Human Coronavirus 229E Papain-Like Proteases Have Overlapping Specificities but Distinct Functions in Viral Replication

Abstract

Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PL pro s). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/ pp1ab region by two paralogous PL pro activities. Reverse-genetics data revealed that replacement of the PL2 pro active-site cysteine was lethal. By contrast, the PL1 pro activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1 pro active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1 pro activity. Further experiments showed that both PL1 pro and PL2 pro were able to cleave the nsp1-nsp2 cleavage site, with PL2 pro cleaving the site less efficiently. The PL1 pro -negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1 pro mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PL pro paralogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2 pro plays a universal and essential proteolytic role that appears to be assisted by the PL1 pro paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed.Expression of positive-strand RNA virus genomes generally starts with the translation of the incoming viral RNA to produce large precursor proteins that are co-and posttranslationally processed by viral and, in some cases, cellular proteases. The proteolytic release of intermediate and mature processing products, which may have diverse functions in different phases of viral replication, is spatially and temporally coordinated. Protease activities can therefore be viewed as key regulators of the life cycles of positive-strand RNA viruses.The positive-strand RNA coronaviruses (CoVs) have evolved a most complex pattern of polyprotein processing and regulation (64, 66). The 5Ј-terminal two-thirds of the giant 30-kb genome is occupied by two overlapping open reading frames (ORFs), called 1a and 1b, which together form the viral replicase gene and whose translation results in the production of two large polyproteins. ORF1a encodes polyprotein 1a (pp1a), and ORFs 1a and 1b encode pp1ab. The biosynthesis of pp1ab involves a ribosomal frameshift at the ORF1a-ORF1b junction during translation (9, 10). As many as 15 or 16 processing end products, which are called nonstructural proteins (nsps), are proteolytically released from pp1a/pp1ab (64,66...

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