The RNA helicase, nucleotide 5′-triphosphatase, and RNA 5′-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

Abstract

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.

Substitution of NS5 N-terminal Domain of Dengue Virus Type 2 RNA with Type 4 Domain Caused Impaired Replication and Emergence of Adaptive Mutants with Enhanced Fitness

“…The N-terminal domain of NS5 possesses methyltransferase (MT) and guanylyltransferase (GT) activities necessary for forming a mature RNA cap structure that protects the viral genome and directs efficient translation (4 -10). The first step in 5Ј-capping of the positive strand progeny viral RNA is hydrolysis of ␥-phosphate of the 5Ј-triphosphorylated RNA, which is catalyzed by NS3 (11,12). Then GT activity of NS5 adds a GMP, hydrolysis product of GTP, to the 5Ј-diphosphorylated RNA to yield GpppN-RNA (13).…”

“…The C-terminal region is an ATP-dependent RNA helicase and has an ATP-independent RNA annealing activity (25). The ATPase active site of NS3 is also shared with that of 5Ј-RNA triphosphatase (11,12). There is evidence for an interdomain interaction between the NS2B-tethered N-terminal NS3 protease domain and the C-terminal RNA helicase domain from the studies of NS3 mutations in the region downstream of the protease domain and upstream of the first conserved motif of RNA helicase domain of NS3 (26,27).…”

Molecular-based identification and phylogeny of genomic and proteomic sequences of mosquito-borne flavivirus

“…The NS protein NS3 and NS5 are the best characterized proteins, with multiple enzyme activities that are required for viral replication. NS3 has three distinct activities: serine protease together with the cofactor NS2B, required for polyprotein processing; helicase/NTPase activity, required for unwinding the doublestranded replicative form of RNA; RNA triphosphatase, required for capping nascent viral RNA (Falgout et al 1991;Zhang et al 1992;Arias et al1993;Li et al 1999;Benarroch et al 2004). NS5 is the largest and most highly conserved flaviviral protein, with more than 75 % sequence identity across all DENV serotypes.…”

Comparative Analysis of Full-Length Genomic Sequences of 10 Dengue Serotype 1 Viruses Associated with Different Genotypes, Epidemics, and Disease Severity Isolated in Thailand over 22 Years

“…Domain III (residues 482-618) influences NTPase and helicase activities, as demonstrated by the mutation of a single Arg residue within helix (Arg513Ala), which slightly decreases NTPase activity and produces a defective helicase. 41 Thus, mutations of residues 350 and 466, and in particular, G466H, which resulted in an amino acid charge change (from neutral to positive) in domain II of the NS3 protein for all four strains in 1990-2001 clade of predominant genotype I may increase the activities of ATPase, RNA triphosphatase, and RNA unwinding, and enable the virus to replicate more efficiently.…”