Nonstructural Proteins NS4B Research Articles

Prediction of drug-binding sites in HCV NS5B polymerase: MD simulation study

Hepatitis C virus (HCV) is a small, single-stranded, positive-sense ribonucleic acid (RNA) virus and is a member of the Flaviviridae family of viruses. Its genome has a length of 9.6 kb and encoding a polyprotein of about 3000 amino acids which is cleaved co- and post-translationally by cellular and viral proteases into 10 different products: core, envelope protein E1, E2, p7, nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. Core, E1 and E2 are the main viral constituents of the HCV particle. NS5B of the hepatitis C virus, a RNA-dependent RNA polymerase, is an important therapeutic target. In order to gain deeper insight into and to know its molecular mechanism, the authors employed molecular dynamics simulation at the nanosecond scale. Initial coordinates were taken from the protein data bank entries with IDs 3IGV and 3GYN. The present study shows the strong binding affinity of the inhibitor with NS5B polymerase. The binding free energies, calculated by the molecular mechanics and Poisson–Boltzmann surface area/generalized Born surface area method, suggest a good connection with the experimental biological activity. The analysis of the combined simulation results has identified that Tyr448, Ser556 and Asp318 are the key amino acid residues in NS5B binding. The results obtained from this study provide some insights into the development of novel strong NS5B inhibitors.

Virulence determinants of West Nile virus: how can these be used for vaccine design?

West Nile virus (WNV), a neurotropic mosquito-borne flavivirus, has become endemic in the USA and parts of Europe since 1999. There is no licensed WNV vaccine for humans. Considering the robust immunity from immunization with live, attenuated vaccines, a live WNV vaccine is an ideal platform for disease control. Animal and mosquito studies have identified a number of candidate attenuating mutations, including the structural proteins premembrane/membrane and envelope, and the nonstructural proteins NS1, NS2A, NS3, NS4A, NS4B and NS5, and the 3' UTR. Many of the mutations that have been examined attenuate WNV using different mechanisms, thus providing a greater understanding of WNV virulence while also identifying specific mutations as candidates to include in a WNV live vaccine.

Zika virus evades interferon-mediated antiviral response through the co-operation of multiple nonstructural proteins in vitro

Type I interferon (IFN) serves as the first line of defense against invading pathogens. Inhibition of IFN-triggered signaling cascade by Zika virus (ZIKV) plays a critical role for ZIKV to evade antiviral responses from host cells. Here we demonstrate that ZIKV nonstructural proteins NS1, NS4B and NS2B3 inhibit the induction of IFN and downstream IFN-stimulated genes through diverse strategies. NS1 and NS4B of ZIKV inhibit IFNβ signaling at TANK-binding kinase 1 level, whereas NS2B-NS3 of ZIKV impairs JAK–STAT signaling pathway by degrading Jak1 and reduces virus-induced apoptotic cell death. Furthermore, co-operation of NS1, NS4B and NS2B3 further enhances viral infection by blocking IFN-induced autophagic degradation of NS2B3. Hence, our study reveals a novel antagonistic system employing multiple ZIKV nonstructural proteins in restricting the innate antiviral responses.

Viral infection: Dengue as mitochondrial landscapers.

This study reports that the DENV non-structural protein NS4B induces elongation of mitochondria, and that this is linked to the formation of convoluted membranes, enhanced viral replication and impaired immune responses.

Interaction between Nonstructural Proteins NS4B and NS5A Is Essential for Proper NS5A Localization and Hepatitis C Virus RNA Replication.

