The Hepatitis C Virus NS5A Protein Activates a Phosphoinositide 3-Kinase-dependent Survival Signaling Cascade

Abstract

Hepatitis C virus (HCV) establishes a persistent infection, with up to 80% of infected individuals proceeding to chronic hepatitis, which in many cases may result in liver cirrhosis and hepatocellular carcinoma (HCC); indeed HCV infection is increasingly associated with the development of HCC. The long time period (up to 30 years) between primary infection and the onset of HCC implies that HCV is not directly oncogenic but in some way predisposes patients to develop tumors, though the mechanism for this is unclear as yet. We report here that NS5A binds directly to the Src homology 3 domain of the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K), and this interaction is mediated by a novel (non-proline-rich) motif within NS5A. Coimmunoprecipitation analysis revealed that NS5A bound native heterodimeric PI3K and enhanced the phosphotransferase activity of the catalytic (p110) subunit both in vitro and in human cell lines harboring a subgenomic HCV replicon or expressing NS5A alone. NS5A-mediated activation of PI3K resulted in increased phosphorylation and activity of Akt/protein kinase B and concomitantly provided protection against the induction of apoptosis in both replicon-harboring cells and cells stably expressing NS5A alone. These data suggest that stimulation of PI3K by NS5A may represent an indirect mechanism for development of HCC in HCV-infected patients and further suggests potential therapeutic strategies to counteract the occurrence of HCV-related HCC. Hepatitis C virus (HCV) establishes a persistent infection, with up to 80% of infected individuals proceeding to chronic hepatitis, which in many cases may result in liver cirrhosis and hepatocellular carcinoma (HCC); indeed HCV infection is increasingly associated with the development of HCC. The long time period (up to 30 years) between primary infection and the onset of HCC implies that HCV is not directly oncogenic but in some way predisposes patients to develop tumors, though the mechanism for this is unclear as yet. We report here that NS5A binds directly to the Src homology 3 domain of the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K), and this interaction is mediated by a novel (non-proline-rich) motif within NS5A. Coimmunoprecipitation analysis revealed that NS5A bound native heterodimeric PI3K and enhanced the phosphotransferase activity of the catalytic (p110) subunit both in vitro and in human cell lines harboring a subgenomic HCV replicon or expressing NS5A alone. NS5A-mediated activation of PI3K resulted in increased phosphorylation and activity of Akt/protein kinase B and concomitantly provided protection against the induction of apoptosis in both replicon-harboring cells and cells stably expressing NS5A alone. These data suggest that stimulation of PI3K by NS5A may represent an indirect mechanism for development of HCC in HCV-infected patients and further suggests potential therapeutic strategies to counteract the occurrence of HCV-related HCC. Hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GA, glutathione-agarose; Grb, growth factor receptor-bound; GST, glutathione S-transferase; HA, hemagglutinin; PI3K, phosphoinositide-3 kinase; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PIPES, 1,4-piperazinediethanesulfonic acid; SH2 and SH3, Src homology domain 2 and 3, respectively. causes acute and chronic hepatitis, often associated with an increased risk of developing chronic liver disease and hepatocellular carcinoma (1Kim W.R. Hepatology. 2002; 36: 30-34Crossref PubMed Google Scholar). The genome of the virus is a single-strand positive sense RNA molecule of ∼9.5 kb in length with a single large open reading frame encoding for a polyprotein of ∼3,000 amino acids (2Choo Q.L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6271) Google Scholar). The open reading frame is flanked by 5′- and 3′-untranslated regions, which have been shown to be involved in initiation of translation and viral replication (3Bartenschlager R. Lohmann V. J. Gen. Virol. 