Interaction of Glycoprotein H of Human Herpesvirus 6 with the Cellular Receptor CD46
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m302373200
ISSN1083-351X
AutoresFabio Santoro, Heather L. Greenstone, Alessandra Insinga, M. Kathryn Liszewski, John Atkinson, Paolo Lusso, Edward A. Berger,
Tópico(s)Viral-associated cancers and disorders
ResumoHuman herpesvirus 6 (HHV-6) employs the complement regulator CD46 (membrane cofactor protein) as a receptor for fusion and entry into target cells. Like other known herpesviruses, HHV-6 encodes multiple glycoproteins, several of which have been implicated in the entry process. In this report, we present evidence that glycoprotein H (gH) is the viral component responsible for binding to CD46. Antibodies to CD46 co-immunoprecipitated an ∼110-kDa protein band specifically associated with HHV-6-infected cells. This protein was identified as gH by selective depletion with an anti-gH monoclonal antibody, as well as by immunoblot analysis with a rabbit hyperimmune serum directed against a gH synthetic peptide. In reciprocal experiments, a monoclonal antibody against HHV-6 gH was found to co-immunoprecipitate CD46. Studies using monoclonal antibodies directed against specific CD46 domains, as well as engineered constructs lacking defined CD46 regions, demonstrated a close correspondence between the CD46 domains involved in the interaction with gH and those previously shown to be critical for HHV-6 fusion (i.e. short consensus repeats 2 and 3). Human herpesvirus 6 (HHV-6) employs the complement regulator CD46 (membrane cofactor protein) as a receptor for fusion and entry into target cells. Like other known herpesviruses, HHV-6 encodes multiple glycoproteins, several of which have been implicated in the entry process. In this report, we present evidence that glycoprotein H (gH) is the viral component responsible for binding to CD46. Antibodies to CD46 co-immunoprecipitated an ∼110-kDa protein band specifically associated with HHV-6-infected cells. This protein was identified as gH by selective depletion with an anti-gH monoclonal antibody, as well as by immunoblot analysis with a rabbit hyperimmune serum directed against a gH synthetic peptide. In reciprocal experiments, a monoclonal antibody against HHV-6 gH was found to co-immunoprecipitate CD46. Studies using monoclonal antibodies directed against specific CD46 domains, as well as engineered constructs lacking defined CD46 regions, demonstrated a close correspondence between the CD46 domains involved in the interaction with gH and those previously shown to be critical for HHV-6 fusion (i.e. short consensus repeats 2 and 3). Human herpesvirus 6 (HHV-6) 1The abbreviations used are: HHV, human herpesvirus; SCR, short consensus repeat; gp, glycoprotein; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cells; gH, glycoprotein H; DAF, decay accelerating factor; EMEM, Eagle's minimum essential medium; PBS, phosphate-buffered saline. is a member of the β-herpesvirus subfamily (reviewed in Ref. 1Dockrell D.H. J. Med. Microbiol. 2003; 52: 5-18Crossref PubMed Scopus (78) Google Scholar). Two major HHV-6 variants, A and B, have been defined that form two segregated clusters with unique genetic, biologic, and immunologic characteristics. Primary infection with HHV-6 B occurs almost universally in early childhood and has been etiologically linked to exanthema subitum. In adult life, HHV-6 infection and/or reactivation have been associated with a wide range of diseases, including multiple sclerosis, but most of these associations have yet to be substantiated by rigorous epidemiological studies. In immunocompromised people, including those infected with human immunodeficiency virus, HHV-6 B has been implicated as an opportunistic agent that may cause life-threatening respiratory tract and central nervous system infections, as well as bone marrow or organ graft failure. Moreover, HHV-6 may act as a cofactor to accelerate progression of human immunodeficiency virus disease. The best studied members of the herpesvirus family enter cells by direct fusion with the plasma membrane in a process involving binding of viral glycoproteins to specific protein receptors on the target cell (reviewed in Ref. 2Spear P.G. Eisenberg R.J. Cohen G.H. Virology. 