NS3 serine protease of bovine viral diarrhea virus: characterization of active site residues, NS4A cofactor domain, and protease-cofactor interactions.
The gene expression of bovine viral diarrhea virus (BVDV), a pestivirus, occurs via translation of a hypothetical polyprotein that is processed cotranslationally and posttranslationally by viral and cellular enzymes. A protease located in the N-terminal region of nonstructural (NS) protein NS3 catalyzes the cleavages, leading to the release of NS4A, NS4B, NS5A, and NS5B. Our study provides experimental evidence that histidine at position 1658 and aspartic acid at position 1686 constitute together with the previously identified serine at position 1752 (S1752) the catalytic triad of the pestiviral NS3 serine protease. Interestingly, a mutant protease encompassing an exchange of the active site S1752 to threonine still showed residual activity. This finding links the NS3 protease of pestiviruses to the capsid protease of Sindbis virus. Furthermore, we observed that the minimal protease domain of NS3 encompasses about 209 amino acids. The NS3 protease was found to be sensitive to N-terminal truncation because a deletion of 6 amino acids significantly reduced the cleavage efficiency at the NS4A/4B site. Larger N-terminal deletions also impaired the activity of the enzyme with respect to the other cleavage sites but to a different degree at each site. The NS3 protease of BVDV has previously been shown to depend on NS4A as cofactor. We demonstrate here that the central region of NS4A represents the cofactor domain. Furthermore, coprecipitation studies strongly suggest an interaction between NS4A and the N-terminal region of NS3. Besides the remarkable similarities observed between the pestiviral NS3 protease and the corresponding enzyme of hepatitis C virus (HCV), our results suggest a common ancestry between these enzymes and the capsid protease of Sindbis virus.
Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität Giebetaen, Giebetaen, D-35392, Germany. Norbet.Tautz@vetmed.uni-giessen.de
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D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D021382:Protein Sorting Signals / Q000378:metabolism; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000956:Antigens, Viral; D021382:Protein Sorting Signals; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C080106:NS4A protein, flavivirus