AbstractCoronaviruses have extremely large RNA genomes and use complex regulatory mechanisms to replicate and express their genomes. This involves the expression of two large polyproteins, pp1a and pp1ab, that are autoproteolytically processed to produce nonstructural proteins (nsp) 1 to 16. In this study, enzymatic activities associated with Human coronavirus 229E (HCoV-229E) nsp3 were characterised.
First, N-terminal processing of HCoV-229E nsp3 by viral papain-like proteases (PLP) 1 and 2 was investigated. The two proteases were previously shown to have partially overlapping substrate specificities and functionally replace each other at specific sites. Using polyclonal antisera, nsp3 and nsp4 were now detected as proteins of 175 and 43 kDa, respectively, in virus-infected cells. Immunofluorescence assays revealed a punctate perinuclear staining pattern, suggesting that nsp3 and nsp4 associate with viral replication complexes. Experiments using recombinant vaccinia viruses expressing wild-type or mutant forms of the HCoV-229E pp1a/pp1ab nsp1-to-nsp4 region revealed that PLP2 is responsible for processing at the predicted nsp3|nsp4 cleavage site while PLP1 does not cleave at this site. The data provide new insight into HCoV-229E PLP substrate specificities and polyprotein processing pathways.
Second, a conserved nsp3 domain called ADRP (ADP-ribose -1”-phosphatase) was investigated using a reverse genetics approach. To better understand the role of the ADRP in viral replication, a set of HCoV-229E ADRP mutants was generated, including viruses with a (i) mutation in the putative ADRP substrate-binding site (N1357A), (ii) deletion of the entire ADRP domain and (iii) substitution of the ADRP domain with that of HCoV-NL63. Characterisation of ADRP mutants revealed defects in RNA synthesis and viral growth in cell culture. Sequence analysis of viable clones following serial passage in cell culture identified a number of second-site mutations, possibly contributing to restoration of viral fitness and suggesting important (though nonessential) functions of the ADRP domain in coronavirus replication in cell culture.
|Date of Award||Jul 2011|
|Supervisor||John Ziebuhr (Supervisor) & Paul Duprex (Supervisor)|