2000; 275: 1-8Crossref PubMed Scopus (419) Google Scholar). Fusion is also facilitated by virus interactions with cell surface glycosaminoglycans. Recently, we identified CD46 (membrane cofactor protein) as a cellular receptor mediating fusion and entry for both HHV-6 variants (3Santoro F. Kennedy P.E. Locatelli G. Malnati M.S. Berger E.A. Lusso P. Cell. 1999; 99: 817-827Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). This glycoprotein also serves as a cellular receptor mediating critical events in the infectious process of other human pathogens, including binding/fusion/entry of measles virus, pilus binding of different pathogenic Neisseriae, and adhesion of group A Streptococci (reviewed in Ref. 4Hourcade D. Liszewski M.K. Krych-Goldberg M. Atkinson J.P. Immunopharmacology. 2000; 49: 103-116Crossref PubMed Scopus (71) Google Scholar). CD46 is a member of the regulators of complement activation (RCA) protein family. It is a type I transmembrane glycoprotein of 45–67 kDa, expressed on most or all human nucleated cell types. The N-terminal region consists of four partially homologous short consensus repeats (SCRs) linked in tandem; the SCRs are followed by a serine-threonine-proline (STP)-rich domain, a short region of unknown function, a transmembrane domain, and a cytoplasmic tail. Four distinct CD46 isoforms are variably expressed on different cell types; these contain the same SCRs but differ in the STP and cytoplasmic domains as a result of alternative RNA splicing. Recently the CD46 determinants required for HHV-6 fusion have been analyzed; our results indicated that the critical determinants reside within SCRs 2 and 3, but not SCRs 1 or 4 (5Greenstone H.L. Santoro F. Lusso P. Berger E.A. J. Biol. Chem. 2002; 277: 39112-39118Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), whereas another study suggested involvement of SCRs 2–4 (6Mori Y. Seya T. Huang H.L. Akkapaiboon P. Dhepakson P. Yamanishi K. J. Virol. 2002; 76: 6750-6761Crossref PubMed Scopus (81) Google Scholar). By contrast, the critical CD46 domains are SCRs 1 and 2 for measles virus, SCR3 and the STP regions for Neisseria gonorrhoeae, SCRs 3 and 4 for group A Streptococcus (4Hourcade D. Liszewski M.K. Krych-Goldberg M. Atkinson J.P. Immunopharmacology. 2000; 49: 103-116Crossref PubMed Scopus (71) Google Scholar). HHV-6 encodes multiple surface glycoproteins, including gB, gH, gL, and gM (1Dockrell D.H. J. Med. Microbiol. 2003; 52: 5-18Crossref PubMed Scopus (78) Google Scholar), for which relatively conserved homologs have been identified in all known mammalian herpesviruses. Of these, gH and gL appear to play prominent roles in the membrane fusion events that initiate HHV-6 infectivity, based on inhibitory activities of specific antibodies (7Foà-Tomasi L. Boscaro A. Digaeta S. Campadelli-Fiume G. J. Virol. 1991; 65: 4124-4129Crossref PubMed Google Scholar, 8Gompels U.A. Carss A.L. Saxby C. Hancock D.C. Forrester A. Minson A.C. J. Virol. 1991; 65: 2393-2401Crossref PubMed Google Scholar, 9Liu D.X. Gompels U.A. Foà-Tomasi L. Campadelli-Fiume G. Virology. 1993; 197: 12-22Crossref PubMed Scopus (61) Google Scholar, 10Qian G. Wood C. Chandran B. Virology. 