Characterizationof the NTPase Activity of Japanese Encephalitis Virus NS3 Protein

111

West Nile virus (WN virus), a member of the family of Flaviviridae, is a small enveloped single-stranded RNA positivestrand virus. The viral genome encodes a monocistronic polyprotein of 3,430 amino acids that is processed into three structural proteins, protein M, capsid protein C, and glycoprotein E, and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (10, 11, 52). The processing of the polyprotein is carried out by the host signal peptidase associated with the endoplasmic reticulum and viral proteases. The polyprotein of WN virus and its processing are similar to those of the pestivirus-and hepatitis C virus (HCV)-related viruses (36,44,55). Sequence analysis of the nonstructural region of WN virus polyprotein revealed numerous conserved motifs specific for serine proteases, RNA helicase with intrinsic RNA-stimulated nucleoside triphosphatase (NTPase) localized in the NS3 protein, and RNA-directed RNA polymerase associated with the NS5 protein (3, 16, 17). These predictions were partially confirmed by verifying the enzymatic properties of a COOH-terminal segment of NS3 released from a membrane fraction of infected cells by subtilisin (54). Further information about the interactions and functions of the viral proteins was obtained by using synthesized recombined proteins of Flaviviridae or HCV-related viruses (19,23,47,49,50).Due to multiple enzymatic and biological activities associated with NS3, this protein appears to be the most promising target for antiviral agents. The protease activity of NS3 is the subject of numerous studies and has been well characterized previously (24, 31). However, despite the importance of enzymes modulating RNA structures in diverse metabolic processes and their critical role in the life cycles of viruses whose genomes are composed of RNA, only limited information on the viral helicases or helicase-like enzymes is available.Helicases are capable of enzymatically unwinding duplex DNA or RNA structures by disrupting the hydrogen bonds that keep the two strands together (18,21). The unwinding reaction is accomplished by the hydrolysis of ␥-phosphate of nucleotide triphosphate (NTP). Based on sequence comparisons, the viral helicases have been divided into three superfamilies. The WN virus helicase is a member of superfamily II (SFII), which includes helicases from bymovirus, potyvirus, pestivirus, herpesvirus, poxvirus, HCV, and other Flaviviridae (22). All of the helicases contain seven highly conserved amino acid sequences (motifs I to VII) that are located on the surfaces of domains 1 and 2 of the three-domain enzymes. The involvement of the motifs in NTP binding, NTP hydrolysis, and the binding of polynucleotide(s) was well explained by resolving the crystal structures of several enzymes (25,57). However, these structures did not elucidate the mechanisms coupling ATP hydrolysis to the unwinding reaction. Although numerous studies about the quantification of the interaction of SFII helicases with NTP and polynucleotides were performed, uniform resul...

Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 69%

Section: Fig 4 Modulation Of the Atpase Activity Of The Wn Virusmentioning

confidence: 99%

See 1 more Smart Citation

Purification and Characterization of West Nile Virus Nucleoside Triphosphatase (NTPase)/Helicase: Evidence for Dissociation of the NTPase and Helicase Activities of the Enzyme

Borowski

Niebuhr

Mueller

et al. 2001

111

“…This was in contrast to the results obtained by Li et al (33) who showed a 9.7-fold increase in V max for a DEN-2 NS3 in the presence of poly(A), using a protein with a similar N-terminal truncation but containing a C-terminal His tag rather than the much larger N-terminal GST tag of our experiments. Other significant differences in NTPase activities among NS3 proteins in the presence of polynucleotides have also been described (4,31,47,48,54). The reasons for the differences have not been identified, but they probably reflect variation in the types, sizes, and locations of fused peptides; the degree of truncation of the enzymes; the methods of expression (e.g., in bacteria, insect, or mammalian cells); the purification procedures; and the assay conditions.…”

