Poxvirus Infection Rapidly Activates Tyrosine Kinase Signal Transduction

Viruses have evolved a number of strategies to gain entry and replicate in host target cells that, for human immunodeficiency virus (HIV) and the poxvirus, myxoma virus, involve appropriating chemokine receptors. In this report we demonstrate that activation of multiple intracellular tyrosine phosphorylation events rapidly ensues following virus adsorption to NIH 3T3.CD4.CCR5 cells and affects the ultimate level of myxoma virus replication. UV-inactivated myxoma virus induces the rapid phosphorylation of CCR5 on tyrosine residues, the association of CCR5 with Jaks and p56lck, and their phosphorylation-activation within minutes of virus adsorption. Additionally, we provide evidence for myxoma virus-inducible signal transducers and activators of transcription (Stat) and insulin receptor substrate (IRS) activation. In contrast to CCR5 activation effected by HIV Env protein, these myxoma virus-inducible phosphorylation events are not sensitive to pertussis toxin treatment. Moreover, in cells that are non-permissive for myxoma virus infection, we provide evidence that myxoma virus fails to invoke this tyrosine phosphorylation cascade. Consistent with the observation that infection of CCR5-expressing cells is blocked by herbimycin A and the Jak 2 inhibitor, tyrophostin AG490, we infer that viral infectivity may be dependent on non-G-protein-coupled signal transduction pathways triggered by the infecting myxoma virus particle. This provides a novel post-binding mechanism by which viruses can co-opt a cellular receptor to permit productive virus infection. Viruses have evolved a number of strategies to gain entry and replicate in host target cells that, for human immunodeficiency virus (HIV) and the poxvirus, myxoma virus, involve appropriating chemokine receptors. In this report we demonstrate that activation of multiple intracellular tyrosine phosphorylation events rapidly ensues following virus adsorption to NIH 3T3.CD4.CCR5 cells and affects the ultimate level of myxoma virus replication. UV-inactivated myxoma virus induces the rapid phosphorylation of CCR5 on tyrosine residues, the association of CCR5 with Jaks and p56lck, and their phosphorylation-activation within minutes of virus adsorption. Additionally, we provide evidence for myxoma virus-inducible signal transducers and activators of transcription (Stat) and insulin receptor substrate (IRS) activation. In contrast to CCR5 activation effected by HIV Env protein, these myxoma virus-inducible phosphorylation events are not sensitive to pertussis toxin treatment. Moreover, in cells that are non-permissive for myxoma virus infection, we provide evidence that myxoma virus fails to invoke this tyrosine phosphorylation cascade. Consistent with the observation that infection of CCR5-expressing cells is blocked by herbimycin A and the Jak 2 inhibitor, tyrophostin AG490, we infer that viral infectivity may be dependent on non-G-protein-coupled signal transduction pathways triggered by the infecting myxoma virus particle. This provides a novel post-binding mechanism by which viruses can co-opt a cellular receptor to permit productive virus infection. human immunodeficiency virus multiplicity of infection 4-amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo(3,4-d)pyrimidine insulin receptor substrate signal transducers and activators of transcription regulated on activation normal T cell expressed and secreted Poxviruses are DNA viruses that replicate autonomously in the cytoplasm of infected cells. They have been the subject of intensive study, based largely on their severe pathogenesis in humans and a variety of domestic animals (1Fenner F. FEMS Microbiol. Rev. 2000; 24: 123-133Crossref PubMed Google Scholar). Until