The Hypervariable Amino-Terminus of P1 Protease Modulates Potyviral Replication and Host Defense Responses

Abstract

The replication of many RNA viruses involves the translation of polyproteins, whose processing by endopeptidases is a critical step for the release of functional subunits. P1 is the first protease encoded in plant potyvirus genomes; once activated by an as-yet-unknown host factor, it acts in cis on its own C-terminal end, hydrolyzing the P1-HCPro junction. Earlier research suggests that P1 cooperates with HCPro to inhibit host RNA silencing defenses. Using Plum pox virus as a model, we show that although P1 does not have a major direct role in RNA silencing suppression, it can indeed modulate HCPro function by its self-cleavage activity. To study P1 protease regulation, we used bioinformatic analysis and in vitro activity experiments to map the core C-terminal catalytic domain. We present evidence that the hypervariable region that precedes the protease domain is predicted as intrinsically disordered, and that it behaves as a negative regulator of P1 proteolytic activity in in vitro cleavage assays. In viral infections, removal of the P1 protease antagonistic regulator is associated with greater symptom severity, induction of salicylate-dependent pathogenesis-related proteins, and reduced viral loads. We suggest that fine modulation of a viral protease activity has evolved to keep viral amplification below host-detrimental levels, and thus to maintain higher long-term replicative capacity.

The P1N-PISPO trans -Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor

“…The functions of potyviral P1s have remained elusive for many years, but recent results are revealing the importance of P1 during potyvirus infection (32,52). In the case of SPFMV and other potyviruses infecting sweet potato, the presence of P1N-PISPO adds a further layer of complexity to efforts to unravel the role(s) of these gene products in the infection context (53).…”

“…pENTR-P1 was used as the template for PCR amplifications of all constructs used for in vitro translation, including unmodified wild-type P1 and variants with P1 interrupted by a stop codon after the slippage site (P1⌬) and P1 with the PISPO sequence interrupted (P1N-PISPO⌬) generated by site-directed mutagenesis with the primers FMP1⌬F and ⌬1PMFR for P1⌬ and FMPISPO⌬F and ⌬OPSIPMFR for P1N-PISPO⌬ (see Table S1 in the supplemental material). T7 promoter, 5= untranslated region (5=UTR), and 3=UTR fragments were prepared as described previously (32). mRNAs were produced by in vitro transcription using the T7-Scribe Standard RNA IVT kit (Cellscript) and purified by organic extraction and ammonium acetate precipitation, and their quality and amount were assessed by NanoDrop (Thermo Fisher Scientific) and gel electrophoresis.…”

Tobacco etch virus Protein P1 Traffics to the Nucleolus and Associates with the Host 60S Ribosomal Subunits during Infection

“…Nonetheless, many viral proteins are multifunctional, and P1 most probably plays multiple roles during infection. A recent work shows that the hypervariable amino terminus of plum pox potyvirus P1 protein modulates replication and the host defense response (66). Also, a transient suppression of host gene expression was reported exclusively at the infection front of pea seed-borne mosaic potyvirus (67).…”

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection

“…P1 enhances viral suppression of the RNA silencing mediated by HC-Pro (16,17) and interacts with host ribosomal proteins to stimulate the translation of viral proteins in infected cells (18). P1 likely modulates viral replication and the host defense response via host-dependent regulation of its viral protease activity (19). Another good example is the multifunctional protein CI, whose functions have been reviewed recently (20).…”