Both amino acid changes in nsP1 of Sindbis virusLM21 contribute to and are required for efficient expression of the mutant phenotype

SVLM21 is a mutant of Sindbis virus which in contrast to the standard virus, SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. Previously, by making use of the infectious Toto plasmids, we had constructed recombinant viruses containing various SVLM21 sequences, and were thereby able to map the mutations associated with the SVLM21 phenotype to the gene for the nonstructural protein nsP1. Two mutations were found in the nsP1 gene of SVLM21. These led to predicted amino acid changes at residue 87 from Arg to Leu, and at residue 88 from Ser to Cys. In the work presented here, we assess the relative contributions of these two mutations to the SVLM21 phenotype using site-directed mutagenesis to obtain virus encoding only the change to Leu at residue 87 of nsP1 (SVMS319), or only the change to Cys at residue 88 (SVMS321). In addition we show that SVLM10, which was isolated during the selection procedure for SVLM21, encodes only the change at residue 88. In addition to its ability to grow in methionine-deprived mosquito cells, SVLM21 differs from SVSTD in two other respects: (1) it shows an increased sensitivity to neplanocin A (NPA) and (2) it generates increased levels of methyltransferase in infected cells. Whether we looked at resistance to low methionine, sensitivity to NPA, or levels of methyltransferase generated, SVMS319, SVMS321, and SVLM10 all expressed only a partial SVLM21 phenotype. Furthermore we were not able in these experiments to distinguish between these three viruses. We conclude therefore that both amino acid changes, i.e., at residues 87 and 88, are required to produce the full SVLM21 phenotype, and that both changes contribute equally.     

SVLM21, a Sindbis virus mutant resistant to methionine deprivation, encodes an altered methyltransferase

The replication of Sindbis virus (SVSTD) in cultured Aedes albopictus mosquito cells is sensitive to methionine deprivation. We have suggested from earlier work that this sensitivity is primarily because of a decreased pool of S-adenosyl methionine (ado met) and the resultant failure to methylate the 5' cap of the viral mRNAs. SVLM21, a strain of Sindbis virus derived in our laboratory from SVSTD by serial passage on mosquito cells maintained after infection in low concentrations of methionine, is resistant to methionine starvation. It was proposed that this adaptation to low methionine, and to the resultant low intracellular levels of ado met, reflected the accumulation of mutations which led to the generation of a viral RNA cap methyltransferase with an increased affinity for ado met. We report here kinetic data which distinguished the enzymes coded for by SVSTD and SVLM21. Using guanylylimidodiphosphate (GIDP) as the methyl acceptor, radioactively labeled ado met as the methyl donor, and lysates from infected BHK cells as the enzyme source, we calculated from our results that SVLM21 generated a methyltransferase with a Km for ado met 10-fold lower than that generated by either SVSTD or the related alphavirus, Semliki Forest virus. In addition, we found that BHK cells infected with SVLM21 generated higher levels of methyltransferase activity than did cells infected with SVSTD and that the SVSTD and SVLM21 enzymes differed with respect to their relative activities at elevated temperatures. We conclude from these results that the SVLM21 phenotype is associated with an altered methyltransferase and suggest that this is the basis of the resistance of SVLM21 to methionine deprivation.     

