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W2108018047 abstract "Cytoskeletal proteins are exploited by many viruses during infection. We report a novel finding that actin can act as a cofactor for the adenovirus proteinase (AVP) in the degradation of cytoskeletal proteins. Transfection studies in HeLa cells revealed AVP localized with cytokeratin 18, and this was followed by destruction of the cytokeratin network. For AVP to cleave cytokeratin 18, a cellular cofactor was shown to be required, consistent with AVP being synthesized as an inactive proteinase. Actin was considered a cellular cofactor for AVP, because the C terminus of actin is homologous to a viral cofactor for AVP. AVP was shown to bind to the C terminus of actin, and in doing so AVP exhibited full enzymatic activity. In vitro, actin was a cofactor in the cleavage of cytokeratin 18 by AVP. The proteinase alone could not cleave cytokeratin 18, but in the presence of actin, AVP cleaved cytokeratin 18. Indeed, actin itself was shown to be a cofactor and a substrate for its own destruction in that it was cleaved by AVP in vitro. Cleavage of cytoskeletal proteins weakens the structure of the cell, and therefore, actin as a cofactor may play a role in cell lysis and release of nascent virions. Cytoskeletal proteins are exploited by many viruses during infection. We report a novel finding that actin can act as a cofactor for the adenovirus proteinase (AVP) in the degradation of cytoskeletal proteins. Transfection studies in HeLa cells revealed AVP localized with cytokeratin 18, and this was followed by destruction of the cytokeratin network. For AVP to cleave cytokeratin 18, a cellular cofactor was shown to be required, consistent with AVP being synthesized as an inactive proteinase. Actin was considered a cellular cofactor for AVP, because the C terminus of actin is homologous to a viral cofactor for AVP. AVP was shown to bind to the C terminus of actin, and in doing so AVP exhibited full enzymatic activity. In vitro, actin was a cofactor in the cleavage of cytokeratin 18 by AVP. The proteinase alone could not cleave cytokeratin 18, but in the presence of actin, AVP cleaved cytokeratin 18. Indeed, actin itself was shown to be a cofactor and a substrate for its own destruction in that it was cleaved by AVP in vitro. Cleavage of cytoskeletal proteins weakens the structure of the cell, and therefore, actin as a cofactor may play a role in cell lysis and release of nascent virions. During viral infections, different properties of actin are exploited (1Cudmore S. Reckmann I. Way M. Trends Microbiol. 1997; 5: 142-148Abstract Full Text PDF PubMed Scopus (133) Google Scholar). Actin has been shown to play a role in the transcription of several paramyxoviridae genomes. Actin stimulates human parainfluenza virus type 3 transcription; depletion of actin abolishes viral mRNA synthesis (2De B.P. Lesoon A. Banerjee A.K. J. Virol. 1991; 65: 3268-3275Crossref PubMed Google Scholar). A hallmark of oncogenic transformation by RNA tumor viruses is the loss of cytoskeletal integrity resulting from the disappearance of actin stress fibers, perturbation of focal adhesions, and aggregation of actin near the ventral surface of the transformed cell (3Boschek C.B. Jockusch B.M. Friis R.R. Back R. Grundmann E. Bauer H. Cell. 1981; 24: 175-184Abstract Full Text PDF PubMed Scopus (135) Google Scholar). In the case of human immunodeficiency virus, the Gag protein, which is both necessary and sufficient for viral budding, is associated with the actin cytoskeleton in vitro (4Rey O. Canon J. Krogstad P. Virology. 1996; 220: 530-534Crossref PubMed Scopus (100) Google Scholar), and their association at the plasma membrane may play a role in the budding of retroviruses. During baculovirus infection byAutographa californica M nuclear polyhedrosis virus, there is a dramatic rearrangement and eventual destruction of the actin cytoskeleton (5Lanier L.M. Slack J.M. Volkman L.E. Virology. 1996; 216: 380-388Crossref PubMed Scopus (45) Google Scholar). The virus encodes a proteinase that specifically degrades actin. Here we reveal another property of actin that is exploited by a virus; actin can act as a cofactor to stimulate a virus-coded proteinase.Throughout an adenovirus infection, the actin, cytokeratin, tubulin, and vimentin networks that make up the cytoskeleton of the cell undergo dramatic changes (6Staufenbiel M. Epple P. Deppert W. J. Virol. 