The hepatitis C virus NS5A protein is tethered to cellular membranes via an amphipathic amino-terminal helix that is inserted in-plane into the outer endoplasmic reticulum (ER)-derived membrane leaflet. The charged face of the helix faces the cytoplasm and may contribute to interactions involved in replicase assembly and function. Using an aggressive charge flip mutagenesis strategy, we identified a number of essential residues for replication on the charged face of the NS5A anchor and identified a double charge face mutant that is lethal for RNA replication but generates suppressor mutations in the carboxy-terminal helix of the NS4B protein. This suppressor restores RNA replication of the NS5A helix double flip mutant (D1979K/D1982K) and, interestingly, seems to function by restoring the proper localization of NS5A to the viral replicase. These data add to our understanding of the complex organization and assembly of the viral replicase via NS4B-NS5A interactions. Information about the functional role of the cytosolic face of the NS5A anchoring helix remains obscure. In this study, we show that while the hydrophobic face of the NS5A anchor helix mediates membrane association, the polar cytosolic face of the helix plays a key role during hepatitis C virus (HCV) replication by mediating the interaction of NS5A with other HCV nonstructural proteins via NS4B. Such an interaction determines the subcellular localization of NS5A by engaging NS5A in the HCV replication process during the formation of a functional HCV replication complex. Thus, collectively, it can be stated that the findings in the present study provide further information about the interactions between the HCV nonstructural proteins during HCV RNA replication and provide a platform to gain more insights about the molecular architecture of HCV replication complexes.

Prevalence and characteristics of naturally occurring sofosbuvir resistance‐associated variants in patients with hepatitis C virus genotype 1b infection

Sofosbuvir (SOF), a nucleotide analog pro-drug, targets hepatitis C virus (HCV) NS5B polymerase and shows potential for treating HCV infection, given its high efficacy and good barrier to resistance. However, in addition to the rare resistant-associated variant (RAV) of non-structural protein NS5B S282T, several new potential RAVs of SOF have been reported, especially related to HCV genotype 1b. However, the prevalence and characteristics of these RAVs have not been clarified. We analyzed the prevalence of variants in the NS3/NS5A/NS5B regions in 96 patients treated with simeprevir (SMV) combination therapy, and the prevalence of RAVs in patients showing treatment failure was determined by direct- or deep-sequencing methods. Associations between these potential RAVs and clinical factors were also analyzed. Prevalence of NS5B RAV C316N was high (46.9%, 45/96), whereas that of NS5B L159F was relatively low (1.04%, 1/96); however, deep sequencing showed that 30.0% of patients with C316N also had NS5B RAV L159F. Additionally, there was no significant relationship between the existence of potential NS5B and NS5A or NS3 RAVs. However, the presence of NS5B C316N was significantly associated with an HCV core amino acid 91 substitution. No significant difference was detected between each RAV and sustained virological response in simeprevir combination therapy. We provide clear evidence of the high prevalence of two potential naturally occurring NS5B RAVs (C316N and L159F) in Japan. It may be important to pay particular attention to these new potential RAVs, especially when using SOF-based therapy in patients with RAVs due to previous direct-acting antiviral therapy failure.

Logical design of anti-prion agents using NAGARA

To accelerate the logical drug design procedure, we created the program “NAGARA,” a plugin for PyMOL, and applied it to the discovery of small compounds called medical chaperones (MCs) that stabilize the cellular form of a prion protein (PrPC). In NAGARA, we constructed a single platform to unify the docking simulation (DS), free energy calculation by molecular dynamics (MD) simulation, and interfragment interaction energy (IFIE) calculation by quantum chemistry (QC) calculation. NAGARA also enables large-scale parallel computing via a convenient graphical user interface. Here, we demonstrated its performance and its broad applicability from drug discovery to lead optimization with full compatibility with various experimental methods including Western blotting (WB) analysis, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) measurements. Combining DS and WB, we discovered anti-prion activities for two compounds and tegobuvir (TGV), a non-nucleoside non-structural protein NS5B polymerase inhibitor showing activity against hepatitis C virus genotype 1. Binding profiles predicted by MD and QC are consistent with those obtained by SPR and NMR. Free energy analyses showed that these compounds stabilize the PrPC conformation by decreasing the conformational fluctuation of the PrPC. Because TGV has been already approved as a medicine, its extension to prion diseases is straightforward. Finally, we evaluated the affinities of the fragmented regions of TGV using QC and found a clue for its further optimization. By repeating WB, MD, and QC recursively, we were able to obtain the optimum lead structure.