Section: Discussionmentioning

confidence: 99%

Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication

Matusan

Pryor

Davidson

et al. 2001

165

154

The protein NS3 of Dengue virus type 2 (DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the Nterminal region and an NTPase-helicase in the remaining 70% of the protein. The latter region has seven conserved helicase motifs found in all members of the family Flaviviridae. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione S-transferase in Escherichia coli. The effects of 10 mutations on ATPase and RNA helicase activity were examined. Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis. The mutations were also tested for their effects on virus replication by incorporation into genomic-length cDNA. Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity. Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R 376 KNGK 380 , abolished helicase activity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication. The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication. For the two nonreplicating viruses encoding clustered changes at R 184 KR 186 and D 436 GEE 439 , we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the viral replication complex. Two of the replicating viruses displayed a temperature-sensitive phenotype. One contained a clustered mutation at D 334 EE 336 and grew too poorly for further characterization. However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37°C) and showed reduced RNA synthesis at the higher temperature.The four serotypes of Dengue virus (types 1 to 4) belong to the family Flaviviridae, which consists of the genera Flavivirus, Pestivirus, and Hepacivirus (52). The dengue virus genome is positive-sense RNA of 11 kb and encodes the proteins C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 in a single open reading frame. Co-and posttranslational polyprotein processing by host and viral proteinases generates three structural proteins, namely, C (capsid), M (membrane associated) and E (envelope), and seven nonstructural (NS) proteins, NS1 through NS5 (reviewed in reference 45). Biochemical functions have been demonstrated for some nonstructural proteins. NS5 possesses RNA-dependent RNA polymerase activity (49). A complex of NS2B and NS3 acts as a chymotrypsin-like serine proteinase; the N-terminal 30% of NS3 is sufficient for this activity (15, 42). The C-terminal 70% of NS3 has seven motifs characteristic of RNA helicases of the DExH subfamily. Recombinant proteins containing the C-terminal helicase region of dengue virus NS3 possess n...

“…The NTPase domain is located in the middle portion of NS3 slightly overlapping the serine protease domain. An RNA-stimulated NTPase activity has been identified in the NS3 proteins of DEN, YF, West Nile, and JE viruses (7,17,18,27,32,34). The domain includes a Walker motif A, involved in binding the ␤ and ␥ phosphates of nucleoside triphosphate (NTP), and a Walker motif B responsible for chelation of Mg 2ϩ in the Mg-NTP complex.…”

mentioning

confidence: 99%

Langat Flavivirus Protease NS3 Binds Caspase-8 and Induces Apoptosis

Prikhod'ko¹,

Prikhod’ko

Pletnev³

et al. 2002

The flavivirus NS3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral RNA. NS3 recruits NS2B and NS5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. We have found that NS3 also induces apoptosis. Expression of the Langat (LGT) virus NS3 protein resulted in a cleavage of cellular DNA and reduced the viability of cells. Coexpression of NS3 with apoptotic inhibitors (CrmA and P35) and addition of caspase peptide substrates (Z-VAD-FMK and Z-IETD-FMK) to NS3-transfected cells blocked NS3-induced apoptosis. In cotransfection experiments, NS3 bound to caspase-8 and enhanced caspase-8-mediated apoptosis. NS3 and caspase-8 colocalized in the cytoplasm of transfected cells. Deletion analysis demonstrated that at least two regions of NS3 contribute to its apoptotic activities. The protease and helicase domains are each able to bind to caspase-8, while the protease domain alone induces apoptosis. The protease domain and tetrahelix region of the helicase domain are required for NS3 to augment caspase-8-mediated apoptosis. Thus, the LGT virus NS3 protein is a multifunctional protein that binds to caspase-8 and induces apoptosis.The Flavivirus genus includes mostly arthropod-borne pathogens, many of which are widely distributed throughout the world and cause human diseases that vary in severity. The four serotypes of dengue (DEN) virus, Japanese encephalitis (JE) virus, and yellow fever (YF) virus cause millions of cases of disease annually in the tropics and subtropics. Tick-borne encephalitis (TBE) flaviviruses grouped within the TBE complex (Russian spring-summer encephalitis or TBE, Kyasanur forest disease, Langat [LGT], Louping ill, Negishi, Omsk hemorrhagic fever, and Powassan viruses) are mainly found in the Northern Hemisphere and, except for LGT virus, cause thousands of human cases every year, which can have a mortality as high as 20 to 30%.LGT virus does not naturally infect humans and has been studied as a potential live virus vaccine to protect against encephalitis caused by viruses of the TBE complex (4).LGT virus, like other flaviviruses, is a small lipid-enveloped, positive-sense RNA virus. During infection, a single polycistronic viral RNA is translated into a polyprotein precursor that is co-and posttranslationally processed by host and viral proteases to produce the virion and replicase components. The structural proteins of flaviviruses, capsid (C), premembrane (preM), and envelope (E), are encoded in the 5Ј quarter of the genome, and signal peptidase appears to mediate their cleavages during elongation of the polyprotein precursor through the translocating channel of the rough endoplasmic reticulum. Nonstructural proteins NS2A and -B, NS3, NS4A and -B, and NS5 are cleaved by viral serine protease NS2B-NS3 after a sequence that usually contains two basic residues followed by a short side chain residue (reviewed in reference 19).NS3 protein exhibits p...