Secondary Structure and Membrane Topology of the Full-Length Dengue Virus NS4B in Micelles

Dengue virus (DV) is a positive sense RNA virus replicating in the cytoplasm in membranous compartments that are induced by viral infection. The non-structural protein (NS) 4A is one of the least characterized DV proteins. It is highly hydrophobic with its C-terminal region (designated 2K fragment) serving as a signal sequence for the translocation of the adjacent NS4B into the endoplasmic reticulum (ER) lumen. In this report, we demonstrate that NS4A associates with membranes via 4 internal hydrophobic regions, which are all able to mediate membrane targeting of a cytosolic reporter protein. We also developed a model for the membrane topology of NS4A in which the N-terminal third of NS4A localizes to the cytoplasm, while the remaining part contains three transmembrane segments, with the C-terminal end localized in the ER lumen. Subcellular localization experiments in DV-infected cells revealed that NS4A resides primarily in ER-derived cytoplasmic dot-like structures that also contain dsRNA and other DV proteins, suggesting that NS4A is a component of the membrane-bound viral replication complex (RC). Interestingly, the individual expression of DV NS4A lacking the 2K fragment resulted in the induction of cytoplasmic membrane alterations resembling virus-induced structures, whereas expression of full-length NS4A does not induce comparable membrane alterations. Thus, proteolytic removal of the 2K peptide appears to be important for induction of membrane alterations that may harbor the viral RC. These results shed new light on the role of NS4A in the DV replication cycle and provide a model of how this protein induces membrane rearrangements and how this property may be regulated.

Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5'-cap structure that is methylated at the guanine N-7 and the ribose 2'-OH positions of the first transcribed nucleotide, adenine (m(7)GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2'-O methylations and therefore is essential for flavivirus 5'-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m(7)GpppA-capped RNAs to m(7)GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m(7)GpppA- and m(7)GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2'-O methylation. Mutagenesis of a K(61)-D(146)-K(182)-E(218) tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2'-O MTase activities. The entire K(61)-D(146)-K(182)-E(218) motif is essential for 2'-O MTase activity, whereas N-7 MTase activity requires only D(146). The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K(61)-D(146)-K(182)-E(218) motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.