its global irradication, smallpox, caused by an orthopoxvirus, was one of the most serious diseases of mankind. Myxoma virus, a member of theLeporipoxvirus genus, is the causative agent of myxomatosis, a lethal disease of the European rabbit (2Nash P. Barret J. Cao J.X. Hota-Mitchell S. Lalani A.S. Everett H. Xu X.M. Robichaud J. Hnatiuk S. Ainslie C. Seet B.T. McFadden G. Immunol. Rev. 1999; 168: 103-120Crossref PubMed Scopus (116) Google Scholar). Myxomatosis is characterized by extensive fulminating lesions and severe immune dysfunction accompanied by supervening Gram-negative bacterial infections of the respiratory tract (3McFadden G.M. Webster R. Granoff A. Encyclpedia of Virology. Academic Press Inc., San Diego, CA1994: 1153-1160Google Scholar). Recently, evidence was provided that myxoma virus may utilize chemokine receptors to initiate infection (4Lalani A.S. Masters J. Zeng W. Barrett J. Pannu R. Everett H. Arendt C.W. McFadden G. Science. 1999; 286: 1968-1971Crossref PubMed Scopus (131) Google Scholar). Viruses have evolved a number of strategies to gain entry and replicate in host target cells that, for HIV1 and myxoma virus, includes appropriating chemokine receptors (4Lalani A.S. Masters J. Zeng W. Barrett J. Pannu R. Everett H. Arendt C.W. McFadden G. Science. 1999; 286: 1968-1971Crossref PubMed Scopus (131) Google Scholar, 5Fauci A. Nature. 1996; 384: 529-534Crossref PubMed Scopus (746) Google Scholar, 6Berger E.A. Murphy P.M. Farber J.M. Annu. Rev. Immunol. 1999; 17: 657-700Crossref PubMed Scopus (1890) Google Scholar). Chemokines and their receptors are critical for the clearance of infectious pathogens. Specifically, chemokines are implicated in directing lymphocyte trafficking to sites of infection and in activating the effector functions of these immune cells to eliminate infectious pathogens (7Rossi D. Zlotnik A. Annu. Rev. Immunol. 2000; 18: 217-242Crossref PubMed Scopus (2109) Google Scholar). Thus, viral subversion of chemokine receptors is an effective way to modulate chemokine-receptor-mediated interactions that would invoke an immune response against the invading virus. Indeed, herpesviruses and poxviruses subvert a host immune response by encoding several candidate chemokine receptor homologues and chemokine mimetics, capable of precluding chemokines from activating their cognate cell surface receptors (8Pease J.E. Murphy P.M. Semin. Immunol. 1998; 10: 169-178Crossref PubMed Scopus (40) Google Scholar, 9Lalani A.S. Barrett J.W. McFadden G. Immunol. Today. 2000; 21: 100-106Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). However, for HIV-1, there is accumulating evidence to suggest that interference with the signaling capacity of CCR5 can compromise its role as an HIV-1 entry coreceptor (10Wang J.M. Oppenheim J.J. J. Exp. Med. 1999; 190: 591-595Crossref PubMed Scopus (17) Google Scholar). Indeed, activation of CCR5 by Env protein of HIV leads to the selective stimulation of distinct signaling pathways that are advantageous to establish a “friendly” cellular environment for the virus (10Wang J.M. Oppenheim J.J. J. Exp. Med. 1999; 190: 591-595Crossref PubMed Scopus (17) Google Scholar, 11Liu Q.-H. Williams D.A. McManus C. Baribaud F. Doms R.W. Schols D. De Clercq E. Kotlikoff M.I. Collman R.G. Freedman B.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4832-4837Crossref PubMed Scopus (135) Google Scholar, 12Cicala C. Arthos J. Ruiz M. Vaccarezza M. Rubbert A. Riva A. Wildt K. Cohen O. Fauci A.S. J. Immunol. 