Dengue Virus Nonstructural Protein 5 Adopts Multiple Conformations in Solution

Abstract

The dengue virus (DENV) nonstructural protein 5 (NS5) is composed of two globular domains separated by a 10-residue linker. The N-terminal domain participates in the synthesis of a messenger RNA cap 1 structure (7MeGpppA2'OMe) at the 5' end of the viral genome, and possesses guanylyltransferase, guanine-N7-methyltransferase, and nucleoside-2'O-methyltransferase activities. The C-terminal domain is an RNA-dependent RNA polymerase responsible for viral RNA synthesis. Although crystal structures of the two isolated domains have been obtained, there are no structural data for the full-length NS5. It is also unclear whether the two NS5 domains interact with each other to form a stable structure in which the relative orientation of the two domains is fixed. To investigate the structure and dynamics of DENV type 3 NS5 in solution, small-angle X-ray scattering (SAXS) experiments were carried out on the full-length protein. NS5 was found to be monomeric and well-folded under the conditions tested. The results of these experiments also suggest that NS5 adopts multiple conformations in solution, ranging from compact to more extended forms wherein the two domains do not seem to interact with each other. We interpret the multiple conformations of NS5 observed in solution as resulting from weak interactions between the two NS5 domains and flexibility of the linker in the absence of other components of the replication complex.

A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities

“…In this model, Lys 46 /Arg 47 /Glu 49 from the MTase domain are in the vicinity of residue 512, giving a rather compact shape to the NS5. However, a single stable conformation for the isolated NS5 protein is not fully consistent with the small angle x-ray scattering data, which suggested that NS5 can adopt a range of conformations from compact to fully extended (5). In this respect, the segment comprising amino acids 263-267 of the NS5 protein (DENV3 numbering; see Fig.…”

“…Both enzymatic activities form attractive targets for antiviral development (1)(2)(3)(4). Based on small angle x-ray scattering studies in solution as well as limited proteolysis studies of NS5, the two globular protein domains appear to be connected by a flexible linker and could adopt a range of orientations with respect to each other (5). Crystal structures of the separate methyltransferase (6,7) and polymerase domains of NS5 (8,9) as well as limited proteolysis studies done on the full-length NS5 protein (NS5FL) (8) suggest that residues 263 HVNAEPETP 271 (using the DENV3 NS5 sequence numbering scheme; see Fig.…”

Nuclear Localization of Dengue Virus Nonstructural Protein 5 Does Not Strictly Correlate with Efficient Viral RNA Replication and Inhibition of Type I Interferon Signaling

“…multiple conformations (38). In this respect, we note that K402, which was altered in mutant 401-402 (the only mutant with impaired RdRp activity), might be involved in interactions between the MTase and the RdRp domain.…”

Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A

“…Full-length DENV3 NS5 with a C-terminal hexahistidine tag was expressed in Escherichia coli BL21-CodonPlus-RIL cells (20). Briefly, the cells were grown at 37°C in Luria broth (LB) containing 34 g/ml kanamycin and 30 mg/ml chloramphenicol to an absorbance at 600 nm of 0.6 to 0.7.…”

“…Additionally, interdomain interactions between monomers in an NS5 dimer suggest the coordination of RdRp and MTase activities across the NS5 monomer and dimer (17). Data from small-angle X-ray scattering (SAXS) studies indicate that NS5 proteins of all four DENV serotypes can adopt a more elongated shape than that in the crystal structure (20,21), suggesting that flexibility between the MTase and RdRp domains is likely essential for the viral replication process. Interactions between the MTase and RdRp domains are also important for the polymerase activity of NS5, since full-length NS5 has higher polymerase activity than does the RdRp domain alone (22).…”