Association of the sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1

SVLM21 is a mutant of Sindbis virus, which in contrast to SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. We have obtained evidence that the basis of this low methionine-resistance (LMR) phenotype is the generation of an altered RNA methyltransferase with an increased affinity for S-adenosylmethionine (ado met). We now report that following the substitution of the nucleotide sequence, 126-504, from SVLM21 cDNA for the corresponding sequence of the Toto 1101 plasmid (infectious Sindbis viral RNA can be transcribed from this plasmid) we were able to generate recombinant Sindbis virus (SVMS-65a) with the LMR phenotype. (SVTOTO virus derived from Toto 1101, like SVSTD, lacks the LMR phenotype.) As was the case with SVLM21, SVMS-65a not only possessed the LMR phenotype but also showed an increased sensitivity to Neplanocin A, a potent inhibitor of S-adenosylhomocysteine (ado hcy) hydrolase. Sequencing of the nucleotide 126-504 region from SVLM21 revealed two mutations; these mutations occurred in adjacent codons and lead to two predicted amino acid changes in the SV nsPl protein; at residue 87, from Arg to Leu, and at residue 88 from Ser to Cys. Since the nucleotide sequence 126-504 lies entirely within the gene for nsP1, we conclude that the RNA methyltransferase activity generated by SV is associated with nsP1. We suggest that residues 87 and 88 in nsP1, where the amino acid changes in SVLM21 nsP1 have occurred, are at or near the binding site for ado met; we also suggest that these changes in nsP1 are responsible for the increased affinity of the SVLM21 RNA methyltransferase for ado met and thereby for the LMR phenotype. Alternatively, it is possible that the binding site for ado met is elsewhere on nsP1 or even on another protein, and that the changes at residues 87 and 88 lead to an alteration of the binding site.     

Expression of Sindbis virus nsP1 and methyltransferase activity in Escherichia coli.

We have constructed two plasmids, pSR5-42 and pSR5-Toto, which under lac control expressed the SVLM21 and the SVToto forms, respectively, of the Sindbis virus nonstructural protein, nsP1. The induced protein, which was the major protein made following induction with IPTG, had an apparent molecular weight of 60,000 and an amino terminal sequence in agreement with that expected for nsP1. Following induction with IPTG, cells carrying pSR5-42 (which contains the SVLM21 gene sequence) generated much higher RNA methyltransferase activity than cells carrying pSR5-Toto (which contains the SVToto gene sequence). This result is in agreement with what is observed when methyltransferase is measured in cells infected with SVLM21 and SVSTD (or SVToto), respectively. These results provide strong evidence that nsP1 has methyltransferase activity in the absence of any other viral nonstructural proteins.     

Sequence analysis of the E2 gene of a hyperglycosylated, host restricted mutant of Sindbis virus and estimation of mutation rate from frequency of revertants

SVap15/21, a strain of Sindbis virus (SV) derived from our standard laboratory strain of SV (SVstd) after repeated passage on Aedes albopictus cells, grows normally in mosquito cells but is host restricted (hr) in vertebrate cells. It is also temperature sensitive (ts) and produces pinpoint plaques on vertebrate cells (sp). E2 glycoprotein of SVstd differs from that of the more widely used SVHR (from which SVstd was derived) by an additional (i.e., third) N-linked glycan. The E2 of SVap15/21, in turn, differs from that of SVstd by the addition of a fourth glycan. We have determined the nucleotide sequence of the E2 genes of SVap15/21 and of SVstd, as well as that of our isolate of SVHR. The nucleotide sequence of the SVstd E2 gene predicted the occurrence of an additional N-linked glycan attachment site, not present in the SVHR E2, at Asn232 (Asp in SVHR). The sequence of the SVap15/21 E2 gene demonstrated three mutations relative to the SVstd gene, including one that predicted the occurrence of another potential N-linked glycan attachment site at Asn275. Sequence analysis of 15 revertants of SVap15/21 which are no longer host-restricted revealed that all had lost the glycosylation site at Asn275, confirming the connection between the hyperglycosylation and the host dependent block in assembly. Most of these revertants had also lost the temperature sensitivity and small plaque traits (i.e., were ts+ and lp). Each revertant of this class was characterized by one of three different mutations in two separate codons (Asn275 and Thr277), resulting in the loss of the glycosylation site at Asn275. A fourth mutation, resulting in an Asn275----Tyr substitution, was associated with a hr+ ts phenotype in three isolates. Finally an additional mutation in a different part of the E2 gene was found in two hr+ ts sp isolates that had also lost the glycosylation site at Asn275 through a mutation resulting in a Thr277----Ile substitution. Knowledge of the nucleotide sequences associated with the ts defect in SVap15/21 and with its reversion permit an estimation of the mutation rate of this virus. This calculation indicates a mutation rate of less than 10(-6) errors per base incorporation.     

