized with a cut off of 10 Å with 40 cycles of steepest descent until the gradient fell below 10 Kcal/mol and 20 cycles of conjugate gradient. The computations were done in vacuum with using GROMOS 96 [42, 43] force field. To generate alternate models, we have used the 3D-JIGSAW [23–25] server, SCRWL  and MODELLER . In this latter, positions of predicted catalytic residues and secondary structure elements were used as spatial restraints.
Docking GTP molecule in GBV-C
The 3D model of the GBV-C RNA polymerase was used as a target for the docking of GTP. We first superimposed the structure of BVDV RNA polymerase/GTP complex (PDB code 1S49) with our 3D model. This step was performed with the program Turbo-Frodo . A docking study was performed to explore the presence or absence of a GTP binding pocket like, as it was described in the BVDV polymerase structure. For the docking procedure, the program AUTODOCK 3.0.5  was used with a grid spacing of 0.375 Å and 40 × 40 × 40 number of points. The grid was centered on the mass center of the GTP molecule. The GA-LS method was adopted using the default settings. Amber united atoms were assigned to the protein using the program AUTODOCK TOOLS. 250 possible binding conformations were generated. The results of AUTODOCK run were clustered using a RMSD tolerance of 1.0 Å. We considered the structure of the first cluster. To validate the use of the AUTODOCK program, the docking study was performed on the BVDV polymerase with GTP as a reference. This program successfully reproduced the experimental binding conformation with acceptable root-mean-square deviation (RMSD) of atom coordinates. Finally, the interaction models of GTP with the binding pocket were produced using the LIGPLOT program .
Table 1 – A listing of Flaviviridae
Viruses used in the study, together with their correspondent VaZyMolO and NCBI accession numbers.
Table 2 – Quality of the model
A: Parameters reflecting the quality of the model checked by « WHAT IF » .
B: Quality of chain of the model. The model is verified at 2Å resolution. Parameter values in the table represent observed values for the GBV-C polymerase model compared with typical values obtained for well refined structures at the same resolution .
The authors thanks Dr. Barbara Selisko, Dr. Sonia Longhi, Dr Yana Khalina and Jean-Marie Bourhis for critical reading of the manuscript. This work was support by Association Nationale de Recherche sur le Sida (ANRS), by the European Community (Flavitherapeutics European Contract N° QLK3-CT-2001-00506) and by the Centre National de Recherche Scientifique (CNRS).
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