African Swine Fever Virus Protease, a New Viral Member of the SUMO-1-specific Protease Family

Abstract

African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-Gly-Xaa 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. African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-Gly-Xaa 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. African swine fever virus kilobase(s) open reading frame benzyloxycarbonyl-leucyl-valyl-glycyl-diazomethylketone l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane 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 (1Kräusslich H.-G. Wimmer E. Annu. Rev. Biochem. 1988; 57: 701-754Crossref PubMed Scopus (424) Google Scholar, 2Dougherty W.G. Semler B.L. Microbiol. Rev. 1993; 57: 781-822Crossref PubMed Google Scholar, 3Gorbalenya A.E. Snijder E.J. Persp. Drug Discov. Design. 1996; 6: 64-86Crossref Scopus (94) Google Scholar). 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 (1Kräusslich H.-G. Wimmer E. Annu. Rev. Biochem. 1988; 57: 701-754Crossref PubMed Scopus (424) Google Scholar). African swine fever virus (ASFV),1 a large and complex virus containing a 170-kb double-stranded DNA molecule with 151 potential genes (4Viñuela E. Curr. Top. Microbiol. Immunol. 1985; 116: 151-170PubMed Google Scholar, 5Yáñez R.J. Rodrı́guez J.M. Nogal M.L. Yuste L. Enrı́quez C. Rodriguez J.F. Viñuela E. Virology. 1995; 208: 249-278Crossref PubMed Scopus (341) Google Scholar), 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 (6Simón-Mateo C. Andrés G. Viñuela E. EMBO J. 1993; 12: 2977-2987Crossref PubMed Scopus (57) Google Scholar, 7Simón-Mateo C. Andrés G. Almazán F. Viñuela E. J. Virol. 1997; 71: 5799-5804Crossref PubMed Google Scholar). 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 (8Andrés G. Simón-Mateo C. Viñuela E. J. Virol. 1997; 71: 2331-2341Crossref PubMed Google Scholar). 2G. Andrés, unpublished results. All the proteolytic cleavages occur after the second Gly of the consensus sequence Gly-Gly-Xaa, which is also recognized as a cleavage site in the maturation of adenovirus structural proteins and in some cellular proteins, including polyubiquitin (9López-Otı́n C. Simón-Mateo C. Martı́nez L. Viñuela E. J. Biol. Chem. 1989; 264: 9107-9110Abstract Full Text PDF PubMed Google Scholar) and ubiquitin-like proteins (10Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). A similar cleavage site (Ala-Gly-Xaa) is used for the maturation of vaccinia virus structural proteins (11VanSlyke J.K. Whitehead S.S. Wilson E.M. Hruby D.E. Virology. 1991; 183: 467-478Crossref PubMed Scopus (70) Google Scholar, 12Lee P. Hruby D.E. J. Biol. Chem. 1994; 269: 8616-8622Abstract Full Text PDF PubMed Google Scholar). Although the adenovirus protease that processes at Gly-Gly-Xaa sites is well characterized (13Webster A. Hay R.T. Kemp G. Cell. 1993; 72: 97-104Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 14Mangel W.F. Toledo D.L. Brown M.T. Martin J.H. McGrath W.J. J. Biol. Chem. 1996; 271: 536-543Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 15Ding J. McGrath W.J. Sweet R.M. Mangel W.F. EMBO J. 1996; 15: 1778-1783Crossref PubMed Scopus (116) Google Scholar), the enzymes involved in the processing of the ASFV polyproteins or in the cleavage of vaccinia virus precursor proteins have not yet been identified. Recently, Li and Hochstrasser (16Li S.-J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (604) Google Scholar) have described a novel cysteine protease from yeast, designated Ulp-1, that catalyzes the release of the ubiquitin-like protein SUMO-1/Smt3 from its precursor or from sumoylated proteins, with the cleavage taking place at Gly-Gly-Xaa sequences. Since the publication of this report, other SUMO-1-specific proteases similar to the yeast enzyme have been identified in vertebrates (17Suzuki T. Ichiyama A. Saitoh H. Kawakami T. Omata M. Chung C.H. Kimura M. Shimbara N. Tanaka K. J. Biol. Chem. 1999; 274: 31131-31134Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 18Gong L. Millas S. Maul G.G. Yeh E.T.H. J. Biol. Chem. 2000; 275: 3355-3359Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 19Kim K.I. Baek S.H. Jeon Y-J. Nishimori S. Suzuki T. Uchida S. Shimbara N. Saitoh H. Tanaka K. Chung C.H. J. Biol. Chem. 2000; 275: 14102-14106Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). These proteases would regulate the SUMO-1-modified state of certain proteins whose function in a variety of cellular processes, such as cell cycle progression and nuclear import, is dependent on this modification (20Yeh E.T.H. Gong L. Kamitani T. Gene (Amst.). 