Intracellular S-adenosylhomocysteine increased levels are associated with DNA hypomethylation in HUVEC

Hyperhomocysteinemia is a risk factor for atherosclerosis and vascular disease; however, the mechanism underlying this association remains poorly understood. Increased levels of intracellular S-adenosylhomocysteine (AdoHcy), secondary to homocysteine-mediated reversal of the AdoHcy hydrolase reaction, have been associated with reduced DNA methylation patterns and pointed as responsible for the hyperhomocysteinemia-related endothelial dysfunction. Methylation is an epigenetic feature of genomic DNA, which leads to alterations in gene expression. So far, the effect of intracellular AdoHcy accumulation on DNA methylation patterns has not yet been fully substantiated by experimental evidence. The present study was designed to evaluate, in cultured endothelial cells, the effect of AdoHcy accumulation on genomic global DNA methylation status. Experimental intracellular accumulation of AdoHcy was induced by adenosine-2,3-dialdehyde (ADA), an inhibitor of AdoHcy hydrolase. Increased concentrations of inhibitor were tested, and unsupplemented medium incubations were used as controls. Cytosolic and nuclear fractions were obtained from trypsinized cells after 72 h of incubation. Total homocysteine concentration was quantified (culture medium and cytosolic fractions) by high-performance liquid chromatography (HPLC). S-Adenosylmethionine and AdoHcy concentrations were measured (cytosolic fractions) by stable-isotope dilution LC-tandem mass spectrometry method. Genomic DNA was obtained from the nuclear fraction, and global DNA methylation status was evaluated by the cytosine extension assay. The results showed that supplementation of the culture medium with ADA had no cytotoxic effect and increased the intracellular AdoHcy concentration in a dose-dependent manner. A significant negative correlation was observed between intracellular AdoHcy and genomic DNA methylation status. These findings strongly point to the importance of AdoHcy as a pivotal biomarker of genomic DNA methylation status.     

Comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children.

BACKGROUND: The quality of clinical specimens is a crucial determinant for virological diagnosis. OBJECTIVES: We compared the viral diagnostic yield for influenza A and respiratory syncytial virus (RSV) from the recently developed nasopharyngeal flocked swabs (NPFS) with nasopharyngeal aspirates (NPA) collected in parallel from 196 hospitalized children with acute respiratory infection during the peak period of influenza A and RSV activity in Hong Kong. Specimens were tested by RT-PCR for influenza A and RSV and viral load determined. They were also tested by direct immunofluorescence (DIF) for influenza A and B, RSV, parainfluenza types 1-3 and adenovirus. RESULTS: Both NPA and NPFS had excellent sensitivity (100%) for detecting influenza A by RT-PCR but NPA was slightly more sensitive than NPFS for detecting RSV by both RT-PCR (100% vs. 92.3%) and DIF (87.2% vs. 84.6%) and for detecting influenza A by DIF (90.2% vs. 82.9%). Viral load for influenza A in NPA and NPFS was not significantly different but that for RSV was higher in NPA. CONCLUSION: NPA remains the optimal specimen for diagnosis of respiratory infections by RT-PCR and DIF. However, collection of NPFS is easier to perform in an out-patient setting, was more acceptable to parents and less likely to generate aerosols than NPA engendering potentially less infection control hazard.     

Comparison of nasopharyngeal aspirate and nasopharyngeal swab specimens for respiratory syncytial virus diagnosis by cell culture, indirect immunofluorescence assay, and enzyme-linked immunosorbent assay.