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE- vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE- replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

Dengue Virus NS Proteins Inhibit RIG-I/MAVS Signaling by Blocking TBK1/IRF3 Phosphorylation: Dengue Virus Serotype 1 NS4A Is a Unique Interferon-Regulating Virulence Determinant.

ABSTRACTDengue virus (DENV) replication is inhibited by the prior addition of type I interferon or by RIG-I agonists that elicit RIG-I/MAVS/TBK1/IRF3-dependent protective responses. DENV infection of primary human endothelial cells (ECs) results in a rapid increase in viral titer, which suggests that DENV inhibits replication-restrictive RIG-I/interferon beta (IFN-β) induction pathways within ECs. Our findings demonstrate that DENV serotype 4 (DENV4) nonstructural (NS) proteins NS2A and NS4B inhibited RIG-I-, MDA5-, MAVS-, and TBK1/IKKε-directed IFN-β transcription (>80%) but failed to inhibit IFN-β induction directed by STING or constitutively active IRF3-5D. Expression of NS2A and NS4B dose dependently inhibited the phosphorylation of TBK1 and IRF3, which suggests that they function at the level of TBK1 complex activation. NS2A and NS4B from DENV1/2/4, as well as the West Nile virus NS4B protein, commonly inhibited TBK1 phosphorylation and IFN-β induction. A comparative analysis of NS4A proteins across DENVs demonstrated that DENV1, but not DENV2 or DENV4, NS4A proteins uniquely inhibited TBK1. These findings indicate that DENVs contain conserved (NS2A/NS4B) and DENV1-specific (NS4A) mechanisms for inhibiting RIG-I/TBK1-directed IFN responses. Collectively, our results define DENV NS proteins that restrict IRF3 and IFN responses and thereby facilitate DENV replication and virulence. Unique DENV1-specific NS4A regulation of IFN induction has the potential to be a virulence determinant that contributes to the increased severity of DENV1 infections and the immunodominance of DENV1 responses during tetravalent DENV1-4 vaccination.

Combination of pharmacophore hypothesis and molecular docking to identify novel inhibitors of HCV NS5B polymerase.

Hepatitis C virus (HCV) infection or HCV-related liver diseases are now shown to cause more than 350,000 deaths every year. Adaptability of HCV genome to vary its composition and the existence of multiple strains makes it more difficult to combat the emergence of drug-resistant HCV infections. Among the HCV polyprotein which has both the structural and non-structural regions, the non-structural protein NS5B RNA-dependent RNA polymerase (RdRP) mainly mediates the catalytic role of RNA replication in conjunction with its viral protein machinery as well as host chaperone proteins. Lack of such RNA-dependent RNA polymerase enzyme in host had made it an attractive and hotly pursued target for drug discovery efforts. Recent drug discovery efforts targeting HCV RdRP have seen success with FDA approval for sofosbuvir as a direct-acting antiviral against HCV infection. However, variations in drug-binding sites induce drug resistance, and therefore targeting allosteric sites could delay the emergence of drug resistance. In this study, we focussed on allosteric thumb site II of the non-structural protein NS5B RNA-dependent RNA polymerase and developed a five-feature pharmacophore hypothesis/model which estimated the experimental activity with a strong correlation of 0.971 & 0.944 for training and test sets, respectively. Further, the Güner-Henry score of 0.6 suggests that the model was able to discern the active and inactive compounds and enrich the true positives during a database search. In this study, database search and molecular docking results supported by experimental HCV viral replication inhibition assays suggested ligands with best fitness to the pharmacophore model dock to the key residues involved in thumbs site II, which inhibited the HCV 1b viral replication in sub-micro-molar range.

Amino acid substitutions in the non-structural proteins 4A or 4B modulate the induction of autophagy in West Nile virus infected cells independently of the activation of the unfolded protein response.