2000; 248: 1-14Crossref PubMed Scopus (418) Google Scholar). Li and Hochstrasser (16Li S.-J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (604) Google Scholar) have also shown that a “core domain” of ∼90 amino acids of the yeast protease, containing the conserved catalytic cysteine and histidine residues, presents some similarity to gene products from several animal viruses, including the adenovirus L3 protease, the I7 products of fowlpox and vaccinia virus, and open reading frame (ORF) S273R of ASFV. This suggested that the S273R product might be the protease involved in the processing of the ASFV polyproteins. We show here, using an expression system in COS cells, the specific processing of polyproteins pp62 and pp220 in the presence of protein pS273R. Furthermore, inhibitor profiling and site-directed mutagenesis results indicate that this protein is a cysteine protease. Studies on the synthesis and localization of protein pS273R during ASFV infection are also presented. The results are consistent with a role for the virus protease in the assembly of the virus core. Vero and COS-7 cells were obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium containing 5% fetal calf serum. The Vero-adapted ASFV strain BA71V was propagated and titrated as described previously (21Enjuanes L. Carrascosa A.L. Moreno M.A. Viñuela E. J. Gen. Virol. 1976; 32: 471-477Crossref PubMed Scopus (177) Google Scholar). Highly purified ASFV was prepared as described (22Carrascosa A.L. del Val M. Santarén J.F. Viñuela E. J. Virol. 1985; 54: 337-344Crossref PubMed Google Scholar). The recombinant vaccinia virus vTF7-3 expressing bacteriophage T7 RNA polymerase (23Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (1874) Google Scholar) was kindly donated by Dr. B. Moss. Plasmid pRSET-S273R carrying the ASFV S273R gene as an N-terminal hexahistidine fusion was obtained as follows. The S273R gene was amplified by polymerase chain reaction using oligonucleotides 273-PCR1 (5′-CGCGCGCTCGAGATGTCTATATTAGAAAAAATTACGT) and 273-PCR2 (5′-GCGCGCGAATTCTTATGCGATGCGAAACAGATGGGT), digested with XhoI and EcoRI, and cloned into the pRSET-A vector (Invitrogen). The S273R gene was subcloned from pRSET-S273R into the T7 promoter containing mammalian expression vector pcDNA 3.1 (Invitrogen), and the recombinant plasmid pcDNA-S273R was used for activity assays in transfection experiments. Site-directed mutagenesis of the S273R gene was by polymerase chain reaction S. M. Gene (Amst.). PubMed Scopus Google Scholar) using as a the pcDNA-S273R The histidine at was to and cysteine was to to the S273R gene were cloned into the pcDNA 3.1 and the presence of the mutation and of were by of the The recombinant plasmid containing which encodes the pp62 polyprotein of amino acids the of the T7 polymerase was obtained as described C. Andrés G. Almazán F. Viñuela E. J. Virol. 1997; 71: 5799-5804Crossref PubMed Google Scholar). The plasmid used for the of polyprotein pp62 and its mature p35 was obtained as follows. The plasmid was digested with with and digested with and in with this recombinant plasmid the amino residues of pp62 This plasmid was digested with XhoI to the of amino residues Plasmid was by the from plasmid (6Simón-Mateo C. Andrés G. Viñuela E. EMBO J. 1993; 12: 2977-2987Crossref PubMed Scopus (57) Google Scholar) and the of the of the ASFV R.J. Rodrı́guez J.M. Nogal M.L. Yuste L. Enrı́quez C. Rodriguez J.F. Viñuela E. Virology. 1995; 208: 249-278Crossref PubMed Scopus (341) Google Scholar) into vector The plasmid the polyprotein the of the T7 polymerase protein cells the pRSET-S273R plasmid were for with and the recombinant protein was purified using the purified recombinant pS273R protein were in The which the ASFV polyprotein pp220 and its mature product p150, was used in the This as well as the and which also polyprotein have been previously characterized (6Simón-Mateo C. Andrés G. Viñuela E. EMBO J. 1993; 12: 2977-2987Crossref PubMed Scopus (57) Google Scholar, G. Simón-Mateo C. Viñuela E. J. Virol. 1997; 71: 2331-2341Crossref PubMed Google A. B. Nogal M.L. Viñuela E. L. J. Virol. 1985; 54: PubMed Google Scholar). The proteins pp62 and p35 was the recombinant protein by plasmid E. cells were for with and for with and the containing the recombinant protein as the was used to in The ASFV polyprotein pp62 and its mature product been described C. Andrés G. Almazán F. Viñuela E. J. Virol. 