1993; 194: 380-386Crossref PubMed Scopus (22) Google Scholar, 11Takeda K. Haque M. Sunagawa T. Okuno T. Isegawa Y. Yamanishi K. J. Gen. Virol. 1997; 78: 2171-2178Crossref PubMed Scopus (30) Google Scholar, 12Anderson R.A. Gompels U.A. J. Gen. Virol. 1999; 80: 1485-1494Crossref PubMed Scopus (13) Google Scholar). As in other herpesviruses, these glycoproteins form a heterodimeric complex, with gL being required for correct folding, intracellular maturation, and surface expression of gH (9Liu D.X. Gompels U.A. Foà-Tomasi L. Campadelli-Fiume G. Virology. 1993; 197: 12-22Crossref PubMed Scopus (61) Google Scholar, 12Anderson R.A. Gompels U.A. J. Gen. Virol. 1999; 80: 1485-1494Crossref PubMed Scopus (13) Google Scholar, 13Hutchinson L. Browne H. Wargent V. Davispoynter N. Primorac S. Goldsmith K. Minson A.C. Johnson D.C. J. Virol. 1992; 66: 2240-2250Crossref PubMed Google Scholar, 14Liu D.X. Gompels U.A. Nicholas J. Lelliott C. J. Gen. Virol. 1993; 74: 1847-1857Crossref PubMed Scopus (65) Google Scholar, 15Roop C. Hutchinson L. Johnson D.C. J. Virol. 1993; 67: 2285-2297Crossref PubMed Google Scholar, 16Westra D.F. Glazenburg K.L. Harmsen M.C. Tiran A. Scheffer A.J. Welling G.W. The T.H. Welling-Wester S. J. Virol. 1997; 71: 2285-2291Crossref PubMed Google Scholar). Moreover, gB (the most highly conserved glycoprotein among herpesviruses) and another glycoprotein, gp82-gp105, (thus far found only in HHV-6 and the related β-herpesvirus, HHV-7) appear to be critical for the fusion/entry process, as specific antibodies against these proteins block both virus infectivity and syncytia formation (17Takeda K. Okuno T. Isegawa Y. Yamanishi K. Virology. 1996; 222: 176-183Crossref PubMed Scopus (32) Google Scholar, 18Pfeiffer B. Thomson B. Chandran B. J. Virol. 1995; 69: 3490-3500Crossref PubMed Google Scholar, 19Pfeiffer B. Berneman Z.N. Neipel F. Chang C.K. Tirwatnapong S. Chandran B. J. Virol. 1993; 67: 4611-4620Crossref PubMed Google Scholar). Recently the gp82-gp105 glycoprotein has been shown to be associated with gH-gL complexes in infected cells and virions, and the designation gQ was proposed (20Mori Y. Akkapaiboon P. Yang X.W. Yamanishi K. J. Virol. 2003; 77: 2452-2458Crossref PubMed Scopus (56) Google Scholar). At present, the precise roles of individual HHV-6 glycoproteins and their possible interplay in the infection process remain poorly understood. In the present study, we sought to identify the HHV-6 glycoprotein(s) that directly interacts with the cellular receptor CD46. Using complementary co-immunoprecipitation approaches, we identified the gH glycoprotein as the CD46-binding component of HHV-6. Mapping studies demonstrated that the CD46 determinants critical for gH binding reside in SCR2 and SCR3, consistent with our previous study (5Greenstone H.L. Santoro F. Lusso P. Berger E.A. J. Biol. Chem. 2002; 277: 39112-39118Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) indicating the importance of the same CD46 domains for HHV-6 fusion. Cells and Culture Conditions—Primary human peripheral blood mononuclear cells (PBMC) were derived from Leukopak preparations obtained from healthy adult donors by gradient centrifugation (Bio-Whittaker, Walkersville, MD). NIH 3T3 (mouse embryo fibroblasts), RK13 (rabbit kidney cells), and HSB-2 (human immature T-lymphoid cells) were obtained from the American Type Culture Collection. NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mml-glutamine, and 10 μg/ml gentamicin). RK13 cells were maintained in EMEM-10 (Eagle's medium containing 10% fetal bovine serum, 2 mml-glutamine, and 10 μg/ml gentamicin). HSB-2 cells and PBMC were maintained in suspension in RPMI-10 (RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mml-glutamine, and 10 μg/ml gentamicin). Cultures were maintained in a humidified tissue culture incubator at 37 °C in 5% CO2. Virus and Infection Procedure—HHV-6 variant A (strain GS) was first propagated in adult human PBMC followed by co-culturing with HSB-2 cells as described (5Greenstone H.L. Santoro F. Lusso P. Berger E.A. J. Biol. Chem. 2002; 277: 39112-39118Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, 20 × 106 PBMC were cultured for 3 days at 2 × 106 cells/ml in RPMI-10 plus 5 μg/ml of phytohemagglutinin (Sigma). Activated cells were centrifuged at 300 × g for 10 min, resuspended in 2 ml of RPMI-10, and infected by incubation for 2 h at 37 °C with cell-free virus stock at an approximate multiplicity of infection of 0.5. The cells were subsequently diluted to 1 × 106 cells/ml and cultured in RPMI-10 for 6–8 days until cytopathic effects were observed. HSB-2 cells were infected with HHV-6 GS by co-cultivation with infected PBMC at a ratio of 5:1. For vaccinia virus infection, NIH 3T3 or RK13 cells were detached by trypsinization, resuspended at 1 × 107 cells per ml in EMEM with 2.5% fetal bovine serum (EMEM-2.5), and infected with wild-type vaccinia virus Western Reserve (WR) or with recombinant vaccinia virus vCB-48 encoding the BC2 isoform of human CD46 (21Nussbaum O. Broder C.C. Moss B. Stern L.B.L. Rozenblatt S. Berger E.A. J. Virol. 1995; 69: 3341-3349Crossref PubMed Google Scholar) at a multiplicity of infection of 10. After a 2-h incubation at 37 °C, the cells were diluted to 5 × 105 cells/ml in EMEM-2.5 and incubated overnight at 31 °C to allow CD46 expression. Plasmids and Transfection Procedure—Transfection protocols were performed in EMEM-2.5. Briefly, monolayers of RK13 were singly transfected with plasmids encoding either wild type CD46 (21Nussbaum O. Broder C.C. Moss B. Stern L.B.L. Rozenblatt S. Berger E.A. J. Virol. 1995; 69: 3341-3349Crossref PubMed Google Scholar) or one of the following variants (5Greenstone H.L. Santoro F. Lusso P. Berger E.A. J. Biol. Chem. 2002; 277: 39112-39118Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar): CD46 deletion mutants Δ1-CD46 and Δ2-CD46 lacking SCR1 and SCR2, respectively (22Manchester M. Valsamakis A. Kaufman R. Liszewski M.K. Alvarez J. Atkinson J.P. Lublin D.M. Oldstone M.B.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2303-2307Crossref PubMed Scopus (119) Google Scholar) or CD46-DAF chimeras X3-DAF or X4-DAF (23Christiansen D. Loveland B. Kyriakou P. Lanteri M. Escoffier C. Gerlier D. J. Gen. Virol. 2000; 81: 911-917Crossref PubMed Scopus (13) Google Scholar) (generously provided by Denis Gerlier, CNRS, Lyon, France) in which the SCR3 or SCR4 domains of CD46 were replaced with SCR3 and SCR4 domains of decay accelerating factor (DAF), respectively. All the recombinant CD46 sequences were cloned into pSC59, which contains a strong synthetic vaccinia promoter (24Chakrabarti S. Sisler J.R. Moss B. Biotechniques. 1997; 23: 1094-1097Crossref PubMed Scopus (332) Google Scholar). As a parallel negative control, cells were transfected with the pSC59 empty vector. The transfection procedure was carried out with DOTAP transfection reagent (Roche Applied Science). After4hat37 °C the transfected cells were detached by trypsinization, infected with vaccinia WR, and incubated overnight at 31 °C as described above. Immunological Reagents—Immunoprecipitation reactions were performed with monoclonal antibodies (mAbs). The following mAbs against human CD46 were used (at 25 μg/ml): J4.48 (25Pesando J.M. Stucki M. Hoffman P. Hum. Immunol. 1987; 19: 235-243Crossref PubMed Scopus (8) Google Scholar) and E4.3 (26Purcell D.F.J. Deacon N.J. Mckenzie I.F.C. Immunol. Cell Biol. 1989; 67: 279-289Crossref PubMed Scopus (9) Google Scholar) to SCR1, purchased from Beckman Coulter, Fullerton, CA and Santa Cruz Biotechnology, Santa Cruz, CA, respectively; mAbs M177 to CD46 SCR2 and M160 to CD46 SCR3 (27Seya T. Hara T. Matsumoto M. Akedo H. J. Immunol. 