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for outbreaks of meningitis and encephalitis. Whereas the activation of autophagy in cells infected with other flaviviruses is well known, the interaction of WNV with the autophagic pathway still remains unclear and there are reports describing opposite findings obtained even analyzing the same viral strain. To clarify this controversy, we first analyzed the induction of autophagic features in cells infected with a panel of WNV strains. WNV was determined to induce autophagy in a strain dependent manner. We observed that all WNV strains or isolates analyzed, except for the WNV NY99 used, upregulated the autophagic pathway in infected cells. Interestingly, a variant derived from this WNV NY99 isolated from a persistently infected mouse increased LC3 modification and aggregation. Genome sequencing of this variant revealed only two non-synonymous nucleotide substitutions when compared to parental NY99 strain. These nucleotide substitutions introduced one amino acid replacement in NS4A and other in NS4B. Using genetically engineered viruses we showed that introduction of only one of these replacements was sufficient to upregulate the autophagic pathway. Thus, in this work we have shown that naturally occurring point mutations in the viral non-structural proteins NS4A and NS4B confer WNV with the ability to induce the hallmarks of autophagy such as LC3 modification and aggregation. Even more, the differences on the induction of an autophagic response observed among WNV variants in infected cells did not correlate with alterations on the activation of the unfolded protein response (UPR), suggesting an uncoupling of UPR and autophagy during flavivirus infection. The findings here reported could help to improve the knowledge of the cellular processes involved on flavivirus–host cell interactions and contribute to the design of effective strategies to combat these pathogens.

A comprehensive functional map of the hepatitis C virus genome provides a resource for probing viral proteins.

ABSTRACTPairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains.

Discovery of a Novel Class of Potent HCV NS4B Inhibitors: SAR Studies on Piperazinone Derivatives

HTS screening identified compound 2a (piperazinone derivative) as a low micromolar HCV genotype 1 (GT-1) inhibitor. Resistance mapping studies suggested that this piperazinone chemotype targets the HCV nonstructural protein NS4B. Extensive SAR studies were performed around 2a and the amide function and the C-3/C-6 cis stereochemistry of the piperazinone core were essential for HCV activity. A 10-fold increase in GT-1 potency was observed when the chiral phenylcyclopropyl amide side chain of 2a was replaced with p-fluorophenylisoxazole-carbonyl moiety (67). Replacing the C-6 nonpolar hydrophobic moiety of 67 with a phenyl moiety (95) did not diminish the GT-1 potency. A heterocyclic thiophene moiety (103) and an isoxazole moiety (108) were incorporated as isosteric replacements for the C-6 phenyl moiety (95), resulting in significant improvement in GT-1b and 1a potency. However, the piperazonone class of compounds lacks GT-2 activity and, consequently, were not pursued further into development.

Hepatitis C virus‐specific cellular immune responses in sustained virological responders with viral persistence in peripheral blood mononuclear cells

Hepatitis C virus (HCV)-RNA detection in peripheral blood mononuclear cells (PBMCs) after recovery from HCV infection, is a type of occult HCV infection although is unclear how the viral persistence in PBMCs affects HCV-specific T-cell responses. The aim of this study was to investigate if cellular immune responses are modified by HCV persistence in PBMCs. HCV-specific CD4(+) and CD8(+) T-cell responses against six HCV peptides, situated within the non-structural (NS) proteins NS3, NS4b and NS5b, were measured by flow cytometry-through intracellular detection of gamma interferon (IFN-γ) or interleukin 4 (IL-4) and CD69 expression- in 27 sustained virological responders (SVR): 13 with and 14 without occult HCV infection in PBMCs, detected by strand-specific real-time PCR. Ten healthy individuals and 14 chronically infected patients with viraemia, were included as controls. SVR without occult infection showed a higher percentage of activated CD4(+) cells against peptides belonging to NS3 (p124, p153) and NS5b (p257, p294), activated CD8(+) cells against NS3 (p124, p153, p158) and NS5b-p294, as well as an elevated percentage of CD4(+) cells releasing IFN-γ+IL-4 against NS3-p153, and by CD8(+) cells against NS3 (p124, p153). SVR without occult infection showed a higher percentage of activation and release of IFN-γ+IL-4 by both cell subpopulations than the two group of controls, in contrast to SVR with occult infection. The lower HCV-specific T-cell response found in SVR with occult infection indicates that the immune response may be impaired when the virus persists in PBMCs.

