Summary for peptidase C63.001: African swine fever virus processing peptidase

Summary Alignment Sequences Sequence features Distribution Literature Substrates


MEROPS NameAfrican swine fever virus processing peptidase
Other namesprotein S273R (African swine fever virus)
Name and HistoryAfrican swine fever virus, the causative agent of a highly lethal disease of domestic pigs, is an enveloped virus with icosahedral morphology and a DNA genome of 170 to 190 kbp containing more than 150 potential genes (Yáñez et al., 1995). The viral particle consists of several concentric domains, which are the central nucleoid, the core shell, the inner envelope, the capsid and the outer envelope (Andrés et al., 1997). The two virus encoded polyproteins, pp220 and pp62 are proteolytically processed to produce six major components of the virus core shell: proteins p150 (150 kDa), p37 (37 kDa), p34 (34 kDa) and p14 (14 kDa) derived from polyprotein pp220, and proteins p35 (35 kDa) and p15 (15 kDa), which are the products of polyprotein pp62 (Andrés et al., 1997, Simon-Mateo et al., 1997, Simon-Mateo et al., 1993, Andrés et al., 2002). Processing occurs after the second glycine of Gly-Gly-Xaa sites and is catalyzed by the ASFV protease, encoded by the viral gene S273R, and named protein pS273R (Andres et al., 2001), being also designated as ASFV processing peptidase. The ASFV protease belongs to clan CE of cysteine proteases that includes a number of cellular cysteine proteases of family C48 (Li & Hochstrasser, 1999), along with the adenovirus L3 protease (Webster et al., 1989, Mangel et al., 1996, Ding et al., 1996) that belongs to family C5 and the vaccinia and fowlpox virus protease encoded by gene I7 (Vanslyke et al., 1991, Lee & Hruby, 1994) (family C57) (Li & Hochstrasser, 1999), as well as by the predicted thiol proteases from mimiviruses (Li et al., 2011).
Domain architecture
MEROPS Classification
Classification Clan CE >> Subclan (none) >> Family C63 >> Subfamily (none) >> C63.001
HolotypeAfrican swine fever virus processing peptidase, Uniprot accession Q65228 (peptidase unit: 168-269), MERNUM MER0027602
History Identifier created: MEROPS 6.20 (24 March 2003)
Catalytic typeCysteine
NC-IUBMBNot yet included in IUBMB recommendations.
Proteolytic eventsCutDB database (6 cleavages)
PreparationProtein pS273R can be obtained from Vero (African green monkey) cells or swine macrophages infected with ASFV or from COS-7 cells transfected with the recombinant plasmid pcDNA-S273R (Andres et al., 2001). The ASFV S273R gene cloned in pGEX-2T or pGEX-6P has been expressed in E. coli as a fusion protein bound to glutathione S-transferase (GST) and the protein has been purified by affinity chromatography on glutathione-Sepharose. The purified recombinant pS273R protein is active in polyprotein processing. The GST tag can be removed by treatment with thrombin or PreScission Protease without loss of activity.
SpecificitySeveral assays have been used to demonstrate that cleavage of the polyproteins by the pS273R protease occurs in a specific and accurate way producing the same intermediates and final products as those found in infected cells. In one of these assays, the S273R gene is co-transfected with the polyprotein genes in COS-7 cells and the processing of the polyproteins is determined by immunoblotting using specific antibodies against the polyprotein products (Andrés et al., 1997, Simon-Mateo et al., 1993, Simon-Mateo et al., 1997, Andres et al., 2001). Processing activity has also been determined in vitro by mixing extracts from COS-7 cells transfected with the protease gene and one of the polyprotein genes and, after incubating the mixture for different times at 30º in 20 mM HEPES, pH 7.4, the samples are analyzed by Western blotting as before (Andres et al., 2001). Correct processing activity has also been demonstrated in vitro using 35S-labeled polyprotein pp62 and purified recombinant pS273R prepared as described below. Processing of the first pp62 cleavage site is abrogated by mutation of the two Gly residues to alanine, but this does not prevent recognition of the second site. In a similar way, when the second site is mutated its processing is blocked without preventing the cleavage at the first site. N-terminal and C-terminal fragments of polyprotein pp62, with the first and second cleavage sites, respectively, and a pp220-derived fragment, with the first cleavage site of this polyprotein, are processed very efficiently by the protease. The ASFV enzyme cannot desumoylate pRan-GAP, one of the substrates of SUMO-1 proteases. The pS273R protease is inhibited by divalent cations (Mg2+, Mn2+, Ca2+, and Zn2+) and by salt (NaCl or KCl) at concentrations of 100 mM or higher (Rubio et al., 2003)]. Also, the protease is inhibited by DTT, this inhibition being reverted by H2O2. The oxidizing agent inhibits irreversibly the protease in the absence of DTT. Mutation of the catalytic residues His168 and Cys232 to Arg and Ser, respectively, abolishes the activity of protein pS273R (Andres et al., 2001). A 90% inactivation of the protease is achieved by removal of the first eleven amino acids, while the deletion of eleven additional N-terminal residues completely abrogates its activity.