1999; 163: 420-426PubMed Google Scholar, 13Davis C.B. Dikic I. Unutmaz D. Hill C.M. Arthos J. Siani M.A. Thompson D.A. Schlessinger J. Littman D.R. J. Exp. Med. 1997; 186: 1793-1798Crossref PubMed Scopus (344) Google Scholar). Accordingly, we undertook studies to examine whether myxoma virus infection of cells requires activation of the signaling capacity of CCR5. Our data suggest that myxoma virus entry into cells does not require the signaling capacity of CCR5 to be intact, yet initiation of infection is dependent on non-G-protein-coupled tyrosine phosphorylation events initiated by the virus binding to the cell surface. Murine fibroblast NIH 3T3.CD4.CCR5 and NIH 3T3.CD4.neo cells were obtained from D. Littman (New York University) and were maintained in DMEM (Life Technologies, Inc.), supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. vMyxlac is a myxoma virus (strain Lausanne) derivative containing a β-galactosidase marker cassette driven by a late viral promoter in an intergenic location (4Lalani A.S. Masters J. Zeng W. Barrett J. Pannu R. Everett H. Arendt C.W. McFadden G. Science. 1999; 286: 1968-1971Crossref PubMed Scopus (131) Google Scholar). 109 plaque-forming units/ml of vMyxlac was UV inactivated using the Stratagene StratLinker for 20–30 min, as indicated. The StratLinker delivers a dose of 1.2 × 105μJ/cm2. 4-Amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1) was purchased from Biomol and tyrophostin B42 (AG490) from Calbiochem. Cell surface expression of CCR5 and CD4 was quantified by flow cytometry using the monoclonal antibodies 2D7 (PharMingen) and SIM.4 (National Institutes of Health AIDS Research and Reference Reagent Program), respectively. Cells were gated based on forward and side scatter. CCR5 and CD4 expression was determined using an anti-mouse biotin conjugated secondary antibody detected with Cy5 conjugated streptavidin fluorescent tertiary reagent. Staining was according to the manufacturer's protocol, and flow cytometric data were acquired using FACScan (Becton Dickinson Immunocytometry) as described previously (14Ghislain J. Lingwood C.A. Fish E.N. J. Immunol. 1994; 153: 3655-3663PubMed Google Scholar). CELLQuest software was used to analyze data. Approximately 107cells were either exposed to 108 live virus particles, or the equivalent of 108 infectious units of UV-inactivated virus particles, or medium alone, for the indicated times. Cells were washed twice with cold phosphate-buffered saline and lysed as described previously (16Ahmad S. Alsayed Y. Druker B.J. Platanias L.C. J. Biol. Chem. 1997; 272: 29991-29994Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Immunoprecipitations and immunoblotting using enhanced chemiluminescence were performed as described previously (16Ahmad S. Alsayed Y. Druker B.J. Platanias L.C. J. Biol. Chem. 1997; 272: 29991-29994Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Cells in individual wells of a 96-well microtiter plate were either left untreated, or treated with varying doses of PP1 or AG490, then exposed to vMyxlac at a m.o.i. of 10. After 16 h cells were lysed using a buffer composed of 10% Nonidet P-40 and 50 mm Tris, pH 7.5. Following one freeze-thaw cycle, an aliquot of the lysate was transferred to a well in another 96-well microtiter plate, containing 100 mm NaH2PO4, 10 mmKCl, 1 mm MgSO4, and 50 mmβ-mercaptoethanol. The mixture was incubated for 5 min at 37 °C. To measure β-galactosidase activity, 4 mg/ml of substrateo-nitrophenyl-β-d-galactopyranoside was added in 100 mm NaH2PO4, pH 7.5. The breakdown ofo-nitrophenyl-β-d-galactopyranoside by β-galactosidase results in a yellow color reaction. After 1 h the reaction was terminated by the addition of 1 mNa2CO3. Absorbance was measured spectrophotometrically at 420 nm using a THERMOmax microplate reader. To determine whether infection by myxoma virus mediates CCR5 activation, thereby rendering a cell permissive for viral replication, we undertook studies to examine CCR5 mediated signal transduction in two distinct cell lines that are differentially sensitive to viral infection. NIH 3T3.CD4.CCR5 cells, that stably express human CD4 and human CCR5 (Fig. 1 A), are fully permissive for viral replication (Fig. 1 B). For these studies, viral gene expression was monitored using a recombinant myxoma virus that expresses β-galactosidase under the control of a late viral promoter that drives a lacZ transgene reporter (4Lalani A.S. Masters J. Zeng W. Barrett J. Pannu R. Everett H. Arendt C.W. McFadden G. Science. 1999; 286: 1968-1971Crossref PubMed Scopus (131) Google Scholar). By contrast, NIH 3T3.CD4.neo cells that express low levels of human CD4 and no CCR5 (Fig. 1 A) do not support myxoma virus replication, as revealed in Fig. 1 B. When both cell types are infected at an m.o.i. of 0.01 plaque-forming units/cell, then cells are harvested at 1 and 16 h post-infection for virus titration on permissive BGMK cells, we observe that the NIH 3T3.CD4.neo cells are completely non-permissive for infection (data not shown). By contrast, over this same time period we observe a log increase in plaque-forming units/106 BGMK cells (∼500–5000) from the permissive NIH 3T3.CD4.CCR5 cells (data not shown). Since myxoma virus infection of NIH 3T3.CD4.CCR5 cells can be inhibited by herbimycin A (4Lalani A.S. Masters J. Zeng W. Barrett J. Pannu R. Everett H. Arendt C.W. McFadden G. Science. 1999; 286: 1968-1971Crossref PubMed Scopus (131) Google Scholar), at the outset we examined whether adsorption of myxoma virus induces CCR5 phosphorylation. For these studies we employed UV-inactivated myxoma virus, in which the structural integrity of the viral particles is retained, yet UV-induced damage to the viral genome irreversibly prevents viral replication (17Ramsey-Ewing A. Moss B. Virol. 1998; 242: 138-149Crossref PubMed Scopus (73) Google Scholar). UV-inactivated myxoma virus particles can bind and enter into susceptible cells and are uncoated, with similar kinetics to the infective virus, but virus gene expression is severely compromised (data not shown). When lysates from myxoma virus exposed NIH 3T3.CD4.CCR5 cells were immunoprecipitated with antibodies against phosphotyrosine or CCR5 and immunoblotted to detect either CCR5 or phosphotyrosine, we observed that even 1–5-min exposure to UV-inactivated myxoma virus induces rapid phosphorylation of CCR5 on tyrosine residues (Fig.2, A and B). Moreover, in NIH 3T3.CD4.CCR5 cells exposed to myxoma virus particles, we invariably observed the co-immunoprecipitation of a 56-kDa tyrosine-phosphorylated protein with CCR5 (Fig. 2 C). Immunoblot analysis identified this protein as the Src kinase, p56lck (Fig. 2 D). Our data would suggest that p56lck associates with CCR5 in a tyrosine phosphorylation-dependent manner. Notably, we observe inducible phosphorylation of p56lck whether live or UV-inactivated myxoma virus is used (Fig. 2 E). In subsequent experiments we examined the extent of myxoma virus-inducible protein tyrosine phosphorylation in whole cell lysates from the permissive NIH 3T3.CD4.CCR5 and non-permissive NIH 3T3.CD4.neo cells. Immunoprecipitation of tyrosine-phosphorylated proteins followed by Western blot analysis revealed that a number of proteins consistently become tyrosine-phosphorylated following 1–5-min exposure of NIH 3T3.CD4.CCR5 cultures to UV-inactivated myxoma virus (Fig.3 A), yet the non-susceptible NIH 3T3.CD4.neo cells do not show any evidence of virus-inducible tyrosine phosphorylation of cellular proteins (Fig. 3 B). Additionally, BGMK cells become resistant to myxoma virus infection following herbimycin A treatment. 