Structure of the Murray Valley encephalitis virus RNA helicase at 1.9 Angstrom resolution

Abstract

Murray Valley encephalitis virus (MVEV), a mosquito-borne flavivirus endemic to Australia, is closely related to Japanese encephalitis virus and West Nile virus. Nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains, whose activity is central to flavivirus replication and is therefore a possible target for anti-flaviviral compounds. Cloning, purification, and crystal structure determination to 1.9 Angstrom resolution of the NS3 helicase of MVEV and characterization of its enzymatic activity is reported. Comparison with the structures of helicases from related viruses supports a possible mechanism of ATP hydrolysis-driven strand separation.

Figure 1.

(A) Stereo ribbon diagram of MVEVh. The seven helicase motifs are colored using the same scheme as in panel C. (B) Surface representation of MVEVh, d1 (pink) and d2 (brown) are at the bottom and d3 (lime) at the top (the color scheme matches that of the secondary elements of MVE displayed above the sequence alignments in panel C. (C) Structure-based alignment (produced using T-Coffee) (O'Sullivan et al. 2004) of Flaviviridae NS3 helicases. The sequences of Japanese encephalitis virus (JEV, strain JaOArS982, NP_059434), West Nile virus (WNV, strain B956, AAT02759), Yellow Fever virus (YFV, strain 17D, NP_041726), and Hepatitis C virus (HCV, strain H77, NP_671491) were obtained from GenBank. Secondary structure elements of MVEVh are displayed above the sequence alignment. The conserved SF2 motifs are marked red (H1), green (H1a), blue (H2), cyan (H3), magenta (H4), yellow (H5), and orange (H6). (D) d3 of MVEVh (left) structurally compared to d3 of the HCV helicase (right). The structural superimposition was performed using SHP (Stuart et al. 1979). The secondary structural elements are shown in a loop representation (cyan) except for the regions that were best structurally aligned (pink), which are shown in a cartoon representation. (E) The release of inorganic phosphate in the presence of the indicated concentrations of ATP. The solid line corresponds to the fit to the Michaelis–Menten equation. Results of two protein batches were averaged and standard deviations are shown.

Figure 2.

(A) The structures of MVEVh (yellow) and YFVh (blue) superimposed via domain 3 (superimposition performed using SHP) (Stuart et al. 1979). Two regions of the superimposition (top panel: groove between d2 and d3; bottom panel: nucleotide binding groove) are enlarged to highlight the differences between the two structures. (B) Surface representation of MVEVh and YFVh illustrating the opening and closure of the groove between domains 2 and 3. The color scheme matches that of panel A.