2000; 81: 1631-1648Crossref PubMed Scopus (584) Google Scholar). Processing of the precursor polyprotein requires both host and viral proteases to produce the structural (core, E1, E2, and p7) and nonstructural (NS2, NS3/4A, NS4B, NS5A, and NS5B) proteins of the virion (4Bartenschlager R. J. Viral Hepatitis. 1999; 6: 165-181Crossref PubMed Scopus (126) Google Scholar). The nonstructural NS5A protein is generated as a mature protein by the action of the NS3/4A serine protease. NS5A is localized to cytoplasmic and perinuclear regions of the cell and exists as phosphoproteins (p56 and p58) with the degree of serine/threonine phosphorylation accounting for the difference in size (5Tanji Y. Kaneko T. Satoh S. Shimotohno K. J. Virol. 1995; 69: 3980-3986Crossref PubMed Google Scholar). NS5A has generated a large amount of interest because of the observation that it can interact with a wide variety of host signaling proteins and as such may play a role in immune evasion and carcinogenesis. One clue that NS5A may have a potential role in cell signaling is the presence of polyproline motifs with the consensus sequence Pro-Xaa-Xaa-Pro, which are highly conserved throughout most HCV isolates. Such motifs are found in a number of viral and cellular proteins involved in signal transduction and form extended helices that bind to Src homology 3 (SH3) domains found within a diverse group of signal-transducing proteins (6Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2229) Google Scholar). It was shown previously that NS5A interacts directly with the growth factor receptor-bound protein 2 (Grb2) (7Tan S.L. Nakao H. He Y.P. Vijaysri S. Neddermann P. Jacobs B.L. Mayer B.J. Katze M.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5533-5538Crossref PubMed Scopus (208) Google Scholar) and members of the Src family of tyrosine kinases (8Macdonald A. Crowder K. Street A. McCormick C. Harris M. J. Gen. Virol. 2004; 85: 721-729Crossref PubMed Scopus (96) Google Scholar) via these highly conserved proline motifs, in the latter case resulting in the modulation of kinase activity. The implications of these interactions are unclear, although they suggest that NS5A may play a role in the modulation of mitogenic signaling pathways in HCV-infected hepatocytes. Recently it has been reported that NS5A is also able to bind to phosphoinositide-3 kinase (PI3K) (9He Y.P. Nakao H.H. Tan S.L. Polyak P.J. Neddermann P. Vijaysri S. Jacobs B.L. Katze M.G. J. Virol. 2002; 76: 9207-9217Crossref PubMed Scopus (156) Google Scholar). PI3K is a heterodimeric enzyme consisting of a p85 regulatory subunit and a p110 catalytic subunit; the latter phosphorylates the inositol ring of membrane phosphatidylinositol lipids at the 3′-carbon position to produce phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (10Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar). These lipid products act as second messengers, regulating cellular growth and proliferation (10Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar). Activation of PI3K is associated with increased tyrosine phosphorylation of p85. NS5A was shown to interact with the p85 subunit in vitro and in HeLa cells stably expressing NS5A or infected with a vaccinia virus recombinant expressing NS5A (9He Y.P. Nakao H.H. Tan S.L. Polyak P.J. Neddermann P. Vijaysri S. Jacobs B.L. Katze M.G. J. Virol. 2002; 76: 9207-9217Crossref PubMed Scopus (156) Google Scholar). PI3K products activate a number of downstream pathway components. One of these is the serine/threonine protein kinase Akt, recruited to the membrane by binding of its pleckstrin homology domain to PI(3,4,5)P3, where it becomes phosphorylated by phosphoinositide-dependent kinase 1 (10Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar). Activation of Akt leads to changes in glucose transport and synthesis as well as translation initiation and elongation. Akt has also been shown to control apoptosis within cells through a number of means including phosphorylation and subsequent deactivation of the pro-apoptotic Bcl2 homolog Bad and inhibition of caspase 9. An increase in the phosphorylation of p85, Akt, and Bad was demonstrated in NS5A-expressing HeLa cells (9He Y.P. Nakao H.H. Tan S.L. Polyak P.J. Neddermann P. Vijaysri S. Jacobs B.L. Katze M.G. J. Virol. 