Induction of intrahepatic HCV NS4B, NS5A and NS5B-specific cellular immune responses following peripheral immunization.

Numerous studies have suggested that an effective Hepatitis C Virus (HCV) vaccine must induce strong cytotoxic and IFN-γ+ T cell responses targeting the non-structural region of the virus. Most importantly, these responses must be able to migrate into and remain functional within the liver, an organ known to cause T cell tolerance. Using three novel HCV DNA vaccines encoding non-structural proteins NS4B, NS5A and NS5B, we assessed the ability of peripheral immunization to induce functional intrahepatic immunity both in the presence and absence of cognate HCV antigen expression within the liver. We have shown that these constructs induced potent HCV-specific CD4+ and CD8+ T cell responses in the spleen of C57BL/6 mice and that these responses were detected within the liver following peripheral immunization. Additionally, using a transfection method to express HCV antigen within the liver, we showed that intrahepatic HCV-specific T cells remained highly functional within the liver and retained the ability to become highly activated as evidenced by upregulation of IFN-γ and clearance of HCV protein expressing hepatocytes. Taken together, these findings suggest that peripheral immunization can induce potent HCV-specific T cell responses able to traffic to and function within the tolerant environment of the liver.

Identification of three H-2Kd restricted CTL epitopes of NS4A and NS4B protein from Yellow fever 17D vaccine

In this study, 12 nonamers derived from the nonstructural protein NS4A and NS4B of a Chinese attenuated Yellow fever 17D (YF-17D) vaccine strain were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus expressing NS4A or NS4B protein. Three peptides (amino acid residues S(106)YIMLIFFV from NS4A protein and V(43)YVGIVTML and L(174)YLLLALSL from NS4B protein) were identified as CD8(+) cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited a response from a significant numbers of IFN-γ secreting cells compared with the other nonamers of NS4A and NS4B and compared to one irrelevant nonamer derived from infectious bronchitis virus (IBV) H52 strain. Bioinformatics analysis demonstrated that these three nonamers are H-2K(d)-restricted CTL epitopes. The multiple amino acid sequence alignments among different YFV NS4A, and NS4B sequences submitted to GenBank indicated that these three CTL epitopes are strongly conserved; this novel finding will potentially aid further studies on cellular immunity against YF virus and YFV-based expression vectors.

Evidence for a Genetic and Physical Interaction between Nonstructural Proteins NS1 and NS4B That Modulates Replication of West Nile Virus

Flavivirus NS1 is a nonstructural glycoprotein that is expressed on the cell surface and secreted into the extracellular space. Despite its transit through the secretory pathway, NS1 is an essential gene linked to early viral RNA replication. How this occurs has remained a mystery given the disparate localization of NS1 and the viral RNA replication complex, as the latter is present on the cytosolic face of the endoplasmic reticulum (ER). We recently identified an N-terminal di-amino acid motif in NS1 that modulates protein targeting and affected viral replication. Exchange of two amino acids at positions 10 and 11 from dengue virus (DENV) into West Nile virus (WNV) NS1 (RQ10NK) changed its relative surface expression and secretion and attenuated infectivity. However, the phenotype of WNV containing NS1 RQ10NK was unstable, as within two passages heterogeneous plaque variants were observed. Here, using a mutant WNV encoding the NS1 RQ10NK mutation, we identified a suppressor mutation (F86C) in NS4B, a virally encoded transmembrane protein with loops on both the luminal and cytoplasmic sides of the ER membrane. Introduction of NS4B F86C specifically rescued RNA replication of mutant WNV but did not affect the wild-type virus. Mass spectrometry and coimmunoprecipitation studies established a novel physical interaction between NS1 and NS4B, suggesting a mechanism for how luminal NS1 conveys signals to the cytoplasm to regulate RNA replication.

