Novel ATP-Independent RNA Annealing Activity of the Dengue Virus NS3 Helicase

Abstract

The flavivirus nonstructural protein 3 (NS3) bears multiple enzymatic activities and represents an attractive target for antiviral intervention. NS3 contains the viral serine protease at the N-terminus and ATPase, RTPase, and helicase activities at the C-terminus. These activities are essential for viral replication; however, the biological role of RNA remodeling by NS3 helicase during the viral life cycle is still unclear. Secondary and tertiary RNA structures present in the viral genome are crucial for viral replication. Here, we used the NS3 protein from dengue virus to investigate functions of NS3 associated to changes in RNA structures. Using different NS3 variants, we characterized a domain spanning residues 171 to 618 that displays ATPase and RNA unwinding activities similar to those observed for the full-length protein. Interestingly, we found that, besides the RNA unwinding activity, dengue virus NS3 greatly accelerates annealing of complementary RNA strands with viral or non-viral sequences. This new activity was found to be ATP-independent. It was determined that a mutated NS3 lacking ATPase activity retained full-RNA annealing activity. Using an ATP regeneration system and different ATP concentrations, we observed that NS3 establishes an ATP-dependent steady state between RNA unwinding and annealing, allowing modulation of the two opposing activities of this enzyme through ATP concentration. In addition, we observed that NS3 enhanced RNA-RNA interactions between molecules representing the ends of the viral genome that are known to be necessary for viral RNA synthesis. We propose that, according to the ATP availability, NS3 could function regulating the folding or unfolding of viral RNA structures.

AUF1 p45 Promotes West Nile Virus Replication by an RNA Chaperone Activity That Supports Cyclization of the Viral Genome

“…While some of these factors were shown to be essential for the viral life cycle and/or to interact with defined elements of the genomic RNA, their precise functions remained mostly uncertain. The DV NS3 protein (35), the WNV core protein (36), and the cellular La (37) were observed to support flavivirus 5=-3= RNA interactions in a similar way, as it was observed here with AUF1 p45 (Fig. 8B).…”

“…The multifunctional NS3 protease/ helicase is indispensable for viral RNA replication (38) and catalyzes ATP-dependent unwinding and ATP-independent annealing of different cRNA strands. NS3 was accordingly suggested to be involved in folding or unfolding of viral RNA structures in dependency of the ATP concentration (35). The viral core is not essential for RNA replication (39), and recombinant La was found to inhibit RNA synthesis in the in vitro viral replicase assay (40).…”

“…The viral core is not essential for RNA replication (39), and recombinant La was found to inhibit RNA synthesis in the in vitro viral replicase assay (40). As neither NS3, core, nor La were indicated to operate in concert with the viral polymerase, the RNA annealing activities of these proteins were proposed to contribute to virion assembly and morphogenesis (35,36).…”

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

“…24 for a review and references therein). 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).…”

A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production

“…NS3 is a multifunctional protein with activities involved in polyprotein processing, viral RNA replication, and host immune evasion (20,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39). The protein bears an N-terminal serine protease domain (residues 1 to 169) and a Cterminal region containing RNA helicase, nucleoside triphosphatase (NTPase), 5=-RNA triphosphatase (RTPase), and RNA annealing activities (residues 179 to 618) (32,(40)(41)(42). Because of its essential role early in polyprotein processing and RNA replication, genetic studies thus far have provided limited information on the molecular determinants of NS3 responsible for assembly and release of infectious virus particles.…”