2002; 76: 9207-9217Crossref PubMed Scopus (156) Google Scholar). Dysregulation of PI3K, by either overexpression or constitutive activation, has been linked with the malignant transformation of cells (11Djordjevic S. Driscoll P.C. Trends Biochem. Sci. 2002; 27: 426-432Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Additionally, a number of viruses that establish chronic infections express proteins that interact with and activate PI3K to promote cell survival and block apoptosis thus enhancing viral replication. Examples include the hepatitis B virus X protein (12Lee Y.I. Kang-Park S. Do S.I. Lee Y.I. J. Full Text Full Text PDF PubMed Scopus Google the virus membrane protein 1 G. J. Full Text Full Text PDF PubMed Scopus Google and human virus 1 T. R. 2002; PubMed Scopus Google V. K. S. P. A. PubMed Scopus Google Scholar). that HCV establishes a chronic infection and is increasingly associated with the development of hepatocellular carcinoma to the interaction between NS5A and PI3K and to the potential in a cellular the human cell harboring a subgenomic HCV replicon (3Bartenschlager R. Lohmann V. J. Gen. Virol. 2000; 81: 1631-1648Crossref PubMed Scopus (584) Google Scholar). We that NS5A is able to interact with the domain of the p85 subunit via a motif in the of NS5A. interaction of NS5A with the native in and this interaction in stimulation of the lipid kinase activity of PI3K both in vitro and in cells harboring a subgenomic or stably expressing NS5A phosphorylation and activation of Akt are and this with a in the of apoptosis by data are with a role for NS5A in apoptosis in HCV-infected cells by activation of PI3K and that NS5A not to the of HCV but also to the for the development of hepatocellular carcinoma in HCV-infected NS5A HCV M. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google M. M. M. J. 244: PubMed Scopus Google and M. J. 1999; PubMed Scopus Google by as well as of by with sequence and products as the by and either S. PubMed Scopus Google Scholar) or An PI3K p85 subunit was by of V. K. S. P. A. PubMed Scopus Google Scholar). and cells in with 2 and and cells in with amino 2 and cell lines at in a cells in with and at in an the of cells expressing phosphotransferase or with the HCV of NS5A, cells with or and cell with cell lines as previously A. Crowder K. Street A. McCormick C. K. Harris M. J. Full Text Full Text PDF PubMed Scopus Google Scholar). of replicon was and cells to for the of 1 to the as a cell in with cells at 2 cells in and with 2 of the cells in and in 30 and The was by at for at and at The for of and and of the proteins in or cells by PubMed Scopus Google Scholar). to with 1 and at for 3 in and 1 and to proteins bound to for 1 at to in in at the and of the proteins by and with a The proteins bound to at for 1 of cells with in a in and bound proteins by by was used as a protein to the the in and 1 of was bound to the for 2 by and in cells of protein to the and the at a for The with and bound proteins by by with by and to membrane for 1 in in and with either an to NS5A, p85 or p110 and the was with the and PI3K activity was an kinase 1 cells in 2 and an and protein of the immune the in and in PIPES, and in of PIPES, The by the of 1 phosphatidylinositol and and of and at 30 the by the of of and the lipids a The phosphatidylinositol was and by was a in either or growth with in of A. Crowder K. Street A. McCormick C. K. Harris M. J. Full Text Full Text PDF PubMed Scopus Google Scholar) 1 and 1 a in the the protein cell of with of an homology domain Akt to protein for at in Akt kinase 1 1 and this 3 of recombinant Bad protein was with of for at 30 the by the of and by of Akt kinase activity by of Bad by of recombinant Bad protein was by with a of Akt phosphorylation by analysis of Akt with an for Akt of Akt by with a 3 3 activity was the to the 2 cells with the caspase 3 for 1 at of the was a with a and number of cells was with 1 cells and to for apoptosis cells with 1 for in and in in with for cells with which to be as a of of was a of a previously 2002; PubMed Scopus Google Scholar). cells for in to in of cell and of cells by at for by of the with by with 3 and at for in of 1 and at for 2 by at for 3 by with and NS5A to the of the p85 of PI3K via a have shown that NS5A is of with the mitogenic protein (7Tan S.L. Nakao H. He Y.P. Vijaysri S. Neddermann P. Jacobs B.L. Mayer B.J. Katze M.