1997; 71: 5799-5804Crossref PubMed Google Scholar). COS-7 cells were with of cells of the in using the for at In the of pcDNA-S273R of that of the polyprotein the cells were with vTF7-3 at a of infection of the expression of T7 RNA polymerase and of the polyprotein and S273R The infection was in the presence of to vaccinia virus late protein The expression and processing of the polyprotein was at by as COS-7 cells were as described using the the cells were at in containing and a The was at for to and cells, and the was at for at to the from the The activity assays in were using the cytoplasmic as of protein pS273R and polyprotein pp62 and the cytoplasmic as a of polyprotein were at in a of containing of protease and in the the were to and by inhibitor the were for as in the or in the presence of the protease or l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane All the were prepared as a in with the of and prepared in and of cell or were on and to The were with a of the and with a of and the proteins were with the system to the Vero cells were or with ASFV at a of the cells were in the presence of or for RNA was from cells for in the of cell RNA was prepared by the J.M. A.E. R.J. W.J. PubMed Scopus Google Scholar), and RNA was by as described by E. and T. A and Scholar). was as R.J. Rodrı́guez M.L. Viñuela E. J. Gen. Virol. 1993; PubMed Scopus Google Scholar) with of of using as the S273R to to of gene was as described by E. and T. A and Scholar). with the S273R was at to the of RNA and with virus for at The products were in a an DNA sequence Vero cells grown on were or with ASFV at a of infection of and at with at for The cells were with a of and a of as in the to and for at with a of and a of a was were with an Vero cells were with ASFV at a of infection of and at with and in for at the cells were for as by Andrés G. Simón-Mateo C. Viñuela E. J. Virol. 72: PubMed Google Scholar). were for at with a of the by an of at with a of protein were with a the protease activity of protein pS273R, have used a system of COS-7 cells the S273R gene and the genes for polyprotein pp62 and polyprotein which as ASFV polyprotein pp62 is cleaved at a sequence at to produce the structural protein and an precursor of A second cleavage at the site at rise to the mature structural protein p35 of and to an that not been in the cells C. Andrés G. Almazán F. Viñuela E. J. Virol. 1997; 71: 5799-5804Crossref PubMed Google Scholar) processing described in the of also been In this processing place at the cleavage an precursor of of this at the sequence at the two structural proteins and shown in of a specific to the pp62 polyprotein was by using and in from cells with the polyprotein gene and with the polyprotein was with the S273R gene and the pp62 with the of the proteolytic products p35 and which the same as the structural proteins p35 and in the of purified ASFV and In the be with the the protein a specific of which to The expression of gene S273R in the cells is shown in the of B. The protein a of which is consistent with its predicted of J.M. C. Viñuela E. M.L. Virology. PubMed Scopus Google Scholar). shown in protein pS273R was also in purified virus the activity of protein pS273R in from COS-7 cells expressing protein pS273R or polyprotein pp62, prepared as described were and for as in C. the were by using proteins p35 or that in the presence of pS273R polyprotein pp62 is in a and with the formation of and which are to the mature p35 and also be in this the at the at and at the of the mature protein p35 from to of These results as it during ASFV infection C. Andrés G. Almazán F. Viñuela E. J. Virol. 1997; 71: 5799-5804Crossref PubMed Google Scholar), the in the in processing of pp62 by protein pS273R is the of the cleavage site at with the formation of the mature protein and This protein would be cleaved at to produce the mature protein A similar in was to protein pS273R was also to polyprotein pp220 into its mature ASFV polyprotein pp220 is cleaved at the sequence at and the sequence at to rise to the mature product and the precursor protein (6Simón-Mateo C. Andrés G. Viñuela E. EMBO J. 1993; 12: 2977-2987Crossref PubMed Scopus (57) Google Scholar) is cleaved at a sequence at to the mature protein and the precursor which is at a site at to produce the mature products and shown in the