Dissection of a viral autoprotease elucidates a function of a cellular chaperone in proteolysis

Abstract

Replication of positive-strand RNA viruses involves translation of polyproteins which are proteolytically processed into functional peptides. These maturation steps often involve virus-encoded autoproteases specialized in generating their own N or C termini. Nonstructural protein 2 (NS2) of the pestivirus bovine viral diarrhea virus represents such an enzyme. Bovine viral diarrhea virus NS2 creates in cis its own C terminus and thereby releases an essential viral replication factor. As a unique feature, this enzyme requires for proteolytic activity stoichiometric amounts of a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) or its fragment Jiv90. To obtain insight into the structural organization of the NS2 autoprotease, the basis for its restriction to cis cleavage, as well as its activation by Jiv, we dissected NS2 into functional domains. Interestingly, an N-terminal NS2 fragment covering the active center of the protease, cleaved in trans an artificial substrate composed of a C-terminal NS2 fragment and two downstream amino acids. In the authentic NS2, the 4 Cterminal amino acids interfered with binding and cleavage of substrates offered in trans. These findings strongly suggest an intramolecular product inhibition for the NS2 autoprotease. Remarkably, the chaperone fragment Jiv90 independently interacted with protease and substrate domain and turned out to be essential for the formation of a protease͞substrate complex that is required for cleavage. Thus, the function of the cell-derived protease cofactor Jiv in proteolysis is regulation of protease͞substrate interaction, which ultimately results in positioning of active site and substrate peptide into a cleavage-competent conformation.hepacivirus ͉ J-domain chaperone ͉ pestivirus ͉ protease cofactor T he pestiviruses, together with the flaviviruses and hepatitis C virus, form the family Flaviviridae (1). Replication of these positive-strand RNA viruses involves translation of one polyprotein that is processed by cellular and virus-encoded proteases (2). These processing events generate the proteins required for viral RNA replication and the production of infectious progeny.Recently in nonstructural protein 2 (NS2) of the pestivirus bovine viral diarrhea virus (BVDV) an autoprotease was identified (3). In the polyprotein of BVDV strain NCP7, NS2 encompasses amino acids 1137-1589 and thus has a length of 453 aa (4). The N terminus of NS2 is generated by cellular signal peptidases, and the protein remains associated to intracellular membranes, presumably at the endoplasmatic reticulum. The N-terminal half of NS2 is highly hydrophobic and therefore likely to be involved in membrane association. However, so far no experimental data on membrane topology or protein structure are available, in part because of its biochemical properties as well as its toxicity for bacteria. A recent study revealed that NS2 is a cysteine protease (3); the obtained data indicated a catalytic role for histidine at position 1447 (H1447) and cysteine at pos...

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

“…(iii) Furthermore, it indicates that the YFV NS3/4A site is tightly regulated to have a controlled rate of processing in order to generate appropriate levels of uncleaved NS3-4A and cleaved products, NS3 and NS4A, similar to what has been described for cleavage of NS2/3 of BDVD and classical swine fever virus (CSFV) and NS3/4A of Murray Valley encephalitis virus (46,77,79). In this report, we propose that DNAJC14 plays a role in controlling this specific YFV cleavage site, similar to what has been described for BVDV NS2/3 cleavage, although the mechanism and biological relevance may be different (72,77,80). Indeed, to probe for a role of NS3-4A in YFV infection, we abolished the uncleaved NS3-4A form by molecularly engineering the YFV genome to encode the polyprotein in a bicistronic manner with no possibility of NS3-4A expression (NS3-4A SPLIT).…”

Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase

“…5B, lanes 3 and 4). Self-cleavage of wild-type NS2-3 was not detected after expression in BHK cells, as shown earlier for BVDV NS2-3 by Lackner et al (25). To further secure the finding that the uncleaved NS2-3 precursor acts in trans on NS4-5 substrates, the substitution Cys1521Ala was introduced.…”

“…The resulting plasmids were named pCite-NS2-3-4A (aa 1134 to 2336), pCite-NS2-3 (aa 1134 to 2272), pCite-NS3-4A (aa 1590 to 2336), pCite-NS3 (aa 1590 to 2272), pCite-NS4A-4B-5A-5B (aa 2273 to 3898), and pCite-NS4B-5A-5B (aa 2337 to 3898). NS2-3 cleavage was inhibited by the introduction of the mutation C 1512 A (25) in the plasmid pCite-NS2-3 C1512A using outward PCR. C-terminally truncated NS3 constructs pCite-NS3p 1748 (aa 1590 to 1748), pCite-NS3p 1779 (aa 1590 to 1779), pCite-NS3p 1780 (aa 1590 to 1780), pCite-NS3p 1781 (aa 1590 to 1781), and pCite-NS3p 1782 (aa 1590 to 1782) were generated by PCR.…”

Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging

“…It is also known that uncleaved NS2-3 cannot substitute for NS3 in this process, and that generation of the authentic N terminus of NS3 is essential for RNA amplification. In the context of the viral full-length polyprotein, the NS2 autoprotease, in conjunction with a cellular chaperone as its cofactor, regulates the amount of free NS3 and thereby viral RNA replication efficiency (54,64,65 (Fig. 3, compare the decrease in RLuc activity for Bici-388 RLuc NS3-3= WT at the 24-and 30-h time points).…”