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5533-5538Crossref PubMed Scopus (208) Google Scholar) and members of the Src family of tyrosine kinases (8Macdonald A. Crowder K. Street A. McCormick C. Harris M. J. Gen. Virol. 2004; 85: 721-729Crossref PubMed Scopus (96) Google Scholar) via a highly conserved polyproline motif within the C of NS5A. It has also been reported that NS5A interacts with the p85 subunit of PI3K (9He Y.P. Nakao H.H. Tan S.L. Polyak P.J. Neddermann P. Vijaysri S. Jacobs B.L. Katze M.G. J. Virol. 2002; 76: 9207-9217Crossref PubMed Scopus (156) Google although that report the of an polyproline motif in NS5A and the p85 the of interaction within both NS5A and p85 have not been The p85 subunit SH2 domains and a as well as protein interaction domains and a involved in binding to p110 the of interaction within p85 a of proteins either of the p85 SH2 the p85 domain and a the domain and the SH2 These used as to cells with a proteins by with a NS5A shown in NS5A with the p85 domain of PI3K but to bind either of the SH2 domains 2 and NS5A also with the p85 with of binding with the domain the and of the the to bind the p85 domain was conserved of HCV, that NS5A and bound at with NS5A proteins at and and of the by domains bind to polyproline motifs with the consensus sequence R. 1995; Full Text PDF PubMed Scopus Google Scholar). such motifs, an polyproline motif and polyproline motifs the C to as motif in HCV and and highly conserved motif shown previously to bind the role by of these motifs in binding to the p85 generated in which proline by and of these the of NS5A to interact with that NS5A was able to interact with the domain of the PI3K p85 subunit via a novel binding the p85 domain binding within NS5A generated a of and of of the or of NS5A the binding to The both the and motifs and binding to domains including of and kinases not a of the C binding to of the demonstrated that it was to the of NS5A binding to the bound to domains including of and kinases not of was by not These data that the p85 domain binding between and of to this further the C shown in a of the the to bind to this was extended to a in the binding was These data suggest that the for binding to the p85 domain between and and binding is enhanced by the presence of between and The to not a polyproline motif and that NS5A binds to the PI3K p85 domain via a NS5A with the PI3K in the of the in vitro interaction between NS5A and the p85 domain to NS5A was also of with native a in this cells with a expressing an of p85. with a NS5A and the by with either an or a that with NS5A. this observation the and demonstrated that NS5A be in that this result was not the result of overexpression of a further to the interaction of NS5A with the heterodimeric cells either or with with a to the p110 proteins by with to p85, or NS5A. p85 with p110 in NS5A be in the p110 that NS5A is able to with the native PI3K in NS5A in and in further the of the interaction NS5A was able to the activity of PI3K in PI3K was the human cell and the to an in vitro kinase in the presence of and phosphatidylinositol as a NS5A, NS3/4A M. J. Harris M. J. Gen. Virol. PubMed Scopus Google or M. K. J. Gen. Virol. PubMed Scopus Google Scholar) was cells and to the to the shown in the of 1 NS5A resulted in a increase in the phosphotransferase activity of PI3K, NS3/4A or that the activity of PI3K was by the of the and was by the protein kinase a the phosphotransferase activity of PI3K was in the presence of of NS5A. PI3K activity increased in to the of NS5A with a stimulation at NS5A. NS5A PI3K activity within cells generated a of human cells harboring the subgenomic HCV replicon expressing of the HCV nonstructural proteins Lohmann V. R. J. Virol. PubMed Scopus Google Scholar). PI3K was either cells expressing