1986; 60: 1186-1191Crossref PubMed Google Scholar). Chen et al. (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar) have shown that late in an adenovirus infection, cytokeratin 18 is cleaved at two contiguous adenovirus proteinase (AVP) 1The abbreviations used for: AVP, adenovirus proteinase; pVIc, 11-amino acid peptide, GVQSLKRRRCF, that originates from the C terminus of the viral precursor protein pVI; TBS, Tris-buffered saline; GFP, green fluorescent protein. 1The abbreviations used for: AVP, adenovirus proteinase; pVIc, 11-amino acid peptide, GVQSLKRRRCF, that originates from the C terminus of the viral precursor protein pVI; TBS, Tris-buffered saline; GFP, green fluorescent protein.consensus cleavage sequences, leading to the destruction of the cytokeratin network. In cells infected by a temperature-sensitive mutant of adenovirus that lacks proteinase activity at the non-permissive temperature, cytokeratin 18 is not cleaved, and the cytokeratin network remains intact.This observation raises a conundrum. Cleavage of cytokeratin 18 by AVP takes place in the cytoplasm, yet the proteinase is synthesized in an inactive form and is activated in the nucleus by two viral cofactors within immature virions. One cofactor is pVIc, an 11-amino acid peptide that originates from the C terminus of the precursor to protein VI, pVI (8Mangel W.F. McGrath W.J. Toledo D.L. Anderson C.W. Nature. 1993; 361: 274-275Crossref PubMed Scopus (145) Google Scholar, 9Webster A. Hay R.T. Kemp G. Cell. 1993; 72: 97-104Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 10Baniecki M.L. McGrath W.J. McWhirter S.M. Li C. Toledo D.L. Pellicena P. Barnard D.L. Thorn K.S. Mangel W.F. Biochemistry. 2001; 40: 12349-12356Crossref PubMed Scopus (33) Google Scholar), and the other cofactor is adenovirus DNA (8Mangel W.F. McGrath W.J. Toledo D.L. Anderson C.W. Nature. 1993; 361: 274-275Crossref PubMed Scopus (145) Google Scholar, 11McGrath W.J. Baniecki M.L. Li C. McWhirter S.M. Brown M.T. Toledo D.L. Mangel W.F. Biochemistry. 2001; 40: 13237-13245Crossref PubMed Scopus (31) Google Scholar). Once AVP becomes activated, it cleaves the virion precursor proteins used in the assembly of virus particles, thereby rendering the virus particles infectious (12Weber J.M. Tihanyi K. Methods Enzymol. 1994; 244: 595-604Crossref PubMed Scopus (27) Google Scholar). The two cofactors activate AVP by increasing the specificity constant,k cat/K m, for substrate hydrolysis (11McGrath W.J. Baniecki M.L. Li C. McWhirter S.M. Brown M.T. Toledo D.L. Mangel W.F. Biochemistry. 2001; 40: 13237-13245Crossref PubMed Scopus (31) Google Scholar). Compared with AVP alone, thek cat/K m increases 1,130-fold with an AVP-pVIc complex, 110-fold with an AVP-viral DNA complex, and 34,100-fold in the presence of both pVIc and viral DNA. Presumably, if AVP were synthesized in an active form, it would cleave virion precursor proteins before virion assembly thereby aborting the infection (10Baniecki M.L. McGrath W.J. McWhirter S.M. Li C. Toledo D.L. Pellicena P. Barnard D.L. Thorn K.S. Mangel W.F. Biochemistry. 2001; 40: 12349-12356Crossref PubMed Scopus (33) Google Scholar, 13Rancourt C. Keyvani-Amineh H. Sircar S. Labrecque P. Weber J.M. Virology. 1995; 209: 167-173Crossref PubMed Scopus (53) Google Scholar).In this study the conundrum of how AVP may be activated in the cytoplasm is resolved; a new, cellular cofactor for AVP is described, actin. Cytokeratin 18 could not be cleaved by AVP in vitro. However, cytokeratin 18 could be cleaved by AVP in the cytoplasm of HeLa cells in the absence of other viral proteins. This prompted a search for a cellular cofactor. Actin was considered as a cofactor, because its C terminus shares homology with pVIc. In vitro, upon the binding of AVP to the C terminus of actin, the activity of AVP was greatly stimulated. In vitro, cytokeratin 18 could not be cleaved by AVP alone. Most important, in the presence of actin, cytokeratin 18 could be cleaved by AVP.DISCUSSIONThe experiments presented here resolve a conundrum. During an adenovirus infection, how is cytokeratin 18 cleaved by AVP in the cytoplasm since AVP is synthesized in an inactive form that is later activated in the nucleus within immature virions by two viral cofactors? Clearly, the cytokeratin network is destroyed in vivo during an adenovirus infection (23Zhang Y. Schneider R.J. J. Virol. 1994; 68: 2544-2555Crossref PubMed Google Scholar). The transfection experiments with an AVP-GFP chimeric gene showed that AVP destroyed the cytokeratin network in the absence of any other viral components.In vitro, AVP was not able to cleave cytokeratin 18; however, it was able to utilize actin as a cofactor to cleave cytokeratin 18. Thus, AVP can utilize a cellular protein as a cofactor in the cleavage of cytokeratin 18.The rationale for actin being able to serve as a cofactor for AVP is that its C terminus is highly homologous to the viral cofactor pVIc; of the last 8 amino acid residues of actin, 4 are identical and 3 homologous to the last 8 amino acid residues in pVIc. This homology implied that AVP could bind to the C terminus of actin, and it did. Furthermore, this homology implied that actin, like pVIc, could stimulate the activity of AVP; it did so in a concentration-dependent manner.Are other data consistent with the C terminus of actin behaving like pVIc? Of the last 11 amino acids at the C terminus of actin, the 3 at the N terminus, AGP, are not homologous to those in pVIc, GVQ, whereas the next 8 amino acids are homologous. It has been reported that deletion of GVQ in pVIc results in an inactive cofactor (28Cabrita G. Iqbal M. Reddy H. Kemp G. J. Biol. Chem. 1997; 272: 5635-5639Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). However, we have observed that deletion of GVQ from pVIc yielded a peptide that binds to AVP with only a 3-fold higher K d value and exhibits a 3-fold lower k cat value than that of wild-type pVIc. 2M. L. Baniecki and W. F. Mangel, unpublished observations. Alanine-scanning mutagenesis on pVIc indicates the GtoA mutant has a 13-fold higher K d value for binding to AVP; the VtoA mutant has a 7-fold lower k cat value for substrate hydrolysis, and the QtoA mutant behaves like wild-type pVIc (10Baniecki M.L. McGrath W.J. McWhirter S.M. Li C. Toledo D.L. Pellicena P. Barnard D.L. Thorn K.S. Mangel W.F. Biochemistry. 2001; 40: 12349-12356Crossref PubMed Scopus (33) Google Scholar). It is possible that PRODAN bound to Cys-374 enhanced the binding of AVP to the C terminus of actin. However, AVP binds to underivatized actin with an equilibrium dissociation constant of 4 nm as opposed to the equilibrium dissociation of 1.7 μm with PRODAN-labeled actin. 3M. T. Brown and W. F. Mangel, unpublished observations. Thus, PRODAN bound to Cys-347 of actin actually interferes with the binding of AVP to actin. Additionally, there is direct evidence that AVP binds to the C terminus of actin. We have observed that in the presence of DNA a peptide containing the amino acid sequence of the last 11 amino acids of actin specifically behaves as a cofactor in stimulating AVP and that a peptide with the same amino acids but in a randomly chosen sequence does not stimulate AVP.3There is no facile way to determine the relevance of our observation that actin can act as a cofactor for AVP in vitro to what occurs in vivo in an adenovirus-infected cell. Actin is an essential protein; therefore, a deletion mutant of actin will not be viable. However, given that cytokeratin 18 is cleaved by AVP in vivo (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar), that in vitro AVP will not cleave cytokeratin 18 in the absence of a cofactor, that in vitro actin can act as a cofactor for the cleavage of cytokeratin 18, and that theK d value for the binding of actin to AVP is lower than the in vivo G-actin concentration, it seems very likely that in an adenovirus-infected cell, cytokeratin 18 is cleaved by an actin-AVP complex.During an adenovirus infection AVP is exposed to actin in the cytoplasm. In infected cells, proteinase activity can be detected as early as 14 h post-infection with maximal activity beginning at 20 h (29Bhatti A.R. Weber J. Virology. 1979; 96: 478-485Crossref PubMed Scopus (23) Google Scholar). AVP can be detected in the cytoplasm and nucleus at 24 h post-infection by Western blot (30Webster A. Leith I.R. Hay R.T. J. Virol. 