products and and the pp220 polyprotein in pp220 containing for in the of protein pS273R. to of the pp220 polyprotein produced in the cells, were with pS273R containing for and the of polyprotein pp220 in a with the and of proteins of of which was after a These proteolytic products with the mature proteins p150, p34, and present in of purified ASFV a protein was also by the protein and proteins a protein was recognized by the The of proteins is consistent with the formation of the precursors and The other mature product not be in the in assays because of the of protein by in the because of a of processing of precursor These results indicate that the two ASFV polyproteins pp220 and pp62 are cleaved in the presence of protein pS273R to rise to the same mature products and precursors as those produced during ASFV by Li and Hochstrasser (16Li S.-J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (604) Google Scholar) and shown in the of the ASFV protein pS273R its the catalytic core domain of the SUMO-1-specific proteases and the adenovirus protease containing the catalytic residues, in the of the ASFV and which are characteristic of cysteine proteases. the ASFV protein was also a cysteine protease, a of specific for the of proteases were using the in described shown in of proteases or proteases on pp62 On the other hand, two cysteine protease and the or the cleavage of the is of to that which is also a specific inhibitor of cysteine not the processing activity of protein pS273R. In this it be that the adenovirus protease is also to this K. M. A. C. J.M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, S. A. J.M. PubMed Scopus Google Scholar). the pS273R protein as a cysteine protease, the conserved cysteine and histidine residues of the catalytic domain were to and and the activity of the proteins was shown in cleavage was with the or that the conserved cysteine and histidine residues are for the activity of protein pS273R. the of gene S273R during ASFV RNA and were with S273R specific for of this to containing RNA from cells and and and late RNA from cells with ASFV a of as well as other in the late RNA a of that was in the of the late RNA and to of at to the of the of A of or residues been identified as a for formation of ASFV F. Rodrı́guez J.M. Andrés G. Viñuela E. Rodriguez J.F. J. Virol. PubMed Google Scholar). The after S273R is found of the for the virus J.M. C. Viñuela E. M.L. Virology. PubMed Scopus Google Scholar). of at this would produce an RNA of which is consistent with the of the RNA by the presence of a specific at the for protein pS273R in ASFV-infected cells This was at a at which viral DNA replication in Vero cells is M.L. J. J.M. A. Viñuela E. Virology. 1986; PubMed Scopus Google Scholar), and to Furthermore, the protein was not in the presence of an inhibitor of viral DNA replication and late that protein pS273R is a late also be in this protein pS273R in the cells with that present in purified virus the localization of pS273R and ASFV-infected Vero cells were at after infection and by with the serum. shown in a was in the cells used as a In the protein pS273R cytoplasmic of the cells which were identified as virus by with a that the polyprotein precursor pp220 and its mature product (8Andrés G. Simón-Mateo C. Viñuela E. J. Virol. 1997; 71: 2331-2341Crossref PubMed Google Scholar). previously the to proteins also virus the (8Andrés G. Simón-Mateo C. Viñuela E. J. Virol. 1997; 71: 2331-2341Crossref PubMed Google Scholar). Although the also cytoplasmic of the assembly that protein pS273R is into the virus in with the with purified virions. in the localization of ASFV protease the virus factories, an of Vero cells with ASFV for were with the by protein shown in the the of the viral factories, in with the results. the assembly was associated with virus and mature were localized the core of icosahedral that the DNA-containing mature the was associated with the core the protein domain the nucleoid and containing the polyprotein products (8Andrés G. Simón-Mateo C. Viñuela E. J. Virol. 1997; 71: 2331-2341Crossref PubMed Google Scholar). A family of cysteine proteases that SUMO-1-modified proteins at consensus Gly-Gly-Xaa been described (16Li S.