Role of protein conformation in the processing of dengue virus type 2 nonstructural polyprotein precursor.
The dengue virus type-2 (DEN-2) genome is a positive-strand RNA encoding a single polyprotein precursor, C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B- NS5, consisting of 3391 amino acids (aa). The N-terminal region of the polyprotein precursor, C-prM(M)-E, encodes the structural proteins and is processed cotranslationally by the host signal peptidase. The nonstructural region NS1-->NS5 is processed by the viral protease(s), as well as by the signal peptidase. A two-component viral protease consisting of NS2B and the serine protease domain of NS3 has been shown to be required for cleavages having the consensus sequence of dibasic aa (K-R, R-R, R-K, or Q-R). In this study, the region encoding all the nonstructural proteins, NS1-->NS5, was expressed using a recombinant vaccinia virus system. Cleavages at the consensus viral protease recognition sites, 2B-3 at the N terminus and 3-4A at the C terminus, are prerequisites to the release of mature NS3 protease. Although the 2B-3 site was cleaved readily in a variety of polyprotein precursors containing the intact NS2B and the NS3 protease domain, the 3-4A site was most efficiently cleaved, similar to that found in DEN-2-infected cells, only in the polyprotein precursor encoding the entire nonstructural region. Removal of NS1 at the N terminus or of NS5 coding sequences at the C terminus affected the cleavage at the 3-4A site to produce the processing intermediate, NS3-NS4A. These results indicate that the conformation of the nonstructural polyprotein precursor, NS1-->NS5, plays a major role in the efficient cleavage at the 3-4A site.
Zhang
L
L
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66103.
Padmanabhan
R
R
D000818:Animals; D001483:Base Sequence; D004274:DNA, Recombinant; D003716:Dengue Virus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D000882:Haplorhini; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011498:Protein Precursors / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017423:Sequence Analysis, RNA; D012697:Serine Endopeptidases / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics
D004274:DNA, Recombinant; C079340:NS1 protein, Dengue virus type 2; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase; D012697:Serine Endopeptidases