pH optimumThe highest activity is obtained around pH 8.5, but the protease is active over the pH range 6.0-9.5.
Special substrateThe ASFV polyproteins, pp220 and pp62, are the specific substrates of the S273R protease.
Substrate commentsPolyproteins pp220 and pp62 are the only known substrates of the pS273R protease.
Special inhibitorThe decapeptide MILGGADELE, which corresponds to the sequence encompassing the first processing site of polyproteins pp220, specifically inhibits the processing of pp220 by 52% at a concentration of 1 mM, raising the possibility of using this and other small peptide derivatives as inhibitors of the protease activity in vitro and in vivo (Rubio et al., 2003).
Inhibitor commentsThe ASFV enzyme is inhibited by two cysteine protease inhibitors, N-ethylmaleimide (J02.002) and the peptidyl diazomethane Z-Leu-Val-Gly-CHN2. However, the specific inhibitor of cysteine proteases, E64, does not significantly inhibit the processing activity of protein pS273R (Andres et al., 2001, Rubio et al., 2003). Inhibitors of aspartic proteases (pepstatin), metallo-proteases (1,10-phenanthroline), or serine proteases (PMSF) do not inhibit the processing activity of of the ASFV protease (Andres et al., 2001, Rubio et al., 2003). The inhibitor of the vaccinia virus I7 proteinase, TTP-6171, (Byrd et al., 2004) has no effect on the ASFV enzyme up to a concentration of 100 µM.
StructureThe ASFV protease is a protein of 273 amino acids with a determined molecular mass of 31 kDa and a predicted pI of 8.85. The conserved catalytic domain is situated between amino acids 168 and 252 and contains the catalytic triad formed by His168, Asn187, and Cys232, as well as a Gln residue (Gln226) that is required for the formation of the oxyanion hole in the active site in the case of Ulp1 enzymes (Mossessova & Lima, 2000). This domain is preceded by a 167 amino acid region that shows no similarity to the N-terminal sequence of cellular SUMO-1 proteases, the adenovirus protease and the vaccinia I7 protease, lacking the Ulp1 motifs that interact with SUMO-1 substrates (Mossessova & Lima, 2000). However, the N-terminus of the ASFV protease is conserved in the predicted thiol protease encoded by the mimivirus R355 gene (Jeudy et al., 2011).
LocationWithin the cytoplasmic virus factories, the pS273R protease is present in the core region of immature particles and the core shell of mature virions, where the polyproteins and their products are also found. Highly purified virus particles also contain the pS273R protease (Andres et al., 2001). The adenovirus L3 protease and the vaccinia virus I7 protein are also located in the core of mature virus particles. Although no proteolytic activity has been demonstrated yet for the mimivirus R355 gene product, the protein has been shown to be present in the newly formed viral particles (Renesto et al., 2006).
