The Journal of Immunology Human Cytomegalovirus Envelope Glycoproteins B and H Are Necessary for TLR2 Activation in Permissive Cells1 Karl W. Boehme,* Mario Guerrero,*† and Teresa Compton2*† Human CMV (HCMV) is a ubiquitous member of the Herpesviridae family and an opportunistic pathogen that poses significant health risks for immunocompromised patients. HCMV pathogenesis is intimately tied to the immune status of the host, thus characterization of the innate immune response to HCMV infection is critical for understanding disease progression. Previously, we identified TLR2 as a host factor that detects and initiates inflammatory cytokine secretion in response to HCMV independent of viral replication. In this study, we show that two entry-mediating envelope gp, gp B (gB) and gp H (gH), display determinants recognized by TLR2. Neutralizing Abs against TLR2, gB and gH inhibit inflammatory cytokine responses to HCMV infection, suggesting that inflammatory cytokine stimulation by HCMV is mediated by interactions between these envelope gp and TLR2. Furthermore, both gB and gH coimmunoprecipitate with TLR2 and TLR1, indicating that these envelope gp directly interact with TLR2 and that a TLR2/TLR1 heterodimer is a functional sensor for HCMV. Because our previous studies were conducted in model cell lines, we also show that TLR2 is expressed by HCMV permissive human fibroblast cell strains, and that TLR2 is a functional sensor in these cells. This study further elucidates the importance and potency of envelope gp as a class of molecules displaying pathogen-associated molecular patterns that are recognized with immediate kinetics by TLRs in permissive cells. The Journal of Immunology, 2006, 177: 7094–7102. uman CMV (HCMV)3 is a ubiquitous member of the We recently identified TLR2 as a host factor that activates in- Herpesviridae family that causes significant morbidity flammatory cytokine secretion in response to HCMV (17). The H and mortality in immune compromised patients (1). TLRs are a family of pathogen-recognition receptors that initiate Similar to other herpesviruses, HCMV establishes a lifelong rela- innate immune responses to a myriad of invading microbes, in- tionship with its host as a latent infection, and disease can result cluding viruses (18, 19). Eleven mammalian TLRs have been iden- from either primary infection or reactivation from latency (2, 3). tified, and they are predominantly expressed on phagocytic cells HCMV has an extremely broad tissue tropism that allows it to such as dendritic cells and macrophages; however, most cells ex- infect nearly every organ system in the body (4, 5). Consequently, press at least a subset of TLRs (19). The primary consequences of HCMV disease presents itself in a variety of clinical sequelae (1). TLR activation include NF-␬B activation, inflammatory cytokine by guest on October 1, 2021. Copyright 2006 Pageant Media Ltd. It is a major cause of postoperative disease in chemically immu- secretion, dendritic cell maturation, up-regulation of immune co- nosuppressed transplant recipients and greatly increases the risk of stimulatory molecules, and for a subset of TLRs, the production of graft rejection (6–8). HCMV is also a leading cause of congenital type I IFN (19–22). TLRs detect microorganisms on the basis of birth defects, and infection during the first trimester of pregnancy unique molecular structures termed pathogen-associated molecular often results in neurological and cognitive disorders in the devel- patterns (PAMPs). Analysis of the innate response to bacterial oping child (9, 10). Furthermore, HCMV has been implicated as a PAMPs such as LPS, peptidoglycan, and unmethylated CpG DNA factor in coronary artery disease (11–14). There is currently no are a cornerstone of TLR research, and great strides have been vaccine for HCMV, and existing therapeutics exhibit toxicity pre- made in our understanding of the relationship between bacteria and cluding long-term use (15, 16). Thus, an understanding of the viral the innate immune system (23–28). In contrast, the mechanisms by http://classic.jimmunol.org and cellular determinants of the immune response to HCMV is critical for the development of new vaccines and therapies. which the TLR system recognizes and responds to viruses have only begun to be explored. Viral genomic nucleic acids are one major class of PAMP. TLR3 (dsRNA), TLR7 (ssRNA), TLR8 *McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, (ssRNA), and TLR9 (CpG DNA) (29–33) signal from the endo- Madison, WI 53706; and †Department of Biomolecular Chemistry, University of some (34–38) where degradation of virus particles exposes the Wisconsin, Madison, WI 53706 viral genome for detection by this panel of TLRs (29, 31, 32). Downloaded from Received for publication April 17, 2006. Accepted for publication August 29, 2006. Although significantly less well studied, envelope gps that deco- The costs of publication of this article were defrayed in part by the payment of page rate the exterior of the virion are an emerging class of TLR activators charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (18). To date, three envelope gp have been identified as TLR agonists. 