Paired nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens obtained from each of 32 hospitalized infants with X-ray-confirmed pneumonia (91%) or bronchiolitis were tested for respiratory syncytial virus (RSV) infection by virus culture, the indirect immunofluorescent-antibody (IFA) technique, enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostic Systems, Inc.), and spot hybridization with a human genomic probe to quantitate cellular DNA. RSV was isolated in cell cultures from 72% (23 of 32) of patients by using NPA specimens compared with 47% (15 of 32) by using NPS specimens. With tissue culture positivity as the reference test, the sensitivities of the ELISA on NPA and NPS specimens were found to be 69% (16 of 23) and 61% (14 of 23), respectively, with a specificity and a positive predictive value from both sites of 100%. The sensitivities of the IFA technique compared with the cell culture on NPA and NPS specimens were 61% (14 of 23) and 52% (12 of 23) with specificities of 89 and 78% and positive predictive values of 96 and 92%, respectively. Despite the recovery of significantly more cells (as shown by detection of more cellular DNA by using NPA specimens), virus was detected by the IFA technique or ELISA at similar frequencies in paired specimens. However, virus was recovered more often from NPA than NPS specimens by cell culture, and ELISA optical density readings and the number of RSV-positive fluorescing cells were greater for NPA specimens. NPA specimen collection was less traumatic for the patient, was an easier procedure for the physician to perform, and provided a superior laboratory specimen for RSV diagnosis than the NPS technique.     

Respiratory virus detection in nasopharyngeal aspirate versus bronchoalveolar lavage is dependent on virus type in children with chronic respiratory symptoms

BackgroundThe comparative yield of respiratory virus detection from nasopharyngeal aspirate (NPA) versus bronchoalveolar lavage (BAL) is uncertain. Furthermore, the significance of virus detection and its relationship to lower airway neutrophilic inflammation is poorly studied.ObjectivesTo evaluate the sensitivity, specificity and predictive values of NPA for detecting respiratory viruses in BAL; and to determine the relationship between viruses and lower airway neutrophilia in children with non-acute respiratory illness.Study design150 paired NPA and BAL samples were obtained from 75 children aged <18 years undergoing flexible bronchoscopy for investigation of chronic respiratory symptoms. Viral studies were performed using polymerase chain reaction (PCR). Cellularity studies were performed on BALs. Diagnostic parameters of NPA compared to BAL and associations between viruses and lower airway %neutrophils were evaluated.ResultsNPA had a higher yield than BAL for detection of any respiratory virus (52 versus 38, respectively). NPA had a high sensitivity (92%) and low specificity (57%) for detecting HRV in BAL with poor kappa agreement value of 0.398 (95% CI 0.218–0.578, p < 0.001). NPA had a fair sensitivity (69%) and good specificity (90.3%) for detecting HAdV on BAL, kappa agreement was 0.561 (95% CI 0.321–0.801, p < 0.001). HAdV positivity on NPA, compared to negativity, was independently associated with heightened airway neutrophilia [mean difference (95% CI): 18 (1,35); p = 0.042].ConclusionsNPA has a higher yield for respiratory virus detection than BAL, however its diagnostic accuracy is dependent on viral species. Adenovirus positivity is associated with significantly heightened lower airway neutrophilia in children with chronic respiratory symptoms.     

Most human metapneumovirus and human respiratory syncytial virus in infant nasal secretions is cell free.

BACKGROUND: Nasopharyngeal secretions aspirated from infants with bronchiolitis (NPA) are a valuable resource for the study of virus dynamics and local inflammatory responses, however samples are small and difficult to manipulate. OBJECTIVES: To improve yield of NPA from infants. To establish if removal of the cellular component of NPA affects quantification of human metapneumovirus (hMPV) or human respiratory syncytial virus (hRSV) genome. STUDY DESIGN: Weight of NPA collected into traps from 30 infants was compared with that collected in trap plus catheter and washed through with saline from another 30 infants. hMPV (n=33) and hRSV (n=30) genome was measured by reverse-transcribed real-time polymerase chain reaction (RT-RT-PCR) in paired whole and cell-free NPA collected by the improved method. RESULTS: The improved method of NPA collection gave near two-fold greater weight (p = 0.002) of NPA (mean = 0.52 g (S.D. = 0.30 g)) than the traditional method (0.32 g (S.D. 0.19)). There was strong agreement and no significant difference between viral load measured in whole and cell-free fractions of NPA for both viruses (samples (n), correlation coefficient (cc) and significance (p)); hMPV (n=33, cc=0.938, p<0.001) and hRSV (n=30, cc=0.977 and p<0.001). CONCLUSIONS: The majority of hRSV and hMPV in nasal secretions is not associated with cells. Removal of the cellular component of NPA does not interfere with quantification of hRSV and hMPV.     