PhysiologyDuring ASFV infection polyprotein processing is strictly regulated, depending on the correct assembly of the virus particle. This has been demonstrated using ASFV recombinants that conditionally express different viral proteins in studies on virus morphogenesis. Thus, repression of the major capsid protein p72 or of its molecular chaperone pB602L leads to the assembly of unprocessed polyproteins into zipper-like structures that resemble the core shell of the viral particle (García-Escudero et al., 1998, Epifano et al., 2006). Furthermore, tubular particles containing unprocessed polyproteins are generated by repression of gene B438L, encoding a protein necessary for the formation of the icosahedral capsid (Epifano et al., 2006). Blocking of morphogenesis at earlier stages by inhibiting the synthesis of structural membrane proteins p54 and p17 also abolishes the proteolytic processing of polyproteins (Rodríguez et al., 2004, Suárez et al., 2010). In addition, it has been shown that empty icosahedral particles lacking the nucleoid and core shell are generated by repression of polyproteins pp220 and pp62, revealing a crucial role of the polyproteins in the formation of the viral core (Andrés et al., 2002, Suárez et al., 2010). On the other hand, the requirement of polyprotein processing in ASFV infection has been investigated by making use of the ASFV recombinant vS273Ri, which conditionally expresses the viral pS273R protease. Blocking S273R gene expression inhibits the proteolytic processing of polyproteins in infected cells producing noninfectious icosahedral particles with an abnormal core containing the unprocessed polyproteins, indicating that the protease is required for a late maturational stage in the formation of the virus core and infectivity (Alejo et al., 2003). Taking into account these results and the finding of a co-localization of the protease and polyproteins in viral structures within the factories, it can be suggested that polyprotein processing is a key mechanism for the regulation of spatio-temporal interactions between the core components during the formation of the core domain. Since the protease is a structural component of extracellular viral particles it could also be required for some early step of the infection.
Biological aspectsGene S273R is transcribed into an RNA of 4.5 kb at late times of infection, after initiation of the ASFV DNA replication (Andres et al., 2001). Initiation of transcription occurs at position –15 relative to the first nucleotide of the translation start codon. A motif of seven thymidylate residues, which is known to act as a signal for 3"-end formation of ASFV mRNAs, is found 52 nucleotides downstream of the contiguous topoisomerase II gene. Termination of transcription at this motif would produce an RNA of 4497 bases, which agrees with the size of the RNA found by Northern blot. During ASFV infection, protein pS273R is synthesized 8 h post-infection, a time at which virus DNA replication is already occurring, indicating that it is a late protein. The protease accumulates up to 20 h post-infection (Andres et al., 2001). In ASFV-infected cells processing of the pp220 and pp62 polyproteins takes place through a temporally ordered cascade of cleavages at Gly-Gly-X sites. The initial cleavage event gives rise to the mature p150 protein and preprotein pp90. The second cleavage in pp90 produces the mature protein p34 and preprotein pp55. The final processing step produces the p37 and p14 mature proteins. In the case of polyprotein pp62, the first site to be cleaved is the one located closer to the N terminus of the polyprotein, giving rise to mature protein p15 and the intermediate precursor pp46. This is subsequently cleaved to produce mature p15 and a putative polypeptide of about 8 kDa. The mature proteins of each polyprotein are present in equimolecular amounts in the viral particle, and account for about 30% of the virus protein content.
The maturation of major core proteins of adenovirus and poxvirus also occurs by proteolytic processing at Gly-Gly-X or Ala-Gly-X sites, this cleavage being mediated by the adenovirus L3 protease and the vaccinia virus I7L gene product, respectively. The maturation of newly assembled adenovirus virions into infectious particles requires the L3 protease (Weber, 1976), which is also involved in early stages of virus infection (Cotten & Weber, 1995, Greber et al., 1996).
RNA splicingThere is no RNA splicing or alternative initiation site.
KnockoutGene S273R is essential for ASFV replication and, therefore, a S273R knockout virus cannot be generated. A recombinant virus inducibly expressing gene S273R has been obtained and used to study the role of the protease as described under “Physiology”.
Pharmaceutical relevanceThe ASFV protease can be a specific target of small peptide derivatives corresponding to the processing sites of the polyproteins.
Distinguishing featuresThe ASFV proteinase processes polyprotein precursors recognizing several Gly-Gly-Xaa sites in the substrate sequence. A polyclonal antiserum against protein pS273R has been obtained (Andres et al., 2001).
Contributing authorsMarķa L. Salas, Centro de Biologia Molecular 'Severo Ochoa', CSIC-UAM, C/Nicolas Cabrera 1, Universidad Autonoma de Madrid, 28049, Madrid, Spain.