African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-GlyXaa sites as a strategy to produce several major core components of the viral particle. The virus gene S273R encodes a 31-kDa protein that contains a "core domain" with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS cell expression system, it was found that protein pS273R is capable of cleaving the viral polyproteins pp62 and pp220 in a specific way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells. Furthermore, protein pS273R, like adenovirus protease and SUMO-1-specific enzymes, is a cysteine protease, because its activity is abolished by mutation of the predicted catalytic histidine and cysteine residues and is inhibited by sulfhydryl-blocking reagents. Protein pS273R is expressed late after infection and is localized in the cytoplasmic viral factories, where it is found associated with virus precursors and mature virions. In the virions, the protein is present in the core shell, a domain where the products of the viral polyproteins are also located. The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to our knowledge of the emerging family of SUMO-1-specific proteases.Positive strand RNA viruses and retroviruses encode polyproteins, which are proteolytically cleaved by viral proteases to yield the nonstructural and structural proteins required for replication and morphogenesis (1-3). On the other hand, DNA viruses, such as adenoviruses and poxviruses, synthesize precursor proteins whose maturation by proteolytic removal of terminal peptides plays an essential role in virion formation (1).African swine fever virus (ASFV), 1 a large and complex virus containing a 170-kb double-stranded DNA molecule with 151 potential genes (4, 5), is atypical among DNA viruses in that it encodes two polyproteins, pp220 and pp62, which are cleaved to produce six major structural components of the virus particle (6, 7). These proteins, p150, p37, p34, and p14, derived from polyprotein pp220 and p35 and p15, products of polyprotein pp62, are the major components of the core shell, a thick protein layer that surrounds the DNA-containing central nucleoid and that is enwrapped by the inner lipoprotein envelope and the icosahedral capsid (8).2 All the proteolytic cleavages occur after the second Gly of the consensus sequence Gly-GlyXaa, which is also recognized as a cleavage site in the maturation of adenovirus structural proteins and in some cellular proteins, including polyubiquitin (9) and ubiquitin-like proteins (10). A similar cleavage site (Ala-Gly-Xaa) is used for the maturation of vaccinia virus structural proteins (11,12). Although the adenovirus protease that processes at Gly-Gly-Xaa sites is well characterized (13-15), the enzymes involved in the processing of the ASFV polyproteins or in the cleavage of vaccin...

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.

This report shows that African swine fever virus (ASFV)‐‐a large DNA‐containing virus‐‐synthesizes a polyprotein to produce several of its structural proteins. By immunoprecipitation analysis, we have found that ASFV polyprotein is a 220 kDa myristoylated polypeptide (pp220) which, after proteolytic processing, gives rise to four major structural proteins: p150, p37, p34 and p14. Processing of the ASFV polyprotein takes place at the consensus sequence Gly‐Gly‐X and occurs through an ordered cascade of proteolytic cleavages. So far, polyprotein processing as a mechanism of gene expression had been found only in positive‐strand RNA viruses and retroviruses. According to the results presented here, ASFV is the first example of a DNA virus that synthesizes a polyprotein as a strategy of gene expression.

Since the discovery of microRNA (miRNA)-guided processing, a new type of RNA silencing, the possibility that such a mechanism could play a role in virus defense has been proposed. In this work, we have analyzed whether Plum pox virus (PPV) chimeras bearing miRNA target sequences (miR171, miR167, and miR159), which have been reported to be functional in Arabidopsis, were affected by miRNA function in three different host plants. Some of these PPV chimeras had clearly impaired infectivity compared with those carrying nonfunctional miRNA target sequences. The behaviors of PPV chimeras were similar but not identical in all the plants tested, and the deleterious effect on virus infectivity depended on the miRNA sequence cloned and on the site of insertion in the viral genome. The effect of the miRNA target sequence was drastically alleviated in transgenic plants expressing the silencing suppressor P1/HCPro. Furthermore, we show that virus chimeras readily escape RNA silencing interference through mutations within the miRNA target sequence, which mainly affected nucleotides matching the 5-terminal region of the miRNA.