1990; 145: 238-245PubMed Google Scholar), kindly provided by Tsukasa Seya, Osaka Medical Center, Osaka, Japan; and mAb GB24, which recognizes SCR4 (Ref. 28Cho S.W. Oglesby T.J. Hsi B.L. Adams E.M. Atkinson J.P. Clin. Exp. Immunol. 1991; 83: 257-261Crossref PubMed Scopus (52) Google Scholar and unpublished data). The following mAbs against HHV-6 glycoproteins were employed: anti-HHV-6 gp116/64/54 against gB (used at 25 μg/ml), purchased from ABI, Columbia, MD, and mAbs 7A2 against gH (ascites fluid, used at 1:2000 dilution) and 2D6 against gp82-gp105 (tissue culture supernatant, used at 1:4 dilution) (29Balachandran N. Amelse R.E. Zhou W.W. Chang C.K. J. Virol. 1989; 63: 2835-2840Crossref PubMed Google Scholar) (kindly donated by Dr. Bala Chandran, University of Kansas). The negative control mAb HA.11 to influenza hemagglutinin (30Field J. Nikawa J. Broek D. Macdonald B. Rodgers L. Wilson I.A. Lerner R.A. Wigler M. Mol. Cell. Biol. 1988; 8: 2159-2165Crossref PubMed Scopus (733) Google Scholar) (used at 25 μg/ml) was purchased from Covance (Princeton, NJ). For immunoblot analysis, rabbit polyclonal primary antibodies were employed. For CD46 detection, antibody H-294 was purchased from Santa Cruz Biotechnology. For gH detection, a hyperimmune rabbit antiserum was raised against a short peptide spanning the amino terminus of HHV-6A gH from amino acid 79 to amino acid 93 (sequence: ESLTNYEKRVTRFYE). The N-acetylated synthetic peptide linked to keyhole limpet hemacyanin was used for rabbit immunization (Sigma Genosys, The Woodlands, TX). As the secondary antibody, donkey anti-rabbit immunoglobulin conjugated to horseradish peroxidase (Chemicon, Temecula, CA) was employed. Co-immunoprecipitation Experiments—Co-immunoprecipitation experiments were performed either with metabolically labeled cells coupled with autoradiographic detection or with unlabeled cells using immunoblot analysis. In the labeling protocol, HHV-6-infected HSB-2 cells at 3 days postinfection were washed twice with methionine-free Dulbecco's modified Eagle's medium and resuspended at 5 × 105 cells/ml with methionine-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum and 40 μCi/ml of [35S]methionine; uninfected HSB-2 cells were labeled in parallel as a negative control. After a 20-h culture at 37 °C, the cells were washed and suspended in methionine-free Dulbecco's modified Eagle's medium. Mixtures were prepared containing 1 × 106-labeled HSB-2 cells and 1 × 106 unlabeled cells (NIH 3T3 or RK13) expressing vaccinia-encoded CD46 (wild type or variants) in a total volume of 200 μl and incubated for 1 h at 37 °C. The cells were washed in cold PBS and solubilized in 200 μl of lysis buffer containing 100 mm Tris-HCl (pH 8.0), 100 mm NaCl, and 0.5% (v/v) Triton X-100. Cell lysates were incubated on ice for 20 min, and cellular debris was removed by centrifugation at 24,000 × g for 15 min at 4 °C. The lysates were precleared by adding 50 μl of goat anti-mouse IgG agarose (Sigma) and incubating overnight at 4 °C with rotation. Co-immunoprecipitation from the precleared lysates was performed by adding the indicated anti-CD46 mAb (at 25 μg/ml), incubating for 2 h at 4 °C, adding 50 μl of goat anti-mouse IgG agarose, and incubating for4hat4 °C with rotation. The agarose beads were washed four times with 1 ml of lysis buffer; SDS-PAGE sample buffer containing 5% (v/v) 2-mercaptoethanol was added, and the samples were heated for 5 min at 95 °C. Proteins were separated by SDS-PAGE on 4–20% gradient gels (Invitrogen) and blotted onto nitrocellulose membranes (Protran, Schleicher & Schuell, Keene, NH); immunoprecipitated proteins were detected by autoradiography. Where