Enzymatic characterization of a trypsin-like serine protease encoded by the genome of cell fusing agent virus.
Cell fusing agent virus (CFAV) is a positive strand RNA insect virus first isolated from a mosquito cell line. Based on viral morphology, phenotypic and phylogenetic studies, CFAV had been tentatively assigned to the genus Flavivirus (family Flaviviridae). The determination of the CFAV polyprotein complete sequence showed a putative serine protease domain analogue to the flaviviral NS2B/NS3 complex. This complex had been extensively studied, because it represented one of the main targets for antiflavivirus therapy development. We report herein the biochemical characterization of CFAV DeltaNS2B-NS3pro protease complex. CFAV polyprotein sequence was computationally analysed to identify the amino-acid regions involved in protease activity. We designed, expressed and purified a catalytically active protease whose enzymatic properties were determined using fluorogenic substrates. Our results showed that, despite the low level of conservation of its amino-acid sequence, CFAV protease exhibited physico-chemical properties of other flaviviruses (high pH value requirement for optimal activity, inhibition by salt and preference for substrates featuring a basic residue at P(1) position).
Peyrefitte
Christophe N
CN
Unité de virologie tropicale, Institut de médecine tropicale du Service de santé des armées, BP 46, 13 998, Marseille armées, France. publi.viro@laposte.net
Pastorino
Boris A M
BA
Grandadam
Marc
M
Rolland
Dominique
D
Tolou
Hugues J
HJ
Bessaud
Maël
M
D000595:Amino Acid Sequence; D018067:Flaviviridae / Q000145:classification* / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*
C114880:trypsin-like serine protease; D012697:Serine Endopeptidases