The 3C-Like Proteinase of an Invertebrate Nidovirus Links Coronavirus and Potyvirus Homologs

Abstract

ABSTRACT Gill-associated virus (GAV), a positive-stranded RNA virus of prawns, is the prototype of newly recognized taxa (genus Okavirus , family Roniviridae ) within the order Nidovirales. In this study, a putative GAV cysteine proteinase (3C-like proteinase [3CL pro ]), which is predicted to be the key enzyme involved in processing of the GAV replicase polyprotein precursors, pp1a and pp1ab, was characterized. Comparative sequence analysis indicated that, like its coronavirus homologs, 3CL pro has a three-domain organization and is flanked by hydrophobic domains. The putative 3CL pro domain including flanking regions (pp1a residues 2793 to 3143) was fused to the Escherichia coli maltose-binding protein (MBP) and, when expressed in E. coli , was found to possess N-terminal autoprocessing activity that was not dependent on the presence of the 3CL pro C-terminal domain. N-terminal sequence analysis of the processed protein revealed that cleavage occurred at the location 2827 LVTHE↓VRTGN 2836 . The trans -processing activity of the purified recombinant 3CL pro (pp1a residues 2832 to 3126) was used to identify another cleavage site, 6441 KVNHE↓LYHVA 6450 , in the C-terminal pp1ab region. Taken together, the data tentatively identify VxHE↓(L,V) as the substrate consensus sequence for the GAV 3CL pro . The study revealed that the GAV and potyvirus 3CL pro s possess similar substrate specificities which correlate with structural similarities in their respective substrate-binding sites, identified in sequence comparisons. Analysis of the proteolytic activities of MBP-3CL pro fusion proteins carrying replacements of putative active-site residues provided evidence that, in contrast to most other 3C/3CL pro s but in common with coronavirus 3CL pro s, the GAV 3CL pro employs a Cys 2968 -His 2879 catalytic dyad. The properties of the GAV 3CL pro define a novel RNA virus proteinase variant that bridges the gap between the distantly related chymotrypsin-like cysteine proteinases of coronaviruses and potyviruses.