A Single Amino Acid in Nonstructural Protein NS4B Confers Virulence to Dengue Virus in AG129 Mice through Enhancement of Viral RNA Synthesis

Dengue (DEN) is a mosquito-borne viral disease that has become an increasing economic and health burden for the tropical and subtropical world. The lack of an appropriate animal model of DEN has greatly impeded the study of its pathogenesis and the development of vaccines/antivirals. We recently reported a DEN virus 2 (DENV-2) strain (D2Y98P) that lethally infects immunocompromised AG129 mice, resulting in organ damage or dysfunction and increased vascular permeability, hallmarks of severe DEN in patients (G. K. Tan et al., PLoS Negl. Trop. Dis. 4:e672, 2010). Here we report the identification of one critical virulence determinant of strain D2Y98P. By mutagenesis, we showed that a Phe-to-Leu alteration at amino acid position 52 in nonstructural protein NS4B completely abolished the pathogenicity of the D2Y98P virus, as evidenced by a lack of lethality and the absence of histological signs of disease, which correlated with reduced viral titers and intact vascular permeability. Conversely, a Leu-to-Phe alteration at position 52 of NS4B in nonvirulent DENV-2 strain TSV01 led to 80% lethality and increased viremia. The NS4B(Phe52) viruses displayed enhanced RNA synthesis in mammalian cells but not in mosquito cells. The increased viral RNA synthesis was independent of the ability of NS4B to interfere with the host type I interferon response. Overall, our results demonstrate that Phe at position 52 in NS4B confers virulence in mice on two independent DENV-2 strains through enhancement of viral RNA synthesis. In addition to providing further insights into the functional role of NS4B protein, our findings further support a direct relationship between viral loads and DEN pathogenesis in vivo, consistent with observations in DEN patients.

Evolution of attenuating mutations in dengue-2 strain S16803 PDK50 vaccine and comparison of growth kinetics with parent virus

A live-attenuated dengue-2 virus strain S16803 vaccine candidate that is immunogenic and safe in humans was derived by 50 passages in primary dog kidney (PDK) cells. To identify mutations associated with attenuation of the dengue-2 PDK50 vaccine strain, we determined the nucleotide changes that arose during PDK passage of the dengue-2 virus. Thirteen mutations distinguished the PDK50 virus from low-passage parent resulting in amino acid substitutions in the premembrane (E89G), envelope (E202K, N203D), nonstructural proteins NS1 (A43T), NS2A (L181F), NS2B (I26V), and NS4B (I/T108T, L112F). In addition, the PDK50 virus contained a C to T change of nucleotide 57 in the 5' non-coding region and four silent mutations of nucleotides 591, 987, 6471, and 8907. An infectious PDK50 cDNA clone virus was produced and characterized for growth kinetics in monkey (LLC-MK(2), Vero) and mosquito (C6/36) cells. Identification of mutations in the vaccine strain and availability of an infectious clone will permit systematic analysis of the importance of individual or collective mutations on attenuation of dengue virus.

Identification of an NTPase motif in classical swine fever virus NS4B protein

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive-sense single-stranded RNA virus within the Pestivirus genus of the Flaviviridae family. Here, we have identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV non-structural (NS) protein NS4B. Expressed NS4B protein hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NS4B NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. Similar mutations introduced into the genetic backbone of a full-length cDNA copy of CSFV strain Brescia rendered no infectious viruses or viruses with impaired replication capabilities, suggesting that this NTPase activity is critical for the CSFV cycle. Recovered mutant viruses retained a virulent phenotype, as parental strain Brescia, in infected swine. These results have important implications for developing novel antiviral strategies against CSFV infection.