A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2

Following serial passage of Sindbis virus (SV) on Aedes albopictus mosquito cells a mutant (SVap15/21) was isolated which in chick cells produced small plaques and was temperature sensitive (ts). At 34.5 degrees this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SVap15/21 was RNA+ at both 34.5 and 40 degrees and on the basis of complementation tests carried out at 40 degrees, was assigned to complementation group E. The block in the replication of this mutant, like that of ts20, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SVap15/21 were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SVstd). Analysis of revertants of SVap15/21 suggests a causal relationship between PE2 and E2 hyperglycosylation and the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range.     

The total serum homocysteine as an indicator of vitamin B12 and folate status

Presented is a modification of an assay for total serum homocysteine (Hcy) in which the Hcy plus radioactive adenosine is converted enzymatically to labeled S-adenosylhomocysteine (AdoHcy). The modifications included a commerical source for the AdoHcy hydrolase, adenosine labeled with either 14C or 3H, and separation of the AdoHcy by thin layer chromatography. The assay was sensitive to 25 pmol. Hcy levels in sera from 18 controls ranged from 6.9 to 12.1 mumol/L with a mean of 9.1 and a SD of 1.5 mumol/L. The total serum Hcy was increased in vitamin B12 and folate deficiency. The level was high in congenital defects of vitamin B12 metabolism, blocking the methylation of Hcy regardless of the serum vitamin B12 levels, but was normal in the absence of tissue deficiency even if the serum vitamin B12 levels were low. The procedure has been found practical in two years of use and requires only 0.1 mL of serum.     

Comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection

Paired nasopharyngeal aspirate (NPA) and nasal swab (NS) samples from 475 children hospitalized for acute respiratory infection were studied for the detection of influenza virus, parainfluenza virus, respiratory syncytial virus, and adenovirus by immunofluorescence test, viral culture, and multiplex PCR assay. The overall sensitivity of viral detection with NPA specimens was higher than that obtained with NS specimens.     

Practical implementation of a multiplex PCR for acute respiratory tract infections in children

Molecular testing for acute respiratory infections (ARIs) has documented value but limited implementation due to questions that typically slow the acceptance of new tests. This study sought to address these questions and achieve implementation. Rhinovirus was added to a nested multiplex PCR (M-PCR), increasing its diagnostic yield. Over one winter, three hospital pediatric departments used the M-PCR to complement their direct fluorescent-antibody assay (DFA) for respiratory syncytial virus (RSV). Clinicians recorded "pretest probability estimates" (using continuous scales for various pathogen groups) for comparison with test results; treatments and test turnaround times were also recorded. Transnasal and throat swabs, with or without nasopharyngeal aspirate (NPA), were M-PCR tested. NPA-containing sample sets found to be RSV positive by DFA were not further tested. Single PCR for human metapneumovirus (hMPV) was performed retrospectively. Of 178 ARI episodes representing 172 patients, NPA was included in 97 sample sets; 54 (56%) were determined to be RSV positive. The other NPA-containing sample sets (n = 43) yielded 27 findings (63%), and the swab-only sets (n = 81) yielded 47 findings (58%); rhinovirus was found most often. Testing for hMPV yielded seven positive results. M-PCR median turnaround times were 4 days in swab-only samples and 5 days with NPA. Antibiotics were prescribed in 50 episodes, at rates similar for RSV and rhinovirus. Pretest probability estimates of a viral cause were lower in episodes caused by rhinovirus than in episodes caused by RSV. The hospitals continued to use M-PCR for NPA-containing samples found to be RSV negative by DFA. Test implementation is more likely with higher diagnostic yield and a protocol that reflects day-to-day clinical and laboratory operations.     

Modulation of tumor necrosis factor-alpha-mediated cytotoxicity by changes of the cellular methylation state: mechanism and in vivo relevance.