1994; 68: 7292-7300Crossref PubMed Google Scholar). This timing correlates with the disassembly of the cytokeratin system that begins to fall apart at about 16 h post-infection (6Staufenbiel M. Epple P. Deppert W. J. Virol. 1986; 60: 1186-1191Crossref PubMed Google Scholar), disassembly being complete at 36 h (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar).In virus-infected cells, cleavage of cytoskeletal proteins weakens the mechanical structure of the cell, and this may promote cell lysis and release of nascent virions (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar). AVP cleaves cytokeratin 18 within the N-terminal domain yielding a 41-kDa fragment that is incapable of participating in filament elongation. Such fragments significantly inhibit the elongation of cytokeratin filaments, even when the amount of cleaved cytokeratin comprises only 1% of the population. Inspection of the amino acid sequences of other cytoskeletal proteins reveals AVP consensus cleavage sequences in tubulin, vimentin, and even actin itself (Fig. 2 a). The latter observation raised the possibility that actin may be a cofactor for its own destruction, and this was shown to occur.Degradation of cytoskeletal proteins by virus-coded proteinases during lytic infections is not unusual. The rhinovirus 2A proteinase cleaves cytokeratin 8 (31Seipelt J. Liebig H.-D. Sommergruber W. Gerner C. Kuechler E. J. Biol. Chem. 2000; 275: 20084-20089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and other virus-coded proteinases cleave actin (5Lanier L.M. Slack J.M. Volkman L.E. Virology. 1996; 216: 380-388Crossref PubMed Scopus (45) Google Scholar,32Ott D.E. Coren L.V. Kane B.P. Busch L.K. Johnson D.G. Sowder R.C. Chertova E.N. Arthur L.O. Henderson L.E. J. Virol. 1996; 70: 7734-7743Crossref PubMed Google Scholar) and vimentin (33Shoeman R.L. Honer B. Stroller T.J. Kesselmeier C. Miedel M.C. Traub P. Graves M.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6336-6349Crossref PubMed Scopus (141) Google Scholar). What is currently unique about AVP is that it uses actin as a cofactor. During viral infections, different properties of actin are exploited (1Cudmore S. Reckmann I. Way M. Trends Microbiol. 1997; 5: 142-148Abstract Full Text PDF PubMed Scopus (133) Google Scholar). Actin has been shown to play a role in the transcription of several paramyxoviridae genomes. Actin stimulates human parainfluenza virus type 3 transcription; depletion of actin abolishes viral mRNA synthesis (2De B.P. Lesoon A. Banerjee A.K. J. Virol. 1991; 65: 3268-3275Crossref PubMed Google Scholar). A hallmark of oncogenic transformation by RNA tumor viruses is the loss of cytoskeletal integrity resulting from the disappearance of actin stress fibers, perturbation of focal adhesions, and aggregation of actin near the ventral surface of the transformed cell (3Boschek C.B. Jockusch B.M. Friis R.R. Back R. Grundmann E. Bauer H. Cell. 1981; 24: 175-184Abstract Full Text PDF PubMed Scopus (135) Google Scholar). In the case of human immunodeficiency virus, the Gag protein, which is both necessary and sufficient for viral budding, is associated with the actin cytoskeleton in vitro (4Rey O. Canon J. Krogstad P. Virology. 1996; 220: 530-534Crossref PubMed Scopus (100) Google Scholar), and their association at the plasma membrane may play a role in the budding of retroviruses. During baculovirus infection byAutographa californica M nuclear polyhedrosis virus, there is a dramatic rearrangement and eventual destruction of the actin cytoskeleton (5Lanier L.M. Slack J.M. Volkman L.E. Virology. 1996; 216: 380-388Crossref PubMed Scopus (45) Google Scholar). The virus encodes a proteinase that specifically degrades actin. Here we reveal another property of actin that is exploited by a virus; actin can act as a cofactor to stimulate a virus-coded proteinase. Throughout an adenovirus infection, the actin, cytokeratin, tubulin, and vimentin networks that make up the cytoskeleton of the cell undergo dramatic changes (6Staufenbiel M. Epple P. Deppert W. J. Virol. 1986; 60: 1186-1191Crossref PubMed Google Scholar). Chen et al. (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar) have shown that late in an adenovirus infection, cytokeratin 18 is cleaved at two contiguous adenovirus proteinase (AVP) 1The abbreviations used for: AVP, adenovirus proteinase; pVIc, 11-amino acid peptide, GVQSLKRRRCF, that originates from the C terminus of the viral precursor protein pVI; TBS, Tris-buffered saline; GFP, green fluorescent protein. 1The abbreviations used for: AVP, adenovirus proteinase; pVIc, 11-amino acid peptide, GVQSLKRRRCF, that originates from the C terminus of the viral precursor protein pVI; TBS, Tris-buffered saline; GFP, green fluorescent protein.consensus cleavage sequences, leading to the destruction of the cytokeratin network. In cells infected by a temperature-sensitive mutant of adenovirus that lacks proteinase activity at the non-permissive temperature, cytokeratin 18 is not cleaved, and the cytokeratin network remains intact. This observation raises a conundrum. Cleavage of cytokeratin 18 by AVP takes place in the cytoplasm, yet the proteinase is synthesized in an inactive form and is activated in the nucleus by two viral cofactors within immature virions. One cofactor is pVIc, an 11-amino acid peptide that originates from the C terminus of the precursor to protein VI, pVI (8Mangel W.F. McGrath W.J. Toledo D.L. Anderson C.W. Nature. 1993; 361: 274-275Crossref PubMed Scopus (145) Google Scholar, 9Webster A. Hay R.T. Kemp G. Cell. 1993; 72: 97-104Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 10Baniecki M.L. McGrath W.J. McWhirter S.M. Li C. Toledo D.L. Pellicena P. Barnard D.L. Thorn K.S. Mangel W.F. Biochemistry. 2001; 40: 12349-12356Crossref PubMed Scopus (33) Google Scholar), and the other cofactor is adenovirus DNA (8Mangel W.F. McGrath W.J. Toledo D.L. Anderson C.W. Nature. 1993; 361: 274-275Crossref PubMed Scopus (145) Google Scholar, 11McGrath W.J. Baniecki M.L. Li C. McWhirter S.M. Brown M.T. Toledo D.L. Mangel W.F. Biochemistry. 2001; 40: 13237-13245Crossref PubMed Scopus (31) Google Scholar). Once AVP becomes activated, it cleaves the virion precursor proteins used in the assembly of virus particles, thereby rendering the virus particles infectious (12Weber J.M. Tihanyi K. Methods Enzymol. 1994; 244: 595-604Crossref PubMed Scopus (27) Google Scholar). The two cofactors activate AVP by increasing the specificity constant,k cat/K m, for substrate hydrolysis (11McGrath W.J. Baniecki M.L. Li C. McWhirter S.M. Brown M.T. Toledo D.L. Mangel W.F. Biochemistry. 2001; 40: 13237-13245Crossref PubMed Scopus (31) Google Scholar). Compared with AVP alone, thek cat/K m increases 1,130-fold with an AVP-pVIc complex, 110-fold with an AVP-viral DNA complex, and 34,100-fold in the presence of both pVIc and viral DNA. Presumably, if AVP were synthesized in an active form, it would cleave virion precursor proteins before virion assembly thereby aborting the infection (10Baniecki M.L. McGrath W.J. McWhirter S.M. Li C. Toledo D.L. Pellicena P. Barnard D.L. Thorn K.S. Mangel W.F. Biochemistry. 2001; 40: 12349-12356Crossref PubMed Scopus (33) Google Scholar, 13Rancourt C. Keyvani-Amineh H. Sircar S. Labrecque P. Weber J.M. Virology. 1995; 209: 167-173Crossref PubMed Scopus (53) Google Scholar). In this study the conundrum of how AVP may be activated in the cytoplasm is resolved; a new, cellular cofactor for AVP is described, actin. Cytokeratin 18 could not be cleaved by AVP in vitro. However, cytokeratin 18 could be cleaved by AVP in the cytoplasm of HeLa cells in the absence of other viral proteins. This prompted a search for a cellular cofactor. Actin was considered as a cofactor, because its C terminus shares homology with pVIc. In vitro, upon the binding of AVP to the C terminus of actin, the activity of AVP was greatly stimulated. In vitro, cytokeratin 18 could not be cleaved by AVP alone. Most important, in the presence of actin, cytokeratin 18 could be cleaved by AVP. DISCUSSIONThe experiments presented here resolve a conundrum. During an adenovirus infection, how is cytokeratin 18 cleaved by AVP in the cytoplasm since AVP is synthesized in an inactive form that is later activated in the nucleus within immature virions by two viral cofactors? Clearly, the cytokeratin network is destroyed in vivo during an adenovirus infection (23Zhang Y. Schneider R.J. J. Virol. 1994; 68: 2544-2555Crossref PubMed Google Scholar). The transfection experiments with an AVP-GFP chimeric gene showed that AVP destroyed the cytokeratin network in the absence of any other viral components.In vitro, AVP was not able to cleave cytokeratin 18; however, it was able to utilize actin as a cofactor to cleave cytokeratin 18. Thus, AVP can utilize a cellular protein as a cofactor in the cleavage of cytokeratin 18.The rationale for actin being able to serve as a cofactor for AVP is that its C terminus is highly homologous to the viral cofactor pVIc; of the last 8 amino acid residues of actin, 4 are identical and 3 homologous to the last 8 amino acid residues in pVIc. This homology implied that AVP could bind to the C terminus of actin, and it did. Furthermore, this homology implied that actin, like pVIc, could stimulate the activity of AVP; it did so in a concentration-dependent manner.Are other data consistent with the C terminus of actin behaving like pVIc? Of the last 11 amino acids at the C terminus of actin, the 3 at the N terminus, AGP, are not homologous to those in pVIc, GVQ, whereas the next 8 amino acids are homologous. It has been reported that deletion of GVQ in pVIc results in an inactive cofactor (28Cabrita G. Iqbal M. Reddy H. Kemp G. J. Biol. Chem. 1997; 272: 5635-5639Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). However, we have observed that deletion of GVQ from pVIc yielded a peptide that binds to AVP with only a 3-fold higher K d value and exhibits a 3-fold lower k cat value than that of wild-type pVIc. 2M. L. Baniecki and W. F. Mangel, unpublished observations. Alanine-scanning mutagenesis on pVIc indicates the GtoA mutant has a 13-fold higher K d value for binding to AVP; the VtoA mutant has a 7-fold lower k cat value for substrate hydrolysis, and the QtoA mutant behaves like wild-type pVIc (10Baniecki M.L. McGrath W.J. McWhirter S.M. Li C. Toledo D.L. Pellicena P. Barnard D.L. Thorn K.S. Mangel W.F. Biochemistry. 2001; 40: 12349-12356Crossref PubMed Scopus (33) Google Scholar). It is possible that PRODAN bound to Cys-374 enhanced the binding of AVP to the C terminus of actin. However, AVP binds to underivatized actin with an equilibrium dissociation constant of 4 nm as opposed to the equilibrium dissociation of 1.7 μm with PRODAN-labeled actin. 3M. T. Brown and W. F. Mangel, unpublished observations. Thus, PRODAN bound to Cys-347 of actin actually interferes with the binding of AVP to actin. Additionally, there is direct evidence that AVP binds to the C terminus of actin. We have observed that in the presence of DNA a peptide containing the amino acid sequence of the last 11 amino acids of actin specifically behaves as a cofactor in stimulating AVP and that a peptide with the same amino acids but in a randomly chosen sequence does not stimulate AVP.3There is no facile way to determine the relevance of our observation that actin can act as a cofactor for AVP in vitro to what occurs in vivo in an adenovirus-infected cell. Actin is an essential protein; therefore, a deletion mutant of actin will not be viable. However, given that cytokeratin 18 is cleaved by AVP in vivo (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar), that in vitro AVP will not cleave cytokeratin 18 in the absence of a cofactor, that in vitro actin can act as a cofactor for the cleavage of cytokeratin 18, and that theK d value for the binding of actin to AVP is lower than the in vivo G-actin concentration, it seems very likely that in an adenovirus-infected cell, cytokeratin 18 is cleaved by an actin-AVP complex.During an adenovirus infection AVP is exposed to actin in the cytoplasm. In infected cells, proteinase activity can be detected as early as 14 h post-infection with maximal activity beginning at 20 h (29Bhatti A.R. Weber J. Virology. 1979; 96: 478-485Crossref PubMed Scopus (23) Google Scholar). AVP can be detected in the cytoplasm and nucleus at 24 h post-infection by Western blot (30Webster A. Leith I.R. Hay R.T. J. Virol. 1994; 68: 7292-7300Crossref PubMed Google Scholar). This timing correlates with the disassembly of the cytokeratin system that begins to fall apart at about 16 h post-infection (6Staufenbiel M. Epple P. Deppert W. J. Virol. 1986; 60: 1186-1191Crossref PubMed Google Scholar), disassembly being complete at 36 h (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar).In virus-infected cells, cleavage of cytoskeletal proteins weakens the mechanical structure of the cell, and this may promote cell lysis and release of nascent virions (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar). AVP cleaves cytokeratin 18 within the N-terminal domain yielding a 41-kDa fragment that is incapable of participating in filament elongation. Such fragments significantly inhibit the elongation of cytokeratin filaments, even when the amount of cleaved cytokeratin comprises only 1% of the population. Inspection of the amino acid sequences of other cytoskeletal proteins reveals AVP consensus cleavage sequences in tubulin, vimentin, and even actin itself (Fig. 2 a). The latter observation raised the possibility that actin may be a cofactor for its own destruction, and this was shown to occur.Degradation of cytoskeletal proteins by virus-coded proteinases during lytic infections is not unusual. The rhinovirus 2A proteinase cleaves cytokeratin 8 (31Seipelt J. Liebig H.-D. Sommergruber W. Gerner C. Kuechler E. J. Biol. Chem. 2000; 275: 20084-20089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and other virus-coded proteinases cleave actin (5Lanier L.M. Slack J.M. Volkman L.E. Virology. 1996; 216: 380-388Crossref PubMed Scopus (45) Google Scholar,32Ott D.E. Coren L.V. Kane B.P. Busch L.K. Johnson D.G. Sowder R.C. Chertova E.N. Arthur L.O. Henderson L.E. J. Virol. 1996; 70: 7734-7743Crossref PubMed Google Scholar) and vimentin (33Shoeman R.L. Honer B. Stroller T.J. Kesselmeier C. Miedel M.C. Traub P. Graves M.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6336-6349Crossref PubMed Scopus (141) Google Scholar). What is currently unique about AVP is that it uses actin as a cofactor. The experiments presented here resolve a conundrum. During an adenovirus infection, how is cytokeratin 18 cleaved by AVP in the cytoplasm since AVP is synthesized in an inactive form that is later activated in the nucleus within immature virions by two viral cofactors? Clearly, the cytokeratin network is destroyed in vivo during an adenovirus infection (23Zhang Y. Schneider R.J. J. Virol. 1994; 68: 2544-2555Crossref PubMed Google Scholar). The transfection experiments with an AVP-GFP chimeric gene showed that AVP destroyed the cytokeratin network in the absence of any other viral components.In vitro, AVP was not able to cleave cytokeratin 18; however, it was able to utilize actin as a cofactor to cleave cytokeratin 18. Thus, AVP can utilize a cellular protein as a cofactor in the cleavage of cytokeratin 18. The rationale for actin being able to serve as a cofactor for AVP is that its C terminus is highly homologous to the viral cofactor pVIc; of the last 8 amino acid residues of actin, 4 are identical and 3 homologous to the last 8 amino acid residues in pVIc. This homology implied that AVP could bind to the C terminus of actin, and it did. Furthermore, this homology implied that actin, like pVIc, could stimulate the activity of AVP; it did so in a concentration-dependent manner. Are other data consistent with the C terminus of actin behaving like pVIc? Of the last 11 amino acids at the C terminus of actin, the 3 at the N terminus, AGP, are not homologous to those in pVIc, GVQ, whereas the next 8 amino acids are homologous. It has been reported that deletion of GVQ in pVIc results in an inactive cofactor (28Cabrita G. Iqbal M. Reddy H. Kemp G. J. Biol. Chem. 1997; 272: 5635-5639Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). However, we have observed that deletion of GVQ from pVIc yielded a peptide that binds to AVP with only a 3-fold higher K d value and exhibits a 3-fold lower k cat value than that of wild-type pVIc. 2M. L. Baniecki and W. F. Mangel, unpublished observations. Alanine-scanning mutagenesis on pVIc indicates the GtoA mutant has a 13-fold higher K d value for binding to AVP; the VtoA mutant has a 7-fold lower k cat value for substrate hydrolysis, and the QtoA mutant behaves like wild-type pVIc (10Baniecki M.L. McGrath W.J. McWhirter S.M. Li C. Toledo D.L. Pellicena P. Barnard D.L. Thorn K.S. Mangel W.F. Biochemistry. 2001; 40: 12349-12356Crossref PubMed Scopus (33) Google Scholar). It is possible that PRODAN bound to Cys-374 enhanced the binding of AVP to the C terminus of actin. However, AVP binds to underivatized actin with an equilibrium dissociation constant of 4 nm as opposed to the equilibrium dissociation of 1.7 μm with PRODAN-labeled actin. 