-J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (604) Google Scholar). The sequence similarity the of this family is to a of amino acids that contains a core domain that is also present in the adenovirus protease, as well as in the I7 protein and protein pS273R of ASFV. This core domain contains the site of the adenovirus protease, of residues and which in the of this enzyme have a to that of the catalytic of the cysteine protease J. 1976; 15: PubMed Scopus Google Scholar), the of the residues in the chain of this enzyme is and In a of the adenovirus protease is in an to the of involved in the formation of the in the The ASFV protein pS273R the and residues, as in the of an at the of the adenovirus protease This sequence similarity that this protein was the protease in the proteolytic processing of ASFV polyproteins at Gly-Gly-Xaa The results show that protein pS273R processes the virus polyproteins in a specific and way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells, that this protein is the protease involved in polyprotein processing during the Furthermore, and as from the sequence the protein been characterized as a cysteine protease, because its activity is by mutation of the and residues of the conserved catalytic domain and is inhibited by sulfhydryl-blocking reagents. into the the ASFV protease be in the same of cysteine proteases as the SUMO-1-specific proteases and the protease from All of proteins are characterized by the presence of N-terminal of which for the of the In to it is that the ASFV protease is the enzyme of this that processes polyprotein precursors several Gly-Gly-Xaa sites in the during ASFV this processing an of proteolytic which may on in the products or on with other proteins during virus morphogenesis (6Simón-Mateo C. Andrés G. Viñuela E. EMBO J. 1993; 12: 2977-2987Crossref PubMed Scopus (57) Google Scholar, 7Simón-Mateo C. Andrés G. Almazán F. Viñuela E. J. Virol. 1997; 71: 5799-5804Crossref PubMed Google Scholar). The identification of the viral protease will to the the processing of the viral polyproteins. is also that the viral enzyme might be to cellular or viral ubiquitin-like modified This is an that may into the structural and of this emerging family of cysteine proteases. The on the expression and localization of protein pS273R in cells with ASFV a role for this protein in the processing of the viral polyproteins during the virus protein pS273R, as its polyprotein is at late of infection and is localized the cytoplasmic viral where morphogenesis We have previously shown that the mature proteins p150, p37, p34, and p14, products of polyprotein are major components of the core shell, of the protein of the virus particle (8Andrés G. Simón-Mateo C. Viñuela E. J. Virol. 1997; 71: 2331-2341Crossref PubMed Google Scholar). The same been found for the structural proteins p35 and p15, which are derived from polyprotein These with the localization of protein pS273R in the virus a role for the ASFV protease in the assembly of the viral core. proteolytic processing might be an essential for the of the core in such a way that and the assembly In with it be that other complex DNA-containing viruses, such as adenoviruses and poxviruses, proteolytic processing at similar or cleavage for the maturation of major core In the of poxviruses, the cleavage of core precursors is for the formation of virus which not place the processing is B. J. Biol. PubMed Scopus Google Scholar). In the adenovirus protease in virions, for maturation into J. J. Virol. 1976; PubMed Google Scholar). that processing of the viral precursor proteins place after virion this enzyme employs a and complex of its activity by and DNA (13Webster A. Hay R.T. Kemp G. Cell. 1993; 72: 97-104Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 14Mangel W.F. Toledo D.L. Brown M.T. Martin J.H. McGrath W.J. J. Biol. Chem. 1996; 271: 536-543Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). will be to similar to the and activity of the ASFV protease. also been described for the adenovirus protease and the I7 protein of Virology. PubMed Scopus Google Scholar, E.M. S. J. Virol. 1993; PubMed Google Scholar), the ASFV protein pS273R is present in the core of mature virus This a presence of an activity during the of ASFV is that protein pS273R is involved in some of viral This is the for the adenovirus protease that been in virus into the cell and of the virus M. J.M. Virology. 1995; PubMed Scopus Google P. J. A. EMBO J. 1996; 15: PubMed Scopus Google Scholar). the ASFV protease, a role late in the virus might also other viral or cellular in infection is a that to be We J. and C. for reading of the