indicated, direct immunoprecipitation from the metabolically labeled lysate was performed by using a mAb against the designated HHV-6 glycoprotein and processing the samples as described above. For studies involving immunodepletion of specific metabolically labeled HHV-6 glycoproteins, the supernatants recovered from the first immunoprecipitations with the anti-glycoprotein mAbs were subjected to a second identical immunoprecipitation reaction. Only minimal amounts of the glycoproteins were precipitated by the second reaction (data not shown), thus verifying the efficacy of the immunodepletion. The supernatants recovered after immunodepletion were incubated with goat anti-mouse agarose to remove any remaining antibodies and then subjected to co-immunoprecipitation with an anti-CD46 mAb as described above. For the experiments involving detection of unlabeled proteins by immunoblotting, preparation of cell lysates, co-immunoprecipitation reactions, and SDS-PAGE were performed as described above. The resolved proteins were transferred to nitrocellulose membranes, which were then blocked by a 1-h incubation with PBS/0.1% Tween-20 containing 10% milk. The membranes were then washed and incubated with the designated polyclonal antiserum (see above) in PBS/0.1% Tween-20 containing 5% milk. Rabbit antiserum against the gH peptide was used at a 1:700 dilution; rabbit antiserum to CD46 (H-294) was used at a 1:1000 dilution. After a 1-h incubation, the membranes were washed three times with PBS/0.1% Tween-20 and incubated for 1 h with donkey anti-rabbit antibody conjugated to horseradish peroxidase in PBS/0.1% Tween-20 (dilution 1:5000). After three washes the membranes were subjected to enhanced chemiluminescence detection using the Supersignal West Pico chemiluminescent substrate (Pierce). Co-immunoprecipitation of Human CD46 with HHV-6-associated Proteins—To identify the HHV-6 protein(s) interacting with CD46, co-immunoprecipitation studies were performed to test the ability of the cellular receptor to form a stable complex with proteins expressed in HHV-6-infected cells. Mouse fibroblasts (NIH 3T3) were infected with a recombinant vaccinia virus encoding full-length human CD46 (BC2 isoform). These cells were mixed with [35S]methionine-labeled human immature T-lymphoid cells (HSB-2) productively infected with HHV-6, strain GS (variant A). Flow cytometry verified surface expression of HHV-6 glycoproteins (gH, gp82–105, and gB). The cell mixture was lysed and subjected to immunoprecipitation with an anti-human CD46 mAb (J4–48); this mAb is specific for the SCR1 domain, which is dispensable for HHV-6 fusion (5Greenstone H.L. Santoro F. Lusso P. Berger E.A. J. Biol. Chem. 2002; 277: 39112-39118Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 6Mori Y. Seya T. Huang H.L. Akkapaiboon P. Dhepakson P. Yamanishi K. J. Virol. 2002; 76: 6750-6761Crossref PubMed Scopus (81) Google Scholar). The immunoprecipitates were resolved by reducing SDS-PAGE as described under "Experimental Procedures." As shown in Fig. 1A, a major radiolabeled band of ∼110 kDa was co-precipitated with anti-CD46 (lane 2); this band was absent from a control reaction using uninfected HSB-2 cells (lane 1), as well as from another control using CD46-negative NIH 3T3 cells (lanes 3 and 4). The latter result demonstrates that it was the recombinant CD46 from the NIH 3T3 cells rather than the endogenous CD46 from the infected HSB-2 cells that mediated the co-immunoprecipitation. Several minor bands were also observed under all conditions and thus were interpreted as background. Further specificity is illustrated in Fig. 1B, which demonstrates that the ∼110-kDa band was not co-immunoprecipitated by an isotype control mAb HA.11 directed against influenza virus hemagglutinin. Taken together, these results demonstrate that human CD46 interacts with a protein specific for HHV-6-infected cells. The electrophoretic mobility of this protein is consistent with that reported for glycoprotein H (gH) (100–110 kDa, Refs. 9Liu D.X. Gompels U.A. Foà-Tomasi L. Campadelli-Fiume G. Virology. 