3M. T. Brown and W. F. Mangel, unpublished observations. Thus, PRODAN bound to Cys-347 of actin actually interferes with the binding of AVP to actin. Additionally, there is direct evidence that AVP binds to the C terminus of actin. We have observed that in the presence of DNA a peptide containing the amino acid sequence of the last 11 amino acids of actin specifically behaves as a cofactor in stimulating AVP and that a peptide with the same amino acids but in a randomly chosen sequence does not stimulate AVP.3 There is no facile way to determine the relevance of our observation that actin can act as a cofactor for AVP in vitro to what occurs in vivo in an adenovirus-infected cell. Actin is an essential protein; therefore, a deletion mutant of actin will not be viable. However, given that cytokeratin 18 is cleaved by AVP in vivo (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar), that in vitro AVP will not cleave cytokeratin 18 in the absence of a cofactor, that in vitro actin can act as a cofactor for the cleavage of cytokeratin 18, and that theK d value for the binding of actin to AVP is lower than the in vivo G-actin concentration, it seems very likely that in an adenovirus-infected cell, cytokeratin 18 is cleaved by an actin-AVP complex. During an adenovirus infection AVP is exposed to actin in the cytoplasm. In infected cells, proteinase activity can be detected as early as 14 h post-infection with maximal activity beginning at 20 h (29Bhatti A.R. Weber J. Virology. 1979; 96: 478-485Crossref PubMed Scopus (23) Google Scholar). AVP can be detected in the cytoplasm and nucleus at 24 h post-infection by Western blot (30Webster A. Leith I.R. Hay R.T. J. Virol. 1994; 68: 7292-7300Crossref PubMed Google Scholar). This timing correlates with the disassembly of the cytokeratin system that begins to fall apart at about 16 h post-infection (6Staufenbiel M. Epple P. Deppert W. J. Virol. 1986; 60: 1186-1191Crossref PubMed Google Scholar), disassembly being complete at 36 h (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar). In virus-infected cells, cleavage of cytoskeletal proteins weakens the mechanical structure of the cell, and this may promote cell lysis and release of nascent virions (7Chen P.H. Ornelles D.A. Shenk T. J. Virol. 1993; 67: 3507-3514Crossref PubMed Google Scholar). AVP cleaves cytokeratin 18 within the N-terminal domain yielding a 41-kDa fragment that is incapable of participating in filament elongation. Such fragments significantly inhibit the elongation of cytokeratin filaments, even when the amount of cleaved cytokeratin comprises only 1% of the population. Inspection of the amino acid sequences of other cytoskeletal proteins reveals AVP consensus cleavage sequences in tubulin, vimentin, and even actin itself (Fig. 2 a). The latter observation raised the possibility that actin may be a cofactor for its own destruction, and this was shown to occur. Degradation of cytoskeletal proteins by virus-coded proteinases during lytic infections is not unusual. The rhinovirus 2A proteinase cleaves cytokeratin 8 (31Seipelt J. Liebig H.-D. Sommergruber W. Gerner C. Kuechler E. J. Biol. Chem. 2000; 275: 20084-20089Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and other virus-coded proteinases cleave actin (5Lanier L.M. Slack J.M. Volkman L.E. Virology. 1996; 216: 380-388Crossref PubMed Scopus (45) Google Scholar,32Ott D.E. Coren L.V. Kane B.P. Busch L.K. Johnson D.G. Sowder R.C. Chertova E.N. Arthur L.O. Henderson L.E. J. Virol. 1996; 70: 7734-7743Crossref PubMed Google Scholar) and vimentin (33Shoeman R.L. Honer B. Stroller T.J. Kesselmeier C. Miedel M.C. Traub P. Graves M.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6336-6349Crossref PubMed Scopus (141) Google Scholar). What is currently unique about AVP is that it uses actin as a cofactor. We thank Professor Schutt of Princeton University for pointing out the homology between the C terminus of actin and pVIc. We also thank Greg Bowman, Dr. John Dunn, Dr. William J. McGrath, Diana Toledo, and Dr. Kurt Thorn for materials and helpful discussions." @default.
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W2108018047 title "Actin Can Act as a Cofactor for a Viral Proteinase in the Cleavage of the Cytoskeleton" @default.
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