1 This work was supported by National Institutes of Health Grants RO1AI34998 The fusion protein from respiratory syncytial virus and the mouse and R21A154915 (to T.C.) and National Institutes of Health Training Grant mammary tumor virus envelope protein activate TLR4, while the T32GM07215 (to K.W.B. and M.G.). hemagglutinin protein from measles virus activates TLR2 (39–42). 2 Address correspondence and reprint requests to Dr. Teresa Compton, 100 Tech- Interestingly, a shared feature of these gp is that they play critical roles nology Square, Novartis Institute for Biomedical Research, Cambridge, MA 02139. E-mail address: [email protected] in the entry of their respective viruses, and this shared feature suggests 3 Abbreviations used in this paper: HCMV, human CMV; PAMP, pathogen-associ- that the molecular machinery used by viruses for entry is also targeted ated molecular pattern; gB, gp B; gH, gp H; gL, gp L; gO, gp O; NHDF, normal by the innate immune system (43, 44). human dermal fibroblast; HEK, human embryonic kidney; MOI, multiplicity of in- fection; eGFP, enhanced GFP; VSV-G, vesicular stomatitis virus G; HSV-1, herpes Although we demonstrated previously that TLR2 is activated by simplex virus type 1; CHO, Chinese hamster ovary. HCMV, the molecular trigger for TLR2 has not been determined Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 7095 (17). In contrast with the RNA viruses listed above, HCMV dis- centration of 1 mg/ml. The cells were subsequently adapted to suspension plays as many as 12 envelope gp, four of which are required for in chemically defined CHO medium (Invitrogen Life Technologies) sup- entry. gp B (gB) works in concert with a tripartite complex com- plemented with 1% PSF and geneticin at a concentration of 1 mg/ml. For protein isolation, cells were pelleted and lysed by sonication in lysis buffer prised of gp H (gH), gp L (gL), and gp O (gO) to mediate the (50 mM NaPO4, 300 mM NaCl, 0.5% Tween 20, 10 mM imidazole (pH binding and entry of HCMV virons into host cells (45–48). In 8.0)). Cell debris was removed by centrifugation at 27,000 ϫ g for 30 min. addition to their roles in entry, there is a growing body of evidence The supernatants were incubated for2hat4°Cunder rotation with Ni- that gB and gH elicit responses from cells that are reminiscent of NTA-agarose beads (Qiagen). The beads were transferred to a chromatog- raphy column and washed with 10-column volumes of lysis buffer, fol- TLR activation. Abs against gB and gH block the induction of lowed by 10-column volumes of wash buffer (50 mM NaPO4, 300 mM various innate markers, including NF-␬B (49, 50), and cells ex- NaCl, 20 mM imidazole (pH 8.0)). gBs-GFP was eluted in 4 ml of elution ␬ posed to soluble forms of gB activate NF- B and the type I IFN buffer (50 mM NaPO4, 300 mM NaCl, 300 mM imidazole (pH 8.0)) and (49–53). Additionally, an anti-Id bearing the image of gH activates dialyzed overnight in PBS (50 mM NaPO4, 150 mM NaCl (pH 8.0)) at 4°C. NF-␬B (50). Based on these observations, we hypothesized that gB Dialyzed protein was separated from low m.w. contaminants by size-ex- clusion chromatography. Samples were loaded onto a 50-ml column con- and gH are the target of innate sensing by the host cell. In this taining Sephacryl S-200 substrate (Amersham Biosciences) in 1ϫ PBS study, we show that HCMV gB and gH activate TLR2 and asso- (Invitrogen Life Technologies) and run through by gravity flow at 4°C. ciate with TLR1 and TLR2. Abs against gB and gH, but not gL, Collected fractions were stored at Ϫ80°C. inhibit the inflammatory cytokine response to HCMV, and both gB Construction and generation of TLR2⌬C and TLR4⌬C-encoding and gH coimmunoprecipitate with TLR2 and TLR1, indicating that retroviruses the functional sensor for HCMV is a TLR2/TLR1 heterodimer. We also extend our initial studies to HCMV permissive human fibro- The mutants were constructed using full-length FLAG epitope-tagged blast cells and show that TLR2 mediates NF-␬B activation and TLR2 and TLR4 provided by B. Williams (Cleveland Clinic Foundation, Cleveland, OH). The TLR2 and TLR4 cytoplasmic tails were deleted by inflammatory cytokine responses in cells that support productive PCR mutagenesis using a common upstream primer (5Ј-TAA TAT ACC HCMV infection. GGT GCC ACC ATG TCT GCA CTT CTG ATC C-3Ј) incorporating an AgeI restriction site and TLR2-specific (5Ј-TTA AAT GCG GCC GCT Materials and Methods TAT GTA TTT CAT ATA CCA CAG GCC-3Ј) and TLR4-specific (5Ј- TTA AAT GCG GCC GCT TAT GTA GCA GCC AGC AAG AAG C-3Ј) Cell lines, reagents, and virus downstream primers incorporating NotI restriction sites.