A combination of adenosine (Ado) and homocysteine (Homo) enhances tumor necrosis factor (TNF)-alpha cytotoxicity in vitro and in vivo in several tumor cells. Ado and Homo at concentrations that enhanced TNF-alpha-mediated cytotoxicity accumulated S-adenosylhomocysteine (AdoHcy) and as consequence decreased the cellular methylation state, i.e. the ratio of S-adenosylmethionine to AdoHcy. This decrease led to inhibition of the isoprenylcysteine carboxyl methyltransferase (MTase), an enzyme that catalyzes carboxyl methylation of C-terminal cysteine residues on isoprenylated proteins. The effect of Ado and Homo on TNF-alpha cytotoxicity was at least partly mimicked by S-farnesylthioacetic acid, a selective inhibitor of the isoprenylcysteine carboxyl MTase, suggesting involvement of methylations of prenylated proteins in TNF-alpha-mediated cytotoxicity. Blockage of methylation reactions was associated with an enhancement of the TNF-alpha-induced disruption of the mitochondrial membrane potential (delta psi(m)). In nude mice, a combination of Ado, Homo and TNF-alpha led to TNF-alpha-induced hemorrhagic necrosis and growth inhibition of TNF-sensitive L929 tumors, whereas little effect was observed with TNF-alpha alone. Even more important, the TNF-resistant L929 M1 tumors were rendered TNF-sensitive by the combined action of Ado and Homo. We conclude that Ado and Homo together enhance the effectiveness of TNF-alpha in vitro and in vivo, results that may have therapeutic implications.     

Effect of genetic variation in the human S-adenosylhomocysteine hydrolase gene on total homocysteine concentrations and risk of recurrent venous thrombosis

Hyperhomocysteinemia is an independent and graded risk factor for arterial vascular disease and venous thrombosis. It is still debated via which mechanism homocysteine (Hcy) causes vascular disease. S-adenosylhomocysteine hydrolase (AHCY) catalyses the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to Hcy. As an increase in AdoHcy, a strong inhibitor of many methyltransferases, is observed in hyperhomocysteinemic individuals, AdoHcy may play a role in the development of cardiovascular diseases by inhibiting transmethylation reactions. We sequenced the entire coding region and parts of the untranslated regions (UTRs) of the AHCY gene of 20 patients with recurrent venous thrombosis in order to identify genetic variation within this gene. We identified three sequence variants in the AHCY gene: a C > T transition in the 5' UTR (-34 bp C > T), a missense mutation in exon 2, which mandates an amino-acid conversion at codon 38 (112 C > T; Arg38Trp) and a silent mutation in exon 4 (390 C > T; Asp130Asp). We studied the effect of the first two variants on total plasma Hcy and venous thrombosis risk in a case-control study on recurrent venous thrombosis. The two polymorphisms under study seem to have no evident effect on tHcy. The adjusted relative risk of venous thrombosis associated with the 112CT genotype compared with 112CC individuals was 1.27 (95% CI 0.55-2.94), whereas the -34CT genotype confers a risk of 1.25 (95% CI 0.44-3.52) compared with the wild-type genotype at this locus. However, the wide confidence intervals do not allow firm conclusions to be drawn.     

Methyltransferase Activity of the Insect Orbivirus JKT-7400: Photoaffinity Labeling of a Minor Virion Protein,VP4, withS-Adenosylmethionine

JKT-7400 virus is an orbivirus originally isolated fromCulexmosquitoes. In earlier work we had described the viral structural proteins and presented evidence suggesting that a minor protein, VP6, located in the viral core was the viral guanylyltransferase. We now show that gradient-purified JKT-7400 virions possess a methyltransferase (MTase) activity which can use GTP or GDP as the methyl acceptor. The apparentK_mof the MTase forS-adenosylmethionine (AdoMet) was 25 μM.Photoaffinity labeling experiments in which³H-[methyl]-AdoMet was incubated with virions or viral cores demonstrated labeling of VP4, a minor protein present in the viral core, suggesting that this protein is the viral MTase. Labeling of VP4 was inhibited by addition of unlabeled AdoMet orS-adenosylhomocysteine (AdoHcy).     

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product formation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation.