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J. Biol. Chem., Vol. 276, Issue 51, 48371-48375, December 21, 2001

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Poxvirus Infection Rapidly Activates Tyrosine Kinase Signal Transduction^*

Jennefer Masters^§, Anna A. Hinek^§, Shahab Uddin^¶, Leonidas C. Platanias^¶, Wei Zeng, Grant McFadden^**, and Eleanor N. Fish^§§

From the  Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network, Canadian Blood Services Building, Toronto, Ontario M5G 2M1, Canada, the  Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the ^¶ Section of Hematology-Oncology, University of Illinois and West Side Veterans Affairs Hospital, Chicago, Illinois 60607,  The John P. Robarts Research Institute and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario 46G 2V4, Canada

Received for publication, August 20, 2001, and in revised form, October 5, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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 p56^lck, 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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 (1). Until its global irradication, smallpox, caused by an orthopoxvirus, was one of the most serious diseases of mankind. Myxoma virus, a member of the Leporipoxvirus genus, is the causative agent of myxomatosis, a lethal disease of the European rabbit (2). Myxomatosis is characterized by extensive fulminating lesions and severe immune dysfunction accompanied by supervening Gram-negative bacterial infections of the respiratory tract (3).

Recently, evidence was provided that myxoma virus may utilize chemokine receptors to initiate infection (4). 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 (4-6). 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 (7). 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 (8, 9). 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 (10). 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 (10-13). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cells, Viruses, and Reagents-- 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 -galactosidase marker cassette driven by a late viral promoter in an intergenic location (4). 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 × 10⁵ µJ/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.

Analysis of Human CCR5 and CD4 Expression by Flow Cytometry-- 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 (14). CELLQuest software was used to analyze data.

Cell Lysis and Immunoblotting-- Approximately 10⁷ cells 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 (16). Immunoprecipitations and immunoblotting using enhanced chemiluminescence were performed as described previously (16).

-Galactosidase Colorimetric Assay-- 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 mM KCl, 1 mM MgSO₄, and 50 mM -mercaptoethanol. The mixture was incubated for 5 min at 37 °C. To measure -galactosidase activity, 4 mg/ml of substrate o-nitrophenyl--D-galactopyranoside was added in 100 mM NaH₂PO₄, pH 7.5. The breakdown of o-nitrophenyl--D-galactopyranoside by -galactosidase results in a yellow color reaction. After 1 h the reaction was terminated by the addition of 1 M Na₂CO₃. Absorbance was measured spectrophotometrically at 420 nm using a THERMOmax microplate reader.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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. 1A), are fully permissive for viral replication (Fig. 1B). For these studies, viral gene expression was monitored using a recombinant myxoma virus that expresses -galactosidase under the control of a late viral promoter that drives a lacZ transgene reporter (4). By contrast, NIH 3T3.CD4.neo cells that express low levels of human CD4 and no CCR5 (Fig. 1A) do not support myxoma virus replication, as revealed in Fig. 1B. 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).

View larger version (46K): [in this window] [in a new window]   Fig. 1.   NIH 3T3.CD4.neo and NIH 3T3.CD4.CCR5 are differentially permissive for myxoma virus infection. A, cell surface expression of human CCR5 and human CD4 were determined by flow cytometric analysis. Incubation with medium alone or 2o and 3o reagents resulted in the negative cytogram, represented as the filled profiles. Positive cytograms for CCR5 (gray) and CD4 (black) are represented as open profiles. B, susceptibility to myxoma virus infection was determined for both NIH 3T3.CD4.CCR5 and NIH 3T3.CD4.neo cells. Cells were either mock-infected or infected at a m.o.i. of 1 with vMyxlac for 1 h. After 16 h, infected monolayers were fixed and stained for LacZ expression.

Since myxoma virus infection of NIH 3T3.CD4.CCR5 cells can be inhibited by herbimycin A (4), 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 (17). 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. 2C). Immunoblot analysis identified this protein as the Src kinase, p56^lck (Fig. 2D). Our data would suggest that p56^lck associates with CCR5 in a tyrosine phosphorylation-dependent manner. Notably, we observe inducible phosphorylation of p56^lck whether live or UV-inactivated myxoma virus is used (Fig. 2E).

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Myxoma virus adsorption induces CCR5 tyrosine phosphorylation in NIH 3T3.CD4.CCR5 cells. NIH 3T3.CD4.CCR5 cells were either treated with medium alone () or exposed (+) to an equivalent of an m.o.i. of 10 with UV-inactivated (20 min) myxoma virus particles, for the times indicated. Cell lysates were immunoprecipitated (IP) with either an anti-phosphotyrosine antibody (pY20) (A) or anti-CCR5 antibody (B, C, D), and immunoprecipitated proteins were resolved by SDS-PAGE, then sequentially immunoblotted (WB) as shown, for CCR5 (A), phosphotyrosine (B, C), and p56lck (D). E, NIH 3T3.CD4.CCR5 cells were either left untreated or exposed to live (m.o.i of 10) or an equivalent m.o.i of UV-inactivated (30 min) vMyxlac for 5 min. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody 4G10, and 80 µg of immunoprecipitated proteins were resolved by SDS-PAGE, then immunoblotted for p56lck.

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. 3A), yet the non-susceptible NIH 3T3.CD4.neo cells do not show any evidence of virus-inducible tyrosine phosphorylation of cellular proteins (Fig. 3B). Additionally, BGMK cells become resistant to myxoma virus infection following herbimycin A treatment.²

View larger version (52K): [in this window] [in a new window]   Fig. 3.   Myxoma virus-induced tyrosine phosphorylation of cellular proteins is cell-specific. NIH 3T3.CD4.CCR5 (A) and NIH 3T3.CD4.neo (B) cells were either treated with medium alone () or exposed (+) to an equivalent of an m.o.i. of 10 with UV-inactivated myxoma virus, for the times indicated. Cell lysates were immunoprecipitated (IP) with anti-phosphotyrosine (pY20) antibody, and immunoprecipitated proteins were resolved by SDS-PAGE, then immunoblotted (WB) with 4G10 anti-phosphotyrosine antibody. The NIH 3T3.CD4.CCR5 membrane was stripped and re-probed for Jak1 (C) and Jak2 (D) with the respective antibodies. E, NIH 3T3.CD4.CCR5 cells were either left untreated or exposed to live (m.o.i of 10) or an equivalent m.o.i of UV-inactivated (30 min) vMyxlac for 5 min. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody 4G10, and 80 µg of immunoprecipitated proteins were resolved by SDS-PAGE, then immunoblotted with pY20 anti-phosphotyrosine antibody. Stripping and reprobing the immunoblot confirmed the band indicated corresponded to Jak2.

Chemokine-mediated activation of chemokine receptors leads to the rapid phosphorylation of receptor-associated Jaks (18, 19). 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. 3E). We did not observe myxoma virus-inducible Jak3 phosphorylation (not shown). Since p56^lck associated with CCR5 in a phosphorylation-dependent manner (Fig. 2, C and D), we infer that p56^lck may be a substrate for the activated Jaks (20-22). Alternatively, myxoma virus-CCR5 interactions may lead to cross-talk between cell surface receptors that are constitutively associated with CCR5, such as CD4 (23), which results in the subsequent phosphorylation of CCR5 by other tyrosine kinases, like p56^lck. Certainly, the NIH 3T3.CD4.CCR5 cells also stably express ectopic human CD4 (Fig. 1A), and an earlier report described the dissociation of p56^lck from CD4 that accompanies myxoma virus infection of T lymphocytes (24). As with HIV, myxoma virus may interact with both CD4 and CCR5, and the phosphorylation-activation of p56^lck 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 p56^lck 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 (25). Moreover, HIV-1 gp120 association with CD4 results in p56^lck phosphorylation and CX chemokine receptor CXCR4 down-regulation (26). Although the pathophysiological relevance of CXCR4 down-regulation is unclear, this negative regulatory role for CD4 and p56^lck may be associated with inhibition of ligand-CXCR4-inducible events that would lead to viral clearance and/or limiting the number of viral particles that may subsequently infect a single cell. Accordingly, we examined the effect of inhibition of p56^lck on myxoma virus replication in NIH 3T3.CD4.CCR5 cells. The pharmacological inhibitor PP1 was used that exhibits a half-maximal inhibitory concentration (IC₅₀) on p56^lck activation at 5 nM (27). The results in Fig. 4 reveal a dose-dependent PP1-mediated increase in myxoma virus replication, suggestive of a negative regulatory role for p56^lck in myxoma virus infection.

View larger version (8K): [in this window] [in a new window]   Fig. 4.   PP1 treatment of NIH 3T3.CD4.CCR5 cells results in increased myxoma virus replication. NIH 3T3.CD4.CCR5 cells were either left untreated or treated with varying doses of PP1 for 15 min prior to myxoma virus adsorption. Cells were infected at an m.o.i. of 10, then LacZ activity measured 16 h post-infection, using the -galactosidase colorimetric assay described under "Experimenatl Procedures." Results are representative of five different experiments in which lysates were assayed in quadruplicate.

In other cytokine receptor systems, the activation of Jaks results in the engagement of multiple distinct proteins to transduce signals and alter gene expression. Accordingly, we examined whether two of the best characterized downstream targets of Jak activation, the signal transducers and activators of transcription (Stat) proteins (28) and the insulin receptor substrate (IRS)-proteins (29), were phosphorylated in NIH 3T3.CD4.CCR5 cells exposed to myxoma virus. In an earlier published report, we provided evidence for RANTES-CCR-mediated phosphorylation-activation of the Stat proteins Stat1 and Stat3 (30). The results in Fig. 5, A and B, indicate that exposure of NIH 3T3.CD4.CCR5 cells to UV-inactivated myxoma virus leads to the rapid phosphorylation of Stat1 and Stat3 by 1 min, consistent with the rapid kinetics of phosphorylation-activation that we have observed with the cognate ligand, RANTES. The IRS family of proteins includes IRS-1 and IRS-2, which contain multiple tyrosine phosphorylation sites in protein binding motifs for the SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase, the adapter protein Grb-2, SHP-2 phosphatase, and other signaling elements (31). These proteins play a central signaling role for various cytokine receptors by their ability to link these receptors to diverse downstream signaling pathways. We observed that myxoma virus consistently induces dose-dependent increases in the tyrosine phosphorylation of IRS-1 and IRS-2, albeit relatively weakly (Fig. 5, C-E).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Myxoma virus induces Stat and IRS tyrosine phosphorylation. NIH 3T3.CD4.CCR5 cells were either treated with medium alone () or exposed (+) to an equivalent of an m.o.i. of 10 with UV-inactivated myxoma virus, for the times indicated. Cell lysates were immunoprecipitated (IP) with anti-Stat1 (A) or anti-Stat3 (B) antibodies. These immunocomplexed proteins were resolved by SDS-PAGE, then immunoblotted (WB) with 4G10 anti-phosphotyrosine antibody. The blots were stripped and re-probed with Stat1 and Stat3 antibodies, respectively. C and D, cell lysates treated as above were immunoprecipitated with anti-IRS1 (C) or anti-IRS-2 (D) antibodies, resolved by SDS-PAGE, and immunoblotted with 4G10 anti-phosphotyrosine antibody. The blot was stripped and re-probed for IRS-1 and IRS-2, using anti-IRS-1 and anti-IRS-2 antibodies. E, NIH 3T3.CD4.CCR5 cells were either left untreated or exposed to increasing doses of live vMyxlac for 5 min. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody 4G10, and 80 µg of immunoprecipitated proteins were resolved by SDS-PAGE, then immunoblotted with pY20 anti-phosphotyrosine antibody. Stripping and reprobing the immunoblot confirmed the band indicated corresponded to IRS-1.

Our results clearly show that coincident with initiation of a fully productive myxoma virus infection in NIH 3T3.CD4.CCR5 cells, there is activation of a signal transduction cascade. Unlike HIV-1 (10), interference with tyrosine kinase signaling, but not G-protein-coupled receptor signaling, appears to compromise the ability of myxoma virus to infect cells. Interference with the G-protein-coupled signaling capacity of CCR5 using pertussis toxin can compromise the ability of HIV to infect cells, at both entry and post-entry stages (32). Interestingly, for myxoma virus, this activation is not G-protein-coupled, but is dependent on tyrosine phosphorylation events. These data do not rule out the possibility of signal-dependent entry of myxoma virus into cells, mediated by non-tyrosine kinase pathways. Indeed, the intracellular mature infectious form of vaccinia virus, IMV, enters cells by a process that involves protein kinase C phosphorylation, the small GTPase rac1, in addition to tyrosine phosphorylation (33). In preliminary studies, we have evidence that the non-permissive NIH 3T3.CD4.neo cells, as well as a rat basophilic cell line ectopically expressing human CCR5, RBL-5, that is also non-permissive for myxoma virus infection, permit viral particle entry, yet target the virus for lysosomal degradation (data not shown). Notably, myxoma virus fails to induce either CCR5 phosphorylation or other tyrosine phosphorylation events in the RBL-5 cells (data not shown). The implications from our study are that adsorption by myxoma virus, in a ligand-like fashion, activates a rapid tyrosine kinase signal transduction that, at the very least, allows for downstream events required for the completion of the fully productive virus replication cycle.

In a final series of experiments, we examined the influence of Jak2 activation in mediating viral replication. Specifically, when NIH 3T3.CD4.CCR5 cells were treated with tyrophostin AG490, a dose-dependent inhibition of myxoma virus replication was noted (Fig. 6A). AG490 exhibits an IC₅₀ on Jak2 activation at 10 µM. Treatment with 10 µM AG490 reduced myxoma virus-induced Jak2 phosphorylation by 36% (Fig. 6B). Thus, inhibition of Jak kinase activity blocks myxoma virus replication, indicative of a critical role for this kinase in virus infection.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Tyrophostin AG490 inhibits myxoma virus replication in NIH 3T3.CD4.CCR5 cells. A, NIH 3T3.CD4.CCR5 cells were either left untreated or treated with varying doses of AG490 for 16 h prior to myxoma virus adsorption. Cells were infecetd at an m.o.i. of 10, then LacZ measured using the -galactosidase colorimetric assay described under "Experimental Procedures," 16 h post-infection. Results are representative of four experiments in which lysates were assayed in quadruplicate. B, NIH 3T3.CD4.CCR5 cells were either left untreated or treated with 25 µM of AG490 for 16 h, then exposed to UV-inactivated myxoma virus for 5 min. Lysates from 107 cells were immunoprecipitated with 4G10 anti-phosphotyrosine antibody, then 80 µg of immunocomplexed proteins were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine (pY20) antibody. The membrane was stripped and re-probed with anti-Jak2 Ab.

Myxoma virus-dependent recruitment of p56^lck to CCR5 and its concommitant phosphorylation is reminiscent of an earlier observation of Src family kinase phosphorylation of a vaccinia viral protein, A36R, required for viral spread (34). An alternative role for myxoma virus-inducible tyrosine phosphorylation of p56^lck may be associated with either CCR5 or CD4 down-regulation. As described above, in other studies there is evidence that HIV-1 gp120 binding to CD4 on T cells leads to tyrosine phosphorylation of p56^lck and the subsequent down-regulation of cell surface CXCR4 (26). The observation that PP1 treatment leads to enhanced viral replication is supportive of a negative regulatory role for p56^lck in myxoma virus infection. This contrasts with the requirement for Jak2 activation for myxoma virus replication. Apparently, this kinase activity is obligatory for a productive virus infection. The engagement of Stat and IRS proteins by myxoma virus suggests that the virus may regulate host cell gene expression to create the appropriate cellular environment for the later events in viral replication. In any event, the utilization of cellular tyrosine-kinase signaling pathways initiated by virus adsorption represents a novel strategy by which viruses subvert cell surface receptors to mediate host cell tropism.

	FOOTNOTES

^* This work was supported by Canadian Institutes of Health Research Grants MOP-42564 (to E. N. F.) and MOP-37993 (to G. M.), by National Institutes of Health Grants CA73381 and CA77816, and by a Merit Review grant form the Department of Veterans Affairs (to L. C. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

^** Senior Scientist of the Canadian Institutes of Health Research.

^§§ To whom correspondence should be addressed: Dept. of Cell & Molecular Biology, University Health Network, Toronto General Research Inst., Canadian Blood Services Bldg., 67 College St., Rm. 424, Toronto, Ontario M5G 2M1, Canada. Tel.: 416-340-5380; Fax: 416-340-3453; E-mail: en.fish@utoronto.ca.

Published, JBC Papers in Press, October 8, 2001, DOI 10.1074/jbc.M108019200

² J. Masters, A. A. Hinek, G. McFadden, and E. N. Fish, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; m.o.i, multiplicity of infection; PP1, 4-amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo(3,4-d)pyrimidine; IRS, insulin receptor substrate; Stat, signal transducers and activators of transcription; RANTES, regulated on activation normal T cell expressed and secreted.

	REFERENCES

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