phosphotransferase or cells and for phosphotransferase activity as that PI3K in cells activity PI3K in The with an to that this increase in PI3K phosphotransferase activity was not by an increase in the of PI3K in replicon with an that NS5A associated with PI3K in replicon that to establish cells expressing the NS5A to bind to to that the stimulation of PI3K activity in replicon cells was by NS5A cell lines expressing either or the PI3K was these cells and for phosphotransferase activity. that cells expressing NS5A a increase in PI3K activity with control cells expressing NS5A increase in PI3K with the observation that to bind to not be in p85 not although of both NS5A and p85 was by the NS5A-mediated stimulation of PI3K activity in cells is not as as that in this can be by the of PI3K activity in cells with cells with Akt in of the signaling pathway by PI3K activation is the serine/threonine kinase kinase is by binding to and is phosphorylated by phosphoinositide-dependent kinase also recruited to the membrane by interaction of a pleckstrin homology domain with the downstream of NS5A-mediated PI3K activation the of Akt phosphorylation in cell and an cell stably expressing NS5A the replicon was the of HCV used the in these Akt was and was by of with a was in cells in of be in the or cells growth in in and in or with the increased activity of PI3K in these of Akt in cell either in or as by of with a phosphorylation Akt One of the of Akt is the pro-apoptotic Bcl2 homolog phosphorylation of Bad the the that the increase in Akt phosphorylation in NS5A-expressing cells with an increase in Akt activity Akt and the to an in vitro kinase in the presence of and recombinant Bad protein as a with the of shown in in the Bad phosphorylation be in kinase of Bad phosphorylation in the or cells growth in phosphorylation of Bad was in kinase and in or with the increased Akt activity in these We these data that NS5A-mediated stimulation of PI3K activity in a stimulation of Akt phosphorylation and phosphotransferase activity in human NS5A the of in have demonstrated that activation of Akt via the PI3K pathway in the induction of an cell survival Lee PubMed Scopus Google Scholar). NS5A mediated of PI3K resulted in increased of cells to pro-apoptotic of apoptosis in and cells and these with the control apoptosis was by and cell for of caspase 3 activity a that in the presence of of caspase 3 activity in cell caspase 3 activity was in in both and cells an in caspase 3 We the of cells apoptosis by cells with and with that in the of of apoptosis in cell a increase in the number of cells was in the control cells as shown for caspase 3 activation, with control cells the of cells in both the and by the induction of of at the both and cells the control cells although in this case the was that for both caspase 3 activation and of the revealed that was of control in both to establish cells expressing the NS5A We the induction of by in cells expressing either or the NS5A as in NS5A the induction of this case by of in cells expressing the to control these data suggest that NS5A, by binding to and PI3K, is able to a cell survival The data in this that the HCV NS5A protein interacts with the domain of the PI3K p85 regulatory subunit via a novel binding The of both analysis and that this binding not of the polyproline motifs within NS5A. number of have demonstrated binding of to to be the primary amino sequence of these a of consensus has been such as proteins that bind to the at proline S. M. A. G. J. 1999; PubMed Scopus Google such as binding of to the domains of and H. C. A. G. J. 2000; PubMed Scopus Google Scholar) The binding between and not to of the this a of and of the amino either or It is to that although analysis suggests that the binding for the p85 domain is between amino acids and of NS5A, this have an analysis of this the that p85 binding within NS5A. observation that a of NS5A binds to the p85 domain is not with the report that the of NS5A was for binding to p85 (9He Y.P. Nakao H.H. Tan S.L. Polyak P.J. Neddermann P. Vijaysri S. Jacobs B.L. Katze M.G. J. Virol. 2002; 76: 9207-9217Crossref PubMed Scopus (156) Google Scholar). in the involved of NS5A and p85 HeLa that it has been shown previously V. R. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that of an that NS5A to the membrane resulted in of NS5A to the the observation that an of NS5A to with p85 a in The interaction of NS5A with the p85 regulatory subunit the phosphotransferase activity of the p110 catalytic subunit both in vitro and in The mechanism for this is Activation of p110 can result to the thus it with this has been shown to be mediated through binding of p110 directly to or binding of the p85 SH2 domains to tyrosine The observation that NS5A p110 activity in vitro suggests that this mechanism is not in this p85 the catalytic activity of binding of to the regulatory subunit can activate the catalytic for binding of to the SH2 domain of p85 the activity of p85. tyrosine at the p85 SH2 can also the inhibition by an interaction with the SH2 domain R. K. J. Full Text Full Text PDF PubMed Scopus Google Scholar). p110 activity can be in vitro by NS5A, this implies that NS5A binding to the p85 domain is able to the of p85 via a mechanism that not tyrosine the that NS5A also activate PI3K by a tyrosine kinase that with PI3K, resulting in p85 tyrosine PI3K activation leads to a wide of signaling within the some of these are and cell The latter include of the in and cell and the most by PI3K is cell survival is by a inhibition of signaling by PI3K activity and mediated by proteins such as Akt and stimulation of PI3K can result in as cells are to apoptosis in to or oncogenic data that NS5A-mediated stimulation of PI3K in the of cell survival including Akt activation and caspase 3 these in stably cell not that they result because both the replicon and NS5A-expressing cells generated by cells either RNA or the observation that NS5A PI3K and apoptosis in cell and the that this of NS5A is cell and against the that these data can be by be the of NS5A-mediated PI3K activation and of cell survival in the HCV-infected We that such stimulation result in an to oncogenic in HCV-infected and play a role in the between HCV infection and the development of hepatocellular It is to the host to be to stimulation of inhibition of apoptosis to the of One of the of the host to viral infection is the induction of apoptosis of cells of these cells by The of HCV in cell be by the induction of apoptosis by cell signaling pathways downstream PI3K cell and protein It is that or of these HCV replication. of the potential implications of these for HCV replication at this it is to that the PI3K pathway the activity of the cell translation via the activation of the of A. P. 2000; Google Scholar) and kinase (10Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar). with the that activity of the HCV is the cell M. Kaneko S. K. 2000; Full Text Full Text PDF PubMed Scopus Google and of NS5A Y.P. C. Y. M. Katze M.G. J. Gen. Virol. PubMed Scopus Google it is to that of PI3K by NS5A the cellular to Activation of the PI3K pathway is a both RNA and viruses that establish chronic the membrane protein 1 of virus G. J. Full Text Full Text PDF PubMed Scopus Google Scholar) and the of virus PubMed Scopus Google Scholar) bind to the p85 in the latter case the interaction in and the SH2 domains of p85. of these proteins activate PI3K, although the of activation are unclear and may of PI3K to the membrane The hepatitis B virus protein (12Lee Y.I. Kang-Park S. Do S.I. Lee Y.I. J. Full Text Full Text PDF PubMed Scopus Google Scholar) the PI3K although is for interaction with the human virus 1 protein has been shown to bind to p85 both in vitro T. R. 2002; PubMed Scopus Google Scholar) and in T. R. 2002; PubMed Scopus Google V. K. S. P. A. PubMed Scopus Google Scholar). expressing PI3K NS5A, not the activity of PI3K in in vitro T. R. 2002; PubMed Scopus Google with the of these cases viral of PI3K has been shown to promote cell We that data that HCV NS5A can be to the of these viral that via the PI3K The of replicon cell lines and for the of HCV Harris M. J. Gen. Virol. 2002; PubMed Scopus Google Scholar) the analysis of the role of NS5A-mediated PI3K in viral replication and We and of for the of