Equine arteritis virus (EAV) is the prototypic virus of the family Arteriviridae. The EAV genome is a positive-sense single-stranded RNA molecule in which two open reading frames (ORFs) encode the large replicase polyproteins pp1a and pp1ab. Processing of pp1a and pp1ab is mediated by three viral proteases of which a predicted chymotrypsin-like protease residing in nsp4 releases most non-structural proteins from the replicase polyproteins. To obtain more insight in the biochemical properties of this protease, and viral chymotrypsin-like proteases in general, the three-dimensional structure of nsp4 was determined by X-ray crystallography. The nsp4 three-dimensional structure revealed that the enzyme adopts a chymotrypsin-like fold and possesses an additional C-terminal domain (CTD) not found in most other chymotrypsin-like proteases. The structure revealed also that a connecting stretch of amino acids might facilitate movement of the CTD relative to the rest of the molecule. A site-directed mutagenesis study showed that the nsp4 CTD played a crucial role in EAV replicase processing, but that it was not required for proteloytic activity of the protease per se. Furthermore, the formation of an ion pair between Asp-119 and either Arg-4 in the N-terminus or Arg-203 in the C-terminus was suggested, which could play a role in alternate nsp4 conformations needed for e.g. different cis and trans cleavage activities. Mutations targeting the residues involved in these interactions affected the proteolytic activity of nsp4, but the data were inconclusive regarding the importance of ion pair formation. Processing of the C-terminal half of pp1a (the nsp3-8 region) by nsp4 can proceed following either of two alternative proteolytic pathways. To address the importance of both pathways, various cleavage site mutations were engineered, which were expected to block cleavage by nsp4. The experiments showed that all mutations that blocked processing of the corresponding site in the nsp3-8 precursor also blocked or severely inhibited EAV RNA synthesis. Moreover, evidence was obtained for the presence of a novel, internal nsp4 cleavage site in nsp7, which appears to be conserved among arteriviruses. The theoretical chapters in this thesis introduce the reader to (nido)viruses and nidovirus replicase maturation in particular.UBL - phd migration 201