1993; 197: 12-22Crossref PubMed Scopus (61) Google Scholar, 10Qian G. Wood C. Chandran B. Virology. 1993; 194: 380-386Crossref PubMed Scopus (22) Google Scholar, 14Liu D.X. Gompels U.A. Nicholas J. Lelliott C. J. Gen. Virol. 1993; 74: 1847-1857Crossref PubMed Scopus (65) Google Scholar). Depletion of the CD46-interacting ∼110-kDa Protein with an Anti-gH mAb—To assess whether the ∼110-kDa band was gH, we tested whether depletion of the supernatant with an anti-gH mAb would result in loss of this band upon subsequent co-immunoprecipitation with anti-CD46. We also examined the effects of depleting two other HHV-6 glycoproteins, gp82-gp105 and gB; all three glycoproteins are known to play critical roles in viral infectivity, as shown by the neutralizing activities of specific antibodies to gH (7Foà-Tomasi L. Boscaro A. Digaeta S. Campadelli-Fiume G. J. Virol. 1991; 65: 4124-4129Crossref PubMed Google Scholar, 8Gompels U.A. Carss A.L. Saxby C. Hancock D.C. Forrester A. Minson A.C. J. Virol. 1991; 65: 2393-2401Crossref PubMed Google Scholar, 9Liu D.X. Gompels U.A. Foà-Tomasi L. Campadelli-Fiume G. Virology. 1993; 197: 12-22Crossref PubMed Scopus (61) Google Scholar, 10Qian G. Wood C. Chandran B. Virology. 1993; 194: 380-386Crossref PubMed Scopus (22) Google Scholar, 11Takeda K. Haque M. Sunagawa T. Okuno T. Isegawa Y. Yamanishi K. J. Gen. Virol. 1997; 78: 2171-2178Crossref PubMed Scopus (30) Google Scholar, 12Anderson R.A. Gompels U.A. J. Gen. Virol. 1999; 80: 1485-1494Crossref PubMed Scopus (13) Google Scholar), gB (17Takeda K. Okuno T. Isegawa Y. Yamanishi K. Virology. 1996; 222: 176-183Crossref PubMed Scopus (32) Google Scholar), or gp82-gp105 (19Pfeiffer B. Berneman Z.N. Neipel F. Chang C.K. Tirwatnapong S. Chandran B. J. Virol. 1993; 67: 4611-4620Crossref PubMed Google Scholar). We first analyzed the products of direct immunoprecipitation from the lysate of a mixture of HHV-6-infected HSB-2 cells and CD46-expressing NIH 3T3 cells (Fig. 2A). As expected, the anti-gH mAb (7A2) precipitated a major band of ∼110 kDa, which was not observed with the other mAbs. The anti-gp82-gp105 mAb (2D6) precipitated a major protein band of ∼80 kDa as well as several minor bands of various sizes, consistent with previous analyses of gp82-gp105 (18Pfeiffer B. Thomson B. Chandran B. J. Virol. 1995; 69: 3490-3500Crossref PubMed Google Scholar, 29Balachandran N. Amelse R.E. Zhou W.W. Chang C.K. J. Virol. 1989; 63: 2835-2840Crossref PubMed Google Scholar). The anti-gB mAb (anti-HHV-6 gp 116/64/54) precipitated three major proteins of ∼120, 64, and 54 kDa, consistent with previous analyses of gB (17Takeda K. Okuno T. Isegawa Y. Yamanishi K. Virology. 1996; 222: 176-183Crossref PubMed Scopus (32) Google Scholar, 29Balachandran N. Amelse R.E. Zhou W.W. Chang C.K. J. Virol. 1989; 63: 2835-2840Crossref PubMed Google Scholar). We then analyzed the lysates that had been depleted of specific HHV-6 glycoproteins for co-immunoprecipitation with anti-CD46. With the gH-depleted lysate, the ∼110-kDa band was no longer observed, in contrast with the results for the non-depleted supernatants shown above (Fig. 1). However, in both the gp82-gp105-depleted and the gB-depleted lysates, the ∼110-kDa band was still observed. These results indicate that all of the detected radiolabeled protein specifically co-immunoprecipitated with anti-CD46 was specifically recognized by the anti-gH mAb. Confirmation of gH Binding to CD46 by Reciprocal Immunoprecipitation and Immunoblotting—As an additional approach to test for a possible interaction between gH and CD46, we examined the ability of mAbs against one protein to co-immunoprecipitate the other from mixtures of unlabeled HHV-6-infected and CD46-expressing cells; immunoblotting with specific polyclonal antisera was used to detect the co-immunoprecipitated proteins. In Fig. 3A, the J4.48 anti-CD46 mAb was used for the initial immunoprecipitation, and a rabbit antiserum prepared against a synthetic peptide corresponding to residues 79–93 of gH from HHV-6 strain U1102 (variant A) was used for detection of gH (the specific reactivity of this antiserum for gH was verified by immunoblotting; data not shown). A single prominent ∼110-kDa band was observed. This band was absent from a negative control immunoprecipitate from a mixture lacking CD46-expressing cells, thereby verifying that the gH co-immunoprecipitation was mediated by the recombinant CD46 from the 3T3 cells rather than the endogenous CD46 from the HSB-2 cells. This experiment verifies that gH is co-immunoprecipitated with anti-CD46. A reciprocal experiment was performed to test whether mAbs against different HHV-6 glycoproteins could co-immunoprecipitate CD46 from a lysate of HHV-6-infected HSB-2 cells mixed with recombinant CD46-expressing NIH 3T3 cells. Fig. 3B shows that two broad CD46 bands of ∼67 and 50 kDa were co-immunoprecipitated by the anti-gH mAb, similar to the profile of bands directly immunoprecipitated by an anti-CD46 mAb. These bands were absent in a control experiment with NIH 3T3 cells lacking recombinant CD46 (data not shown), again demonstrating that they represented the recombinant CD46 rather than endogenous CD46 on the HSB-2 cells. The sizes of the two CD46 bands are consistent with the previously described processing of the high mannose-containing precursor (lower band) to the mature product (upper band) (31Liszewski M.K. Tedja I. Atkinson J.P. J. Biol. Chem. 1994; 269: 10776-10779Abstract Full Text PDF PubMed Google Scholar). These two bands were previously observed for vaccinia-encoded CD46 (21Nussbaum O. Broder C.C. Moss B. Stern L.B.L. Rozenblatt S. Berger E.A. J. Virol. 1995; 69: 3341-3349Crossref PubMed Google Scholar) and presumably reflect incomplete processing under conditions of vaccinia-mediated overexpression. Importantly, Fig. 3B also shows that the co-immunoprecipitation of CD46 was specific for gH, because it was not observed with the anti-gB mAb or the anti-gp82-gp105 mAb. Correlation between the CD46 Domains Involved in gH Binding and HHV-6 Fusion—In our previous report (5Greenstone H.L. Santoro F. Lusso P. Berger E.A. J. Biol. Chem. 2002; 277: 39112-39118Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) on the CD46 regions required for HHV-6-mediated cell fusion, a critical role was observed for the two central SCR domains, SCR2 and SCR3. To test for a possible relationship between these two regions of CD46 and the co-immunoprecipitation of gH, two types of experiments were performed. Fig. 4A shows gH co-immunoprecipitation studies performed using mAbs directed against each of the four SCR domains of CD46. Marked differences were observed (Fig. 4A, upper panel): co-immunoprecipitation occurred with the mAbs directed against SCR1 or SCR4 but not with those directed against SCR2 or SCR3. This distinction was noted despite the ability of all the mAbs to directly immunoprecipitate CD46 (Fig. 4A, lower panel). As a complementary approach, we tested gH co-immunoprecipitation by variant CD46 constructs differing in their SCR domain compositions. RK13 cells were transfected with plasmids encoding either wild-type CD46, CD46 deletion mu
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