Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 protease activity.
Most proteolytic cleavages in the nonstructural protein (NS) region of the flavivirus polyprotein are effected by a virus-encoded protease composed of two viral proteins, NS2B and NS3. The N-terminal 180-amino-acid-region of NS3 includes sequences with homology to the active sites of serine proteases, and there is evidence that this portion of NS3 can mediate proteolytic cleavages. In contrast, nothing is known about required sequences in NS2B. We constructed a series of deletion mutations in the NS2B portion of plasmid pTM/NS2B-30% NS3, which expresses dengue virus type 4 (DEN4) cDNA encoding NS2B and the N-terminal 184 residues of NS3 from the T7 RNA polymerase promoter. Mutant or wild-type plasmids were transfected into cells that had been infected with a recombinant vaccinia virus expressing T7 RNA polymerase, and the protease activities of the expressed polyproteins were assayed by examining the extent of self-cleavage at the NS2B-NS3 junction. The results identify a 40-amino-acid segment of NS2B (DEN4 amino acids 1396 to 1435) essential for protease activity. A hydrophobicity profile of DEN4 NS2B predicts this segment constitutes a hydrophilic domain surrounded by hydrophobic regions. Hydrophobicity profiles of the NS2B proteins of other flaviviruses show similar patterns. Amino acid sequence alignment of this domain of DEN4 NS2B with comparable regions of other proteins of flaviviruses indicates significant sequence conservation, especially at the N-terminal end. These observations suggest that the central hydrophilic domain of NS2B of these other flaviviruses will also prove to be essential for protease activity.
Molecular Viral Biology Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
B
B
Falgout
R H
RH
Miller
C J
CJ
Lai
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000235:genetics*; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D005416:Flavivirus / Q000235:genetics; D005816:Genetic Complementation Test; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D016415:Sequence Alignment; D017384:Sequence Deletion; D013045:Species Specificity; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*
C080102:NS2B protein, flavivirus; D009838:Oligodeoxyribonucleotides; D017361:Viral Nonstructural Proteins; D010450:Endopeptidases