2J. Masters, A. A. Hinek, G. McFadden, and E. N. Fish, unpublished observation. Chemokine-mediated activation of chemokine receptors leads to the rapid phosphorylation of receptor-associated Jaks (18Opermann M. Mack M. Proudfoot A.E.I. Olbrich H. J. Biol. Chem. 1999; 274: 8875-8885Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 19Wong M. Uddin S. Majchrzak B. Huynh T. Proudfoot A.E.I. Platanias L.C. Fish E.N. J. Biol. Chem. 2001; 276: 11427-11431Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). PAGE analysis revealed myxoma virus-inducible phosphorylation of proteins that were candidate Jaks. Stripping and reprobing these antiphosphotyrosine immunoblots with antibodies to Jaks confirmed the identity of tyrosine-phosphorylated Jak1 and Jak2 (Fig. 3, C and D). Moreover, identical results were obtained whether live or UV-inactivated myxoma virus was used (Fig. 3 E). We did not observe myxoma virus-inducible Jak3 phosphorylation (not shown). Since p56lck associated with CCR5 in a phosphorylation-dependent manner (Fig. 2, C and D), we infer that p56lck may be a substrate for the activated Jaks (20Uddin S. Sher D.A. Alsayed Y. Pons S. Colamonici O.R. Fish E.N. White M.F. Platanias L.C. Biochem. Biophys. Res. Commun. 1997; 235: 83-87Crossref PubMed Scopus (44) Google Scholar, 21Uddin S. Grumbach I.M. Yi T. Colamonici O.R. Platanias L.C. Br. J. Haematol. 1998; 101: 446-449Crossref PubMed Scopus (13) Google Scholar, 22Reddy E.P. Korapati A. Chaturvedi P. Rane S. Oncogene. 2000; 19: 2532-2547Crossref PubMed Scopus (192) Google Scholar). Alternatively, myxoma virus-CCR5 interactions may lead to cross-talk between cell surface receptors that are constitutively associated with CCR5, such as CD4 (23Xiao X. Wu L. Stantchev T.S. Feng Y.R. Ugolini S. Chen H. Shen Z. Riley J.L. Broder C.C. Sattentau Q.J. Dimitrov D.S. Proc. Natl. Acad. Sc. U. S. A. 1999; 96: 7496-7501Crossref PubMed Scopus (170) Google Scholar), which results in the subsequent phosphorylation of CCR5 by other tyrosine kinases, like p56lck. Certainly, the NIH 3T3.CD4.CCR5 cells also stably express ectopic human CD4 (Fig. 1 A), and an earlier report described the dissociation of p56lck from CD4 that accompanies myxoma virus infection of T lymphocytes (24Barry M. Lee S.F. Boshkov L. McFadden G. J. Virol. 1995; 69: 5243-5251Crossref PubMed Google Scholar). As with HIV, myxoma virus may interact with both CD4 and CCR5, and the phosphorylation-activation of p56lck that we observe may be a direct consequence of myxoma virus interaction with CD4. It is possible that neither of these scenarios is mutually exclusive, since the kinetics of Jak and p56lck phosphorylation in the NIH 3T3.CD4.CCR5 cells would suggest that both tyrosine kinases are rapidly phosphorylated within the first few minutes of virus adsorption. In a recent study, evidence was provided for the reciprocal desensitization of CCR5 and CD4 by their respective ligands (25Mashikian M.V. Ryan T.C. Seman A. Brazer W. Center D.M. Cruikshank W.W. J. Immunol. 1999; 163: 3123-3130PubMed Google Scholar). Moreover, HIV-1 gp120 association with CD4 results in p56lckphosphorylation and CX chemokine receptor CXCR4 down-regulation (26Su S.B. Gong W. Grimm M. Utsunomiya I. R. Oppenheim J.J. J.M. J. Immunol. 1999; Google Scholar). the of CXCR4 down-regulation is this role for CD4 and p56lck may be associated with of events that would lead to viral clearance the number of viral particles that may a Accordingly, we examined the of of p56lck on myxoma virus replication in NIH 3T3.CD4.CCR5 cells. The PP1 was used that a on p56lck activation at 5 J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The results in Fig. 4 a increase in myxoma virus replication, of a role for p56lck in myxoma virus infection. In other receptor the activation of Jaks results in the of multiple distinct proteins to and gene Accordingly, we examined whether two of the characterized of Jak activation, the signal transducers and activators of transcription (Stat) proteins C. Exp. Cell Res. 1999; PubMed Scopus (116) Google and the insulin receptor substrate M.F. Res. 1998; Google Scholar), were phosphorylated in NIH 3T3.CD4.CCR5 cells exposed to myxoma virus. In an earlier we provided evidence for phosphorylation-activation of the proteins and M. Fish E.N. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The results in Fig. A and that exposure of NIH 3T3.CD4.CCR5 cells to UV-inactivated myxoma virus leads to the rapid phosphorylation of and by 1 min, with the rapid kinetics of phosphorylation-activation that we have observed with the cognate The of proteins includes and which multiple tyrosine phosphorylation sites in protein binding for the of the of the protein and other signaling W. T. M. Biochem. 1998; PubMed Scopus Google Scholar). proteins a signaling role for receptors by their to these receptors to signaling We observed that myxoma virus consistently induces in the tyrosine phosphorylation of and (Fig. Our results show that with initiation of a fully productive myxoma virus infection in NIH 3T3.CD4.CCR5 cells, there is activation of a signal transduction cascade. HIV-1 (10Wang J.M. Oppenheim J.J. J. Exp. Med. 1999; 190: 591-595Crossref PubMed Scopus (17) Google Scholar), interference with tyrosine but not receptor to compromise the of myxoma virus to cells. with the signaling capacity of CCR5 using pertussis toxin can compromise the of HIV to cells, at both entry and M. H. T. M. J. Exp. Med. 1999; 190: PubMed Scopus Google Scholar). for myxoma virus, this activation is not but is dependent on tyrosine phosphorylation data do not the of entry of myxoma virus into cells, mediated by Indeed, the intracellular infectious of virus, cells by a that protein C the in addition to tyrosine phosphorylation A. S. G. H. R. G. Biol. 2000; PubMed Scopus Google Scholar). In studies, we have evidence that the non-permissive NIH 3T3.CD4.neo cells, as well as a cell human CCR5, that is also non-permissive for myxoma virus infection, permit viral yet target the virus for (data not shown). Notably, myxoma virus fails to either CCR5 phosphorylation or other tyrosine phosphorylation events in the cells (data not shown). The from are that adsorption by myxoma virus, in a a rapid tyrosine signal transduction that, at the for events for the of the fully productive virus replication In a of we examined the of Jak2 activation in viral replication. Specifically, NIH 3T3.CD4.CCR5 cells were treated with tyrophostin AG490, a of myxoma virus replication was (Fig. an on Jak2 activation at 10 with 10 myxoma Jak2 phosphorylation by (Fig. B). Thus, of Jak myxoma virus replication, of a critical role for this in virus infection. Myxoma of p56lck to CCR5 and its phosphorylation is of an earlier observation of Src phosphorylation of a viral protein, for viral F. S. S. I. G. M. Nature. 1999; PubMed Scopus Google Scholar). role for myxoma virus-inducible tyrosine phosphorylation of p56lck may be associated with either CCR5 or CD4 As described in other studies there is evidence that HIV-1 gp120 binding to CD4 on T cells leads to tyrosine phosphorylation of p56lck and the subsequent down-regulation of cell surface CXCR4 (26Su S.B. Gong W. Grimm M. Utsunomiya I. R. Oppenheim J.J. J.M. J. Immunol. 1999; Google Scholar). The observation that PP1 leads to enhanced viral replication is of a role for p56lck in myxoma virus infection. This with the for Jak2 activation for myxoma virus replication. this is for a productive virus infection. The of and proteins by myxoma virus that the virus may host cell gene expression to the cellular environment for the events in viral replication. In any the of cellular signaling pathways initiated by virus adsorption a novel by which viruses subvert cell surface receptors to host cell

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