A reverse genetics study of the Human Coronavirus 229E ADP-ribose-1"-phosphatase

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. <br><br>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.