Leporipoxvirus Cu,Zn-Superoxide Dismutase (SOD) Homologs Are Catalytically Inert Decoy Proteins That Bind Copper Chaperone for SOD

Abstract

Many Chordopoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD). The biological function of these proteins is unknown, although the proteins encoded by Leporipoxviruses have been shown to promote a slow decline in the level of superoxide dismutase activity in virus-infected cells. To gain more insights into their function, we have further characterized the enzymatic and biochemical properties of a SOD homolog encoded by Shope fibroma virus. Shope fibroma virus SOD has retained the zinc binding properties of its cellular homolog, but cannot bind copper. Site-directed mutagenesis showed that it requires at least four amino acid substitutions to partially restore copper binding activity, but even these changes still did not restore catalytic activity. Reciprocal co-immunoprecipitation experiments showed that recombinant Shope fibroma virus SOD forms very stable complexes with cellular copper chaperones for SOD and these observations were confirmed using glutathione-S-transferase tagged proteins. Similar viral SOD/chaperone complexes were formed in cells infected with a closely related myxoma virus, where we also noted that some of the SOD antigen co-localizes with mitochondrial markers using confocal fluorescence microscopy. About 2% of the viral SOD was subsequently detected in gradient-purified mitochondria extracted from virus-infected cells. These poxviral SOD homologs do not form stable complexes with cellular Cu,Zn-SOD or affect its concentration. We suggest that Leporipoxvirus SOD homologs are catalytically inert decoy proteins that are designed to interfere in the proper metallation and activation of cellular Cu,Zn-SOD. This reaction might be advantageous for tumorigenic poxviruses, since higher levels of superoxide have been proposed to have anti-apoptotic and tumorigenic activity. Many Chordopoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD). The biological function of these proteins is unknown, although the proteins encoded by Leporipoxviruses have been shown to promote a slow decline in the level of superoxide dismutase activity in virus-infected cells. To gain more insights into their function, we have further characterized the enzymatic and biochemical properties of a SOD homolog encoded by Shope fibroma virus. Shope fibroma virus SOD has retained the zinc binding properties of its cellular homolog, but cannot bind copper. Site-directed mutagenesis showed that it requires at least four amino acid substitutions to partially restore copper binding activity, but even these changes still did not restore catalytic activity. Reciprocal co-immunoprecipitation experiments showed that recombinant Shope fibroma virus SOD forms very stable complexes with cellular copper chaperones for SOD and these observations were confirmed using glutathione-S-transferase tagged proteins. Similar viral SOD/chaperone complexes were formed in cells infected with a closely related myxoma virus, where we also noted that some of the SOD antigen co-localizes with mitochondrial markers using confocal fluorescence microscopy. About 2% of the viral SOD was subsequently detected in gradient-purified mitochondria extracted from virus-infected cells. These poxviral SOD homologs do not form stable complexes with cellular Cu,Zn-SOD or affect its concentration. We suggest that Leporipoxvirus SOD homologs are catalytically inert decoy proteins that are designed to interfere in the proper metallation and activation of cellular Cu,Zn-SOD. This reaction might be advantageous for tumorigenic poxviruses, since higher levels of superoxide have been proposed to have anti-apoptotic and tumorigenic activity. Cytosolic Cu,Zn-superoxide dismutase (Cu,Zn-SOD) 1The abbreviations used are: Cu,Zn-SOD, cytosolic Cu,Zn-superoxide dismutase; CCS, copper chaperone for superoxide dismutase; hCCS, human CCS; GST, glutathione S-transferase; MYX, myxoma virus; PBS, phosphate-buffered saline; SFV, Shope fibroma virus; BGMK, Buffalo green monkey kidney; SIRC, Statens Seruminstitut rabbit cornea; Mn-SOD, manganese-superoxide dismutase; DTT, dithiothreitol; TNF, tumor necrosis factor. is a homodimeric enzyme that catalyzes the dismutation of superoxide radical (1McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). This activity protects cells from oxidative stress and the importance of the enzyme is illustrated by the effects of Cu,Zn-SOD expression levels on aging (2Parkes T.L. Elia A.J. Dickinson D. Hilliker A.J. Phillips J.P. Boulianne G.L. Nat. Genet. 1998; 19: 171-174Crossref PubMed Scopus (696) Google Scholar) and the association of SOD1 mutations with cases of human familial amyotrophic lateral sclerosis (3Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. et al.Nature. 1993; 362: 59-62Crossref PubMed Scopus (5568) Google Scholar). Superoxide dismutases are also expected to serve some role in modulating innate immunity, because O2·¯ is one of several toxicants employed by the cellular arm of the immune system to kill bacteria and virus-infected cells (4Weiss S.J. King G.W. LoBuglio A.F. Am. J. Hematol. 1978; 4: 1-8Crossref PubMed Scopus (23) Google Scholar, 5Mills E.L. Debets-Ossenkopp Y. Verbrugh H.A. Verhoef J. Infect. Immun. 1981; 32: 1200-1205Crossref PubMed Google Scholar). 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Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). in the do not formation J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar) but might be expected to some or of the binding in these proteins. The proteins encoded by fibroma virus and myxoma virus and by the are also was very to the of these by many the or the virus to have on in or on in F. Tscharke D.C. Smith G.L. J. Virol. 2001; 75: 7018-7029Crossref PubMed Scopus (48) Google Scholar, 16Cao J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). The biological that has been noted is that Leporipoxvirus Cu,Zn-SOD homologs SOD” and the activity of cellular Cu,Zn-SOD in J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). This is to Leporipoxvirus SOD homologs, but and the or proteins might the activity of cellular Cu,Zn-SOD is These observations several poxviral Cu,Zn-SOD homologs that insights into the function of these proteins. these have process of and has a of a catalytically inactive are the mutations for The to a superoxide dismutase at in the been of some for these it is from a of the of viral and cellular Cu,Zn-SOD homologs, the many amino acid substitutions and have on the binding properties of these proteins. the viral the to bind and function as poxviral Cu,Zn-SODs many of the amino that form the cellular Cu,Zn-SOD and Cu,Zn-SOD and these poxviral proteins have retained the to with cellular Cu,Zn-SOD CCS, a mechanism by the level of cellular superoxide dismutase activity. the that we that Leporipoxvirus SOD homologs have the to bind copper but the to bind the copper chaperone and bind These biochemical properties suggest a in myxoma and Shope fibroma viruses might be using Cu,Zn-SOD homologs to of O2·¯ and promote the of virus-infected cells. and BGMK, and were in with J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). virus and virus were from the and a was from P. of The of a recombinant myxoma virus a of Cu,Zn-SOD and a virus Cu,Zn-SOD the of a is J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). were on or cells. of recombinant Cu,Zn-SOD in cells was by or cells with at a of of virus J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). and Site-directed J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar) the Cu,Zn-SOD with glutathione in mutagenesis and were used to amino acid substitutions into the SOD of the the used and the amino acid amino acid substitutions were in the proteins and The was also into the a were to the of the mutations and to that mutations were a to human copper chaperone for SOD (CCS) in was from J. of D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). expression used forms of proteins by of acid in a and of proteins were a were to of one at in the of and with and were to of and the cells were by The was with phosphate-buffered and with a in and The was by and proteins were by using a were with glutathione in proteins were at in with one of of proteins were of at was using a and a and the copper and zinc were using a Cu,Zn-SOD was used as a and for The of was also and we detected of zinc and copper of Cu,Zn-SOD dismutase activity was by the of of by J. 2000; PubMed Scopus Google Scholar). the and in of PBS, was to of and The was for at and was of activity is the of to the of by used from bacteria a About of was to of of and at for The were by with PBS, and with of in of at for The were with PBS, and the complexes were by of DTT, 2% and at for Cu,Zn-SOD was used as a used from virus-infected cells. The cells were by with a and and the was by The was at with rabbit and of and by The of the was and of was with of rabbit or SOD and at for were used to the complexes that were with were from the by for in of These were with and were using Evans D.H. J. Virol. 1993; PubMed Google Scholar) and to a as by the of the The was with in and with SOD or in for at The was with and with to in for at The were and was detected using a and The SOD and SOD homologs but not cellular Cu,Zn-SOD J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). The rabbit were by J. D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). human and P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and do not with cellular Cu,Zn-SOD. Cu,Zn-SOD was from and also did not with viral SOD homologs not confocal was used to the of SOD cells were on infected with myxoma virus at a of of and was to the at and the was for at The cells were for with in with PBS, and a at with in and were at The cells were for with and for with a SOD 2% The cells were and for with a 2% and in a and of cells were infected with virus, at a of of and of at was to and the cells were with a on were by a of and was with These were a a mitochondrial and a mitochondrial The was the were further gradient-purified using the of and Fridovich A. Fridovich I. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) as by of was with in the to were by of of of and of in The mitochondrial was in of and at for at in a formed at the and of mitochondria was from the by A. Fridovich I. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The of these was by was used as a cytosolic as a rabbit as a and as a mitochondrial SOD was detected using rabbit and from of and Cu,Zn-SOD a of the amino acid of the proteins encoded by with the of Cu,Zn-SOD and the of human This the in the that to have the of and These be expected to the shown from of Cu,Zn-SOD S. B. M. J. Biol. PubMed Scopus Google Scholar) to form the zinc binding and in of virus to a of a the and to have retained many of the structural of including the amino that form the SOD SOD has also retained homologs of the amino that in the A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 751-755Crossref PubMed Scopus (253) Google Scholar) form and Cu,Zn-SOD The for binding zinc have also been in but at least one of the large The to form also to have been by a and by the of the and is expected to in S. B. M. J. Biol. PubMed Scopus Google Scholar). Similar changes the encoded by myxoma virus and to viruses to the the encoded by is to cellular Cu,Zn-SOD to for it catalytically and M. et A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 751-755Crossref PubMed Scopus (253) Google Scholar) noted that a SOD·CCS heterodimer to of and in the SOD is in the SOD·CCS by the SOD and of and that amino acid in the Leporipoxvirus SOD homologs a very to that of in and to the of the cellular SOD homologs encode a that be to form with viral be to and the of the where it be to form with Site-directed and of gain some insights into the of these amino acid substitutions upon the enzymatic and properties of we a of amino acid mutations into the in to the to more closely a catalytically superoxide The are shown in I. The to the was to restore some for with the at upon of the of the copper binding in the and substitutions were expected to restore the and the was to restore a that of the Cu,Zn-SOD catalytic These with the were in as using and with to the proteins These proteins were as proteins in and for the The of the was upon the expression of but was by and to the SOD as a Superoxide to four amino acid mutations into we were to restore dismutase activity to the used a in to that the of the activity of or human Cu,Zn-SOD J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). a more we that the of the activity of the enzyme changes in level of activity were with the of of the amino acid even in the also to the level of activity or SOD was from the and the proteins with on the activity not We that at least and probably mutations have the of the and these mutations the encoded inactive as a superoxide SOD but was used to the copper and zinc of the recombinant proteins These experiments be by the of levels of was to the recombinant proteins and copper or were that detected of and of Cu,Zn-SOD, and we was to the from the or be noted that we also several of including the copper and zinc of the The of the proteins to and some that the are not to in the of These showed that SOD of zinc of SOD and of was by and substitutions into the to the Cu,Zn-SOD the Leporipoxvirus proteins copper with of the forms binding of copper of the for copper of these dismutase activity it is that the also activity even a of the was in a Cu-Zn The Cu,Zn-SOD, it of We detected of zinc and of copper of for these properties of the is that copper still not be into the it be zinc from the and binding the of activity. Leporipoxviruses Cu,Zn-SOD with experiments showed that Leporipoxvirus SOD homologs have retained the of cellular and proteins also form J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar) their cellular These observations that the proteins might have partially the and properties of Cu,Zn-SODs even not have retained the catalytic activity the of viral We were to and proteins a of cellular the to form stable complexes with copper chaperones for SOD of and co-immunoprecipitation showed that is binding in and in the of experiments we cells with recombinant viruses and a encoded the the of a in the of a virus the expression of large of SOD J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). were from these cells and with a of to and on The was with glutathione and using and a was using a for SOD The extracted SOD from of the was from infected did bind to SOD to form complexes with To further the of and or were on and with recombinant proteins were from the and by and a was using cells have a monkey the monkey from and virus-infected The SOD from a virus-infected but not from the the SOD SOD from virus-infected and the monkey from the was not detected in from infected the of the SOD more was detected in virus-infected cells in infected cells and We do not the for but it to be a of virus to these The recombinant SOD is using to the that we were a binding reaction we for the and of SOD or not We also for in cells infected with myxoma virus where the level of expression of SOD is expected to more closely a We to myxoma virus for these because SOD is to SOD proteins amino acid and a myxoma virus J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar) was for as a The SOD with SOD in cells and the monkey from the The SOD did not from cells infected with virus. the the from of the but SOD from cells infected with virus these that Leporipoxvirus Cu,Zn-SOD homologs form and stable with cellular even in Leporipoxvirus Cu,Zn-SOD or the of This and have to still bind human and monkey CCS, it the these proteins also form stable with cellular Cu,Zn-SOD. We were to complexes formed SOD and cellular Cu,Zn-SOD as by We also noted that virus in the level of Cu,Zn-SOD the that is with a decline in the activity of cellular Cu,Zn-SOD J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). We these do are to cellular Cu,Zn-SOD or cannot the do these viruses affect the of Cu,Zn-SOD in infected of Leporipoxvirus SOD in experiments where viral Cu,Zn-SOD homologs might be their effects on the copper The of the myxoma was using confocal and the SOD experiments detected a of mitochondrial This was confirmed using a where infected cells were with the SOD and a some SOD antigen was the by a of the SOD was to with the mitochondrial and was the not SOD antigen was detected in cells infected with the virus or in infected cells. We also to cellular using the a and in and the gain to the the of experiments not it was that the characterized viral Cu,Zn-SOD homologs and cellular CCS, in the further the of viral SOD in infected we used and to including mitochondria A. Fridovich I. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). from the were using and to be of by and The still detected of the antigen in of the from virus-infected cells. these it is to be that proteins diffusion from their the of from the cells and on these we that 2% of the viral SOD was in the mitochondrial SOD was detected in cells infected with the virus, the or of SOD on the of cellular Cu,Zn-SOD detected in the mitochondria from cells infected with or encode a of proteins that serve to the of many of these virus encode homologs of cellular and of these cellular have been retained by the viral insights into its is the many encoded by poxviruses, the properties of their cellular homologs but the and Genet. 1999; PubMed Scopus Google Scholar). These proteins function as in the that kill infected cells. also encode a of binding by binding a of interfere in the of to of or McFadden G. J. Biol. 2002; Google Scholar). and to or to kill infected cells and it is not that might encode homologs of the Cu,Zn-SODs that a role in are stable in because encode DNA J. Biol. Chem. Full Text PDF PubMed Google Scholar). and F. Tscharke D.C. Smith G.L. J. Virol. 2001; 75: 7018-7029Crossref PubMed Scopus (48) Google Scholar, 16Cao J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar) although have retained forms of SOD homologs several of these have not of the dismutase at least four probably amino acid substitutions have and Cu,Zn-SOD homologs catalytically These mutations substitutions that interfere in copper binding and be expected to have a on activity the role of in these are not the catalytically mutations is by the although the be partially in forms of the not restore catalytic activity SOD was to the properties of its cellular homologs of the Cu,Zn-SOD homologs have the We suggest that the zinc atom the structural role in SOD as it cellular Cu,Zn-SOD and that was at some in the of the proteins. might a encode forms of and SOD homologs form complexes with cellular Cu,Zn-SODs or affect the level of but do with their cellular Cu,Zn-SOD homologs the to bind cellular copper chaperone for SOD We suggest that Cu-Zn homologs are that have retained a to bind to cellular CCS, and in interfere with metallation This virus and cellular for the of Cu,Zn-SOD and a decline in dismutase activity even levels of Cu,Zn-SOD antigen SOD homologs are and in the in F. Tscharke D.C. Smith G.L. J. Virol. 2001; 75: 7018-7029Crossref PubMed Scopus (48) Google Scholar, 16Cao J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar), and Cu,Zn-SOD activity is in virus-infected a one a in the activity of Cu,Zn-SOD the of viral J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar). The of SOD homologs are also with one at the of and it is that many amino are in the with is to in the formation of a viral SOD homolog to the A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 751-755Crossref PubMed Scopus (253) Google Scholar). is probably not for stable formation A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 751-755Crossref PubMed Scopus (253) Google Scholar), the of be one more formation of complexes at the of cellular Cu,Zn-SOD. 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Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) proposed that cells the level of superoxide dismutase activity by that activity depend upon a proteins. have a SOD into cellular and we are a system that the of on the chaperone activity of The properties of these proteins suggest that gain some from with the dismutation of superoxide radical and cellular might the of O2·¯ a several the to the formed by and in P.J. The of the Google Scholar). Superoxide have effects on upon the but it is that O2·¯ a the of superoxide and I. J. PubMed Scopus Google Scholar, S. 2002; PubMed Scopus Google Scholar). it is that some of the SOD co-localizes with the mitochondrial has been that it is the form of cellular Cu,Zn-SOD that is into the mitochondrial where it is by a copper reaction L.T. Culotta V.C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar). is a viral SOD homolog to the might be expected to the of Cu,Zn-SOD the mitochondrial and have effects on in the biological for these viruses to encode a of O2·¯ in virus-infected because that promote the cellular of Leporipoxvirus be noted that we not effects of on the of myxoma virus J.X. Teoh M.L. Moon M. McFadden G. Evans D.H. Virology. 2002; 296: 125-135Crossref PubMed Scopus (28) Google Scholar), but a tumor might not have been to using is a to and a and with in its myxoma a very with large P.J. The of the Google Scholar), and these might the effects of a be a in that more closely the in its and we are the that a role in using We J. for the of J. Phillips for and and for very Smith and Y. and on and confocal microscopy.