Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries.
Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.
Li
Jun
J
Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, USA.
Lim
Siew Pheng
SP
Beer
David
D
Patel
Viral
V
Wen
Daying
D
Tumanut
Christine
C
Tully
David C
DC
Williams
Jennifer A
JA
Jiricek
Jan
J
Priestle
John P
JP
Harris
Jennifer L
JL
Vasudevan
Subhash G
SG
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D003716:Dengue Virus / Q000145:classification / Q000235:genetics*; D020869:Gene Expression Profiling; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D019151:Peptide Library; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D012703:Serotyping; D013379:Substrate Specificity; D000085242:Viral Proteases
D017931:DNA Primers; D009842:Oligopeptides; D019151:Peptide Library; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases