Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins.
The cleavages at the junctions of the flavivirus nonstructural (NS) proteins NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 share an amino acid sequence motif and are presumably catalyzed by a virus-encoded protease. We constructed recombinant vaccinia viruses expressing various portions of the NS region of the dengue virus type 4 polyprotein. By analyzing immune precipitates of 35S-labeled lysates of recombinant virus-infected cells, we could monitor the NS2A/NS2B, NS2B/NS3, and NS3/NS4A cleavages. A polyprotein composed of NS2A, NS2B, and the N-terminal 184 amino acids of NS3 was cleaved at the NS2A/NS2B and NS2B/NS3 junctions, whereas a similar polyprotein containing only the first 77 amino acids of NS3 was not cleaved. This finding is consistent with the proposal that the N-terminal 180 amino acids of NS3 constitute a protease domain. Polyproteins containing NS2A and NS3 with large in-frame deletions of NS2B were not cleaved at the NS2A/NS2B or NS2B/NS3 junctions. Coinfection with a recombinant expressing NS2B complemented these NS2B deletions for NS2B/NS3 cleavage and probably also for NS2A/NS2B cleavage. Thus, NS2B is also required for the NS2A/NS2B and NS2B/NS3 cleavages and can act in trans. Other experiments showed that NS2B was needed, apparently in cis, for NS3/NS4A cleavage and for a series of internal cleavages in NS3. Indirect evidence that NS3 can also act in trans was obtained. Models are discussed for a two-component protease activity requiring both NS2B and NS3.
Falgout
B
B
Molecular Viral Biology Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Pethel
M
M
Zhang
Y M
YM
Lai
C J
CJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D004279:DNA, Viral; D003716:Dengue Virus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D020365:RNA Helicases; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases; D015534:Trans-Activators / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004279:DNA, Viral; D010450:Endopeptidases; D020365:RNA Helicases; D012697:Serine Endopeptidases; D015534:Trans-Activators; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases