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The Journal of Immunology, 2008, 180, 4965-4977
Copyright © 2008 by The American Association of Immunologists, Inc.
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Differential Initiation of Innate Immune Responses Induced by Human Cytomegalovirus Entry into Fibroblast Cells1

Laura K. Juckem*, Karl W. Boehme*, Adam L. Feire*,{dagger} and Teresa Compton2,*

* McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical School, Madison, WI 53706; and {dagger} Novartis Institute for Biomedical Research, Cambridge, MA 02139


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection of permissive fibroblasts with human CMV (HCMV, AD169) is accompanied by a robust activation of innate immune defense. In this study, we show that inflammatory cytokine (IC) secretion and activation of the type I IFN pathway ({alpha}β IFN) are initiated through distinct mechanisms. HCMV is recognized by TLR2 leading to the NF-{kappa}B activation and IC secretion. However, the IFN response to HCMV is not a TLR2-dependent process, as a dominant negative TLR2 does not affect the antiviral response to infection. Additionally, bafilomycin, an endosomal acidification inhibitor, has no effect on HCMV-induced IFN responses suggesting that IFN signaling is independent of endosomal resident TLRs. By contrast, disruption of lipid rafts by depletion of cellular cholesterol inhibits both HCMV entry as well as IFN responses. Cholesterol depletion had no effect on the induction of ICs by HCMV, illustrating a biological distinction at the cellular level with the initiation of innate immune pathways. Furthermore, HCMV entry inhibitors block IFN responses but not IC signaling. In particular, blocking the interaction of HCMV with β1 integrin diminished IFN signaling, suggesting that this virus-cell interaction or subsequent downstream steps in the entry pathway are critical for downstream signal transduction events. These data show that HCMV entry and IFN signaling are coordinated processes that require cholesterol-rich microdomains, whereas IC signaling is activated through outright sensing via TLR2. These findings further highlight the complexity and sophistication of innate immune responses at the earliest points in HCMV infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human CMV (HCMV),3 a member of the Herpesviridae family, is a ubiquitous opportunistic pathogen. The outcome of HCMV infection typically correlates with the immune status of the host. Infection of healthy individuals is often asymptomatic; however, infection of immunocompromised hosts, including organ and stem cell transplant recipients and AIDS patients, can be devastating (1, 2). Neonates are also susceptible to HCMV disease, especially when primary infection of the mother occurs during pregnancy; these infections can result in deafness, mental retardation and mortality (3, 4). In addition, persistent HCMV infection has been associated with chronic diseases such as coronary artery disease and diabetes (5, 6).

HCMV infection results in the secretion of inflammatory cytokines (ICs) and type I IFNs from host cells (7, 8, 9). Both classes of molecules are hallmarks of innate immunity that contribute significantly to control infections (10, 11). ICs, such as TNF-{alpha}, IL-1, IL-6, IL-8, IL-12, and IL-18, have a wide range of biological effects on tissues and cells and are critical for the recruitment and activation of phagocytic leukocytes (12). Type I IFNs consist of IFN-β and multiple forms of IFN-{alpha} and are produced in response to many virus and intracellular bacterial infections (13). IFNs elicit the expression of IFN-stimulated genes (ISGs), a subset of cellular factors that inhibit viral replication (7, 8, 11, 14). Together these responses serve to limit viral replication and spread at early times following infection and also activate and promote adaptive immune responses that will ultimately contain or clear the infection. HCMV does not require virus replication or cellular protein synthesis for the robust initial induction of innate immune responses, suggesting that structural components of the virus are responsible for the up-regulation of these genes during virus entry into cells (7, 9).

The IC branch of innate immunity is defined by activation of NF-{kappa}B, which is responsible for the transcription of genes encoding many proinflammatory cytokines and chemokines (15). In response to stimuli such as cytokines or viruses, activation of the canonical NF-{kappa}B pathway occurs via signal transduction cascades that promote the phosphorylation and degradation of I{kappa}Bs by ubiquitination, thereby releasing the NF-{kappa}B heterodimer. Once released, the activated heterodimer comprised of p50 and p65/RelA is able to translocate to the nucleus and drive expression of target genes (16). Fibroblasts and monocytes infected with HCMV exhibit activated NF-{kappa}B as evidenced by its nuclear translocation and increased DNA binding activity (17, 18).

One means by which viruses elicit ICs and IFNs is through TLRs that detect and initiate innate immune response to microbial pathogens. TLRs are a class of pattern recognition receptors (19). To date, 12 members of the TLR family have been identified in humans (20). TLRs are expressed at high levels on phagocytic cells such as dendritic cells and macrophages. However, all cell types appear to express at least a subset of these receptors (21, 22). TLRs recognize microbial pathogens on the basis of structural motifs, termed pathogen-associated molecular patterns (PAMPs), which differ from those found in host cells (23). Examples of PAMPs include dsRNA (TLR3), LPS (TLR4), ssRNA (TLR7/8), and unmethylated CpG DNA (TLR9). A growing body of evidence indicates that viral envelope glycoproteins can trigger TLR-mediated innate immune signaling. For example, the fusion protein from respiratory syncytial virus and the envelope protein of mouse mammary tumor virus are sensed by TLR4 (24, 25). Similarly, HCMV envelope glycoprotein B (gB) and glycoprotein H (gH) elicit IC secretion through TLR2 (26, 27).

Induction of type I IFN responses to virus infection can be divided into two phases, the activation phase and the amplification phase. Virus infection activates an initial activation phase through a key regulatory transcription factor, IFN regulatory factor 3 (IRF3). Incompletely characterized signal transduction pathways lead to virus-induced phosphorylation of IRF3 on C-terminal serine residues by related kinases TANK-binding kinase 1 and I{kappa}B kinase {epsilon} (28, 29, 30). This results in the homodimerization and translocation of IRF3 to the nucleus where it can interact with the coactivators, CREB binding protein and p300, and form a complex that drives the transcription of IFN-β and a subset of ISGs (31). The nascent IFN is then able to act in an autocrine and paracrine manner to initiate signaling through the cellular {alpha}β IFN receptor leading to activation of a JAK and STAT signal transduction cascade. This amplification phase induces the robust expression of a broad panel of ISGs, which further assists the cell in establishing an antiviral state (11).

The mechanisms by which HCMV activates IFN responses remain largely undefined. HCMV and a soluble version of gB are able to activate IRF3 and the transcription of ISGs (32, 33, 34). Recent evidence using several strategies to deplete cellular IRF3 confirms its requirement and proposes that IRF3 is the primary transcription factor mediating HCMV-induced IFN signaling (35). The ability of a soluble version of gB to activate IFN signaling suggests that gB binding to a cell surface receptor during virus entry is sufficient for antiviral responses. Interestingly, a small molecule HCMV entry inhibitor that targets gB, as well as neutralizing Abs to both gB and gH, inhibit ISG accumulation (36). These studies suggest that glycoprotein-cell interactions during the entry process are important for HCMV-induced IFN activation.

HCMV has evolved many mechanisms to attenuate the host immune response to create an environment that is conducive to virus replication and persistence. The means by which HCMV modulates host immune responses is largely attributed to newly synthesized viral proteins. The immediate early gene product 1 (IE72) has been shown to prevent the association of STAT1, STAT2 and IRF9 with the promoters of IFN-responsive genes (37). In addition, the immediate early gene product 2 (IE86) has been shown to interfere with NF-{kappa}B binding to the IFN-β promoter (38). HCMV also evades host immune responses through the molecular mimicry of many immune molecules including, but not limited to, the expression of viral chemokines, a MHC class I homolog, chemokine binding proteins, and virus encoded G protein-coupled receptors (39, 40, 41, 42, 43). Due to the ability of live HCMV to modulate the host innate immune responses, UV-inactivated virus was used in the current study to focus on the initiation of HCMV-induced innate immune responses, whereas live virus was used to study virus entry through tegument delivery and the initiation of viral gene expression.

Understanding the coordination of innate immune activation and virus entry is a daunting task due in part to the complexity associated with HCMV entry. Multiple envelope glycoprotein complexes decorate HCMV virions and interact with several cellular receptors to mediate entry into cells. HCMV glycoprotein M and gB interact with cell surface heparan sulfate proteoglycans to tether the virion to the cell surface (44). This allows for more stable docking with additional cellular receptors including β1 and β3 integrins that interact with gB and gH, respectively (45, 46). Epidermal growth factor receptor has also been proposed as an HCMV entry receptor, although its role remains controversial (47, 48, 49). HCMV entry may occur in a pH neutral or pH-dependent manner depending on the cell type (50, 51). The envelope glycoproteins gB, gH, and glycoprotein L are essential for entry and critical in mediating fusion (52, 53). It is during this entry process that the cell gets its first glimpse at the invading pathogen and the first opportunity to contain it.

We report that HCMV activates IC and IFN responses through discrete mechanisms during virus entry. HCMV activates IC responses via TLR2 apparently independent of events needed for productive infection. By contrast, HCMV entry and IFN signaling are intimately linked processes critically dependent on the organization of cholesterol-rich microdomains. These findings enhance our understanding of the complex interplay between virus entry and host detection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines, reagents, and viruses

Normal human dermal fibroblasts (NHDFs; Clonetics) were grown at 37°C in 5% CO2 in DMEM (Invitrogen Life Technologies) supplemented with 10% FBS (HyClone Laboratories), 1% glutamine, and 1% penicillin-streptomycin-amphotericin B (Fungizone; BioWhittaker). The NHDF cells were serum starved for 24 h before all treatments. The AD169 strain of HCMV was propagated in NHDF cells and titered by immediate early gene product 1 (IE1, IE72) and gene product 2 (IE2, IE86) expression by indirect immunofluorescence (54). HCMV AD169 IE2-GFP virus was provided by D. H. Spector (University of California, San Diego, CA) (55). Where noted, purified AD169 virions isolated by density gradient centrifugation were used (50). UV inactivation of HCMV was performed as previously described (26). Replication competent recombinant vesicular stomatitis virus (VSV) expressing the enhanced gene encoding GFP was provided by J. Yin (University of Wisconsin, Madison, WI) and propagated in baby hamster kidney BHK cells.

Reagents and Abs

NHDFs were transduced with retroviral vectors containing a TLR2 dominant negative construct with Toll/IL-1R domain cytoplasmic deletions as previously described (27). IFN treatments were performed using a combination of {alpha} and β recombinant human IFNs (BioSource International). Recombinant human IL-1β was obtained from R&D Systems. Pam3CSK4 was obtained from EMC Microcollections and polyinosinic-polycytidylic acid potassium salt (poly(I:C)) was obtained from Sigma-Aldrich. A mAb to pp65 was obtained from the Rumbaugh-Goodwin Institute for Cancer Research (Plantation, FL). A mAb to IRF3 was obtained from Active Motif. Rabbit polyclonal Abs to I{kappa}B{alpha} and p65 (RelA) were obtained from Santa Cruz Biotechnology. The anti-P56 Ab was a gift from G. Sen (The Cleveland Clinic Foundation, Cleveland, OH) (56). The second generation β-peptide 19, a potent inhibitor of HCMV entry (57), is designated β-peptide (+) in this study, and a control β-peptide that does not block HCMV entry is designated β-peptide (–).

VSV plaque reduction assay

Subconfluent NHDFs were washed with PBS, mock infected, treated with {alpha}β IFNs (100 IU/ml), or infected with HCMV as indicated. All treatments were performed in serum-free medium. Following a 6-h incubation, the cells were washed once with PBS and infected with 100 PFU/ml VSV (New Jersey strain). VSV was absorbed for 1 h at 37°C, the innoculum was removed, and the cells overlaid with 2 ml of a 60/40 mixture of 2x Eagle’s MEM (BioWhittaker) and 1% agarose. The cells were incubated at 37°C, and plaques were visualized by crystal violet staining (0.5x PBS, 0.07% crystal violet, 5.5% formaldehyde) at 48 h of postinfection.

Inhibition of endosome acidification

NHDF cells were pretreated for 30 min with bafilomycin (1 µM). HCMV AD169 IE2-GFP at a multiplicity of infection (MOI) of 1 was directly added to the bafilomycin-containing medium and adsorbed for 2 h. Nonpenetrated virus was inactivated with a low pH citrate buffer wash (40 mM citric acid, 10 mM KCl, 135 mM NaCl (pH 3.0)) (58). The medium was replaced, and cells were incubated in the presence of bafilomycin at 37°C.

Cholesterol depletion and replenishment treatments

NHDFs were incubated with 8 mM methyl-β-cyclodextrin (MβCD; Sigma-Aldrich), as determined by a dose-response curve, and diluted in serum-free medium for 2 h at 37°C. After two washes with serum-free medium, the cellular cholesterol was replenished by incubating with water-soluble cholesterol (2.5–20 µg/ml; Sigma-Aldrich) or serum-free medium alone for an additional 2 h. Cells were washed an additional two times with serum-free medium and infected with HCMV (MOI = 1) for 2 h. Cells were washed with citrate buffer and incubated in serum-free medium.

Flow cytometry

At 18–24 h postinfection, cells were released from the tissue-culture plate with trypsin, resuspended in complete medium, pelleted, and suspended in PBS. Propidium iodide (Molecular Probes) was added and samples were analyzed using a FACScan flow cytometer (BD Biosciences) using a standard filter set. Samples were gated for propidium iodide exclusion (live cells) and assayed for GFP expression.

Virus attachment assay

NHDF cells were depleted of membrane cholesterol as described and cooled at 4°C for 30 min. HCMV (MOI = 2) was incubated at 4°C for 2 h. Cells were washed three times in cold serum-free medium and harvested in lysis buffer. DNA was extracted using QIAamp Mini Elute Virus Spin kit (Qiagen) according to the manufacturer’s instructions. DNA was eluted in 50 µl of nuclease free water (Ambion). Viral genomes were quantitated using the primer pair, pp549s and pp821as, and UL83 FAM-TAMRA probe as previously described (59). Unknown samples were determined based on a standard curve of known UL83 copy numbers using pCGN-HA-pp65, which was a gift from R. Kalejta (University of Wisconsin, Madison, WI) (60). PCR contained 2.5 µl of 50 µl extracted DNA, 50 nM primers and probe, 12.5 µl of TaqMan Universal PCR Master mix (Applied Biosystems), and nuclease free water to a total volume of 25 µl. Real-time PCR was run on an ABI 7900HT, and data were analyzed using the SDS 2.2.1 program.

Immunofluorescence

NHDF cells were seeded on glass coverslips and treated as described. For the detection of IRF3 and p65 (RelA), infections were performed in the presence of cycloheximide (100 µg/ml; Sigma-Aldrich). Cells were fixed in 3% paraformaldehyde for 20 min, permeabilized in 0.1% Triton X-100 for 10 min, and blocked in 20% purified goat serum (Pierce). Cells were washed, incubated with anti-IRF3 Ab, anti-pp65 Ab, or anti-p65 (RelA) Ab, followed by AlexaFluor 594 goat anti-mouse secondary Ab (IRF3 and p65) or AlexaFlour 488 goat anti-rabbit secondary Ab (p65/RelA; Molecular Probes). Nuclei were counterstained with DAPI (4',6'-diamidino-2-phenylindole), washed, and viewed on a Nikon TE-2000S inverted fluorescence microscope.

Detection of RNA accumulation of ISGs, ICs, and TLRs by RT-PCR

Total cellular RNA was harvested at 6 h postinfection using RNA-STAT 60 ("B"; Tel-Test), according to the manufacturer’s instructions. Briefly, cells were lysed by addition of guanidinium thiocyanate-phenol and chloroform extracted, and RNA was isopropanol precipitated. RT-PCR was performed on 200 ng of recovered total cellular RNA using Recombinant Thermus thermophilus rTth DNA Polymerase (Applied Biosystems). The primer pairs used were as follows: IFN-β sense 5'-CACTACAGCTCTTTCCATGA, antisense 5'-AGGATTTCCACTCTGACTCTGACTATGGTCC; ISG56 sense 5'-CAT CAG GTC AAG GAT AGT CTG GAG C, antisense 5'-GGA TTC AGG GTT TTC AGG GTC C; IL-6 sense 5'-TGTGTGAAAGCAGCAAAGAGGC, antisense 5'-TTGGGTCAGGGGTGGTTATTG; and GAPDH sense 5'-GAGCCAAAAGGGTCATC antisense 5'-GTGGTCATGAGTCCTTC. Endosomal-localized TLR transcripts were detected using the Multigene-12 RT-PCR profiling kit (Superarray), according to the manufacturer’s instructions.

Western blot analysis

NHDF cells were harvested at 3 or 7 h postinfection for immunoblot analysis of I{kappa}B{alpha} or P56 detection, respectively. Experiments to detect I{kappa}B{alpha} were conducted in the presence of cycloheximide (100 µg/ml). At the time of harvest cells, were washed in PBS and lysed in harvest buffer (50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 50 mM NaF, 5 mM EDTA, 0.1% Nonidet P-40). Total cellular protein was quantified using a Bradford assay, and 10 µg of protein was resolved by 10% or 9% reducing SDS-PAGE for I{kappa}B{alpha} or P56 detection, respectively. Proteins were transferred to a nitrocellulose membrane, and blotted with an anti-I{kappa}B{alpha} Ab or an anti-P56 Ab followed by goat anti-rabbit HRP secondary Ab (Pierce). Proteins bound with Ab were visualized using ECL (PerkinElmer). Densitometry was performed using ImageQuant software system (Amersham Biosciences).

Cytokine ELISA

Subconfluent NHDFs were infected with HCMV (MOI = 0.1). At 18 h postinfection, medium samples were collected, and levels of IL-6 were determined by ELISA (OptEIA Set Human IL-6; BD Biosciences) according to the manufacturer’s instructions.

HCMV fusion inhibitor treatments

Soluble proteins, gB-DLD and gB-651, were incubated with NHDFs at a concentration of 250 µg/ml for 1 h at 37°C. Cells were washed three times with buffer, twice with serum-free medium, and subsequently infected with HCMV-IE2-GFP (MOI = 1). β-peptides (50 µM) were incubated with HCMV (MOI = 1) in serum-free medium for 5 min before the addition of the entire complex to cells. Infection was allowed to proceed for 1 h at 37°C, after which cells were citrate washed and incubated at 37°C until harvest.

Expression and purification of gB-DLD and gB-651 soluble proteins

A fragment corresponding to aa 57–146 of gB (gB-DLD) was amplified from pCAGGS-gB (61) by PCR with the following primer: gB-DLD sense 5'-GGA ATT CCA TAT GGT AAC GTC TTC TGA AGC C-3', antisense 5'-CGG GAT CCT TAA ACC TTT TGG TAG ACC CG-3'. The amplified fragment was cloned into the NdeI and BamHI sites of the bacterial expression vector pET-28a (Novagen) with an N-terminal His-6 tag fragment corresponding to aa 651–718 of gB (gB-651) was also amplified from pCAGGS-gB by PCR with the following primers: OML30 5'-CGG GAT CCA TGG ATA TCG ACC CGC TGG AA-3' and OML31 5'-CG AGA TCT TCG AAT TAC TAC TAC TAC TAC TAC TGA AGG AGC ACC TTG TTC GTC CGG CGA GTA CTC CAG CAG-3'. The amplified fragment was cloned into the NcoI and HindIII sites of the bacterial expression vector pTriEx-1.1 (Novagen) with a C-terminal His-6 tag. Both vectors were transformed into Escherichia coli DH5{alpha}. To produce recombinant protein, gB-DLD and gB-651 plasmids were isolated by FastPlasmid Mini per the manufacturer’s instructions (Eppendorf) and transformed into E. coli BL21 (DE3) strain for protein expression. E. coli containing the pET-28a:gB-DLD was grown at 37°C in Luria-Bertani medium containing kanamycin (50 µg/ml) and pTriEx-1.1:gB-651 in Luria-Bertani medium containing ampicillin (50 µg/ml) to an A600 of 0.6. To induce recombinant protein, isopropyl β-D-thiogalacto pyranoside (Roche) was added to a final concentration of 1 mM and incubated for 4 h at 37°C. The cells were chilled on ice and harvested by centrifugation at 4000 rad/min for 10 min at 4°C. Protein was isolated from inclusion bodies as previously described (62). Protein was then solubilized in 8 M urea/300 mM NaCl/10 mM imidazole/50 mM Tris (pH 7.9). Affinity purification of the proteins was accomplished through Ni-NTA agarose columns (Qiagen) per the manufacturer’s instructions. Eluate from the Ni-NTA column was placed onto an S-200 sizing column, and 1-ml fractions collected. Fractions containing protein were determined by measuring the absorbance of each fraction at 214 nm using a SpectraMax 190 spectrophotometer (Molecular Devices). Fractions that corresponded to absorption peaks were analyzed by SDS-PAGE to determine the size of the protein. The absorption peak fractions that contained a protein of the same size as the gB-DLD or gB-651 were pooled and concentrated using a Ni-NTA column we described. The concentrated fractions of gB-DLD were dialyzed extensively against 55 mM MES (pH 5.5) and 300 mM NaCl, whereas gB-651 fractions were dialyzed against 55 mM Tris (pH 8.3) and 300 mM NaCl.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HCMV induces IFN responses in a TLR2-independent manner

HCMV activates TLR2 through a physical interaction with envelope glycoproteins gB and gH, allowing the cell to mount an IC response (26, 27). TLR2 has also been shown to mediate IC signaling in response to HSV type 1 (HSV-1) and varicella-zoster virus (63, 64). To date, no link has been established between TLR2 and the induction of IFN responses (65). However, this hypothesis has not been tested using viral ligands for TLR2. To determine whether TLR2 can activate the IFN pathway following HCMV infection, we used a signaling defective, dominant negative TLR2 construct (TLR2{Delta}C) that lacks the cytoplasmic Toll/IL-1R domain common to all TLRs. NHDFs were transduced with a bicistronic retrovirus vector encoding TLR2{Delta}C and GFP. GFP-positive cells were collected by FACS. To confirm that TLR2-dependent processes were inhibited by TLR2{Delta}C, IL-8 secretion was measured by ELISA (Fig. 1A). The IL-8 response to the TLR-independent cytokine IL-1β was unaffected by the expression of TLR2{Delta}C. However, the cytokine responses to the TLR2-specific ligand Pam3CSK4 and UV-inactivated HCMV (UV-HCMV) were markedly reduced in the TLR2{Delta}C-expressing cells (Fig. 1A). The incomplete block of IL-8 secretion in response to UV-HCMV suggests that TLR2 is not the only mechanism by which UV-HCMV can activate NF-{kappa}B. These data confirm that TLR2{Delta}C specifically inhibits TLR2-dependent IC responses.


Figure 1
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  		FIGURE 1. HCMV-induced antiviral state is not reliant on TLR2. A, NHDF cells were mock transduced or transduced with a TLR2 dominant negative construct (TLR2{Delta}C). Cells were treated with IL-1β (25 pg/ml), Pam3CSK4 (100 ng/ml), or UV-HCMV (MOI = 0.1). At 18 h posttreatment, the supernatants were harvested and IL-8 levels were determined by ELISA. Error bars indicate SD for triplicate samples. B, Control (NHDF) and TLR2{Delta}C-expressing cells were infected at the indicated MOIs with UV-HCMV, or treated with recombinant type I IFN (100 U/ml). At 6 h poststimulation, the treatments were removed and the monolayers were challenged with VSV (~100 PFU/well). Plaque formation was visualized by crystal violet staining at 48 h postinfection.

 
To test the hypothesis that TLR2 mediates type I IFN responses to HCMV, we determined the capacity of UV-HCMV to elicit a functional antiviral response from the TLR2{Delta}C-expressing NHDF cells. Plaque formation by vesicular stomatitis virus (VSV), a virus sensitive to the effects of IFNs, was assessed following treatment with type I IFNs or infection with UV-HCMV (34). As shown in Fig. 1B, both control and TLR2{Delta}C-expressing NHDFs were protected from VSV infection by recombinant type I IFNs or HCMV virions. These data indicate that HCMV can induce a functional antiviral state in the absence of a signaling-competent TLR2 molecule. Furthermore, these data suggest that HCMV initiates IFN and IC responses through distinct cellular mechanisms.

Endosomal acidification is not required for HCMV-induced IFN responses

The observation that TLR2 function does not mediate HCMV-induced IFN signaling does not rule out other members of the TLR family from serving in this capacity. TLR3, TLR7, TLR8, and TLR9 are a subset of TLRs known to activate IFN responses and are localized to endosomal compartments (66, 67, 68). This subset of intracellular localized TLRs requires a low pH trigger to initiate signaling and allow for the rapid recognition of viral ligands during uncoating or degradation processes (69, 70, 71). Interestingly, several members of the Herpesviridae family are reported to induce IFN responses through TLR9, which senses unmethylated CpG 2'-deoxyribo dinucleotides in DNA (72). The CpG rich genome of HSV-1 and HSV type 2 and mouse CMV are recognized by TLR9 in plasmacytoid dendritic cells or dendritic cells in vitro (73, 74, 75). NHDF cells express high levels of TLR3 and TLR8, whereas there is a very low level of TLR7 and no detectable expression of TLR9 (Fig. 2C).


Figure 2
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  		FIGURE 2. Endosomal maturation is not required for HCMV-induced IFN signaling. NHDF cells were incubated with bafilomycin (1 µM) for 30 min before infection with UV-HCMV (MOI = 1) or live VSV (MOI = 150). A, Cells were fixed at 6 h postinfection and assayed for IRF3 localization by immunofluorescence assay. Cell nuclei were stained with DAPI. B, RNA was isolated at 6 h postinfection, and IFN-β, ISG56, IL-6, and GAPDH transcript levels were determined by RT-PCR. PCR products were analyzed on a 1% agarose gel and visualized by ethiduim bromide staining. C, Endosomal TLR transcript levels were determined by RT-PCR from RNA isolated from NHDF cells. PCR products were analyzed on a 1% agarose gel and visualized by ethiduim bromide staining.

 
To determine whether this subset of TLRs activate the IFN response to HCMV, we examined the effect of bafilomycin, a macrolide antibiotic that specifically inhibits the vacuolar type H+-ATPase and prevents the acidification and maturation of endosomal compartments (76). In the presence of bafilomycin, the ability of UV-HCMV to activate IRF3, as evidenced by its translocation to the nucleus, was unaffected as compared with the response in the untreated control (Fig. 2A, panel d). In addition, the accumulation of representative ISG transcripts, IFN-β and ISG56, as well as of a representative IC, IL-6, was unaffected in bafilomycin-treated cells in response to UV-HCMV (Fig. 2B). To verify that bafilomycin blocked endosome maturation, cells were infected with VSV, which activates IFN signaling through its replication intermediates (77). As predicted, the activation of IRF3 by VSV was blocked by bafilomycin treatment due to the low pH requirement of VSV for entry (Fig. 2A, panel f) (78). Bafilomycin treatment also blocked induction of ISG and IC induction by VSV (Fig. 2B). Consistent with previously published data, VSV but not HCMV entry into fibroblasts was blocked in the presence of bafilomycin relative to untreated controls (data not shown) (50, 79). These experiments suggest that IFN and IC responses induced by UV-HCMV do not require endosome acidification and maturation, therefore they are not initiated by endosomal localized TLRs in NHDF cells.

HCMV entry is dependent on cholesterol-rich microdomains

Lipid rafts are cholesterol rich, detergent insoluble microdomains within the plasma membrane hypothesized to serve as platforms for molecules involved in cell signaling, cell adhesion, and membrane trafficking (80, 81). It is becoming increasingly evident that many viruses, including HCMV, use lipid rafts as portals for virus entry (46, 82, 83, 84, 85, 86). To determine whether lipid rafts are required for HCMV entry, NHDFs were treated with the cholesterol-depleting reagent, MβCD, to disrupt lipid rafts before infection with a recombinant HCMV with GFP fused to the viral immediate early protein 2 (HCMV IE2-GFP) (87, 88). Live HCMV was used to measure virus entry, and the GFP-positive cells were scored at 24 h postinfection as a surrogate marker of infection (Fig. 3A). The average total percentage of cells infected for the three independent experiments conducted was 67% at MOI = 1. In cells pretreated with MβCD, viral gene expression was reduced by approximately 90% compared with expression in untreated control; the effects of MβCD could be reversed in a dose-dependent manner with the addition of water-soluble cholesterol (Fig. 3A).


Figure 3
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  		FIGURE 3. HCMV entry is dependent on cholesterol-rich microdomains. NHDF cells were pretreated with 8 mM MβCD to deplete membrane cholesterol, and indicated wells were subsequently incubated with water-soluble cholesterol. Cells were washed and infected with live HCMV (MOI = 1). A, At 18 h postinfection, GFP expression was assessed by flow cytometry as a surrogate marker of infection. No cellular toxicity was observed by propidium iodide staining (data not shown). Data represent the average of three independent experiments. B, Delivery of the HCMV tegument protein pp65 was measured by indirect immunofluorescence at 1 h postinfection. At least six random fields were counted per treatment. Graph is representative of two independent experiments. C, Bound HCMV genomes was measured by quantitative PCR in mock NHDF cells, infected with live HCMV (MOI = 2), pretreated with 8 mM MβCD and infected with live HCMV or with live HCMV treated with soluble heparin (30 µg/ml). Error bars represent SDs.

 
Due to the important role that lipid rafts play in cell signaling, it is possible that the depletion of lipid rafts disrupts signal transduction cascades required for the initiation of viral gene expression. Therefore we assessed virus payload delivery by measuring the nuclear localization of the tegument protein pp65, which traffics to the nucleus almost immediately upon infection with HCMV. Similar to the results obtained with the IE2-GFP reporter virus, MβCD pretreatment resulted in a 70% decrease in the number of pp65-positive nuclei compared with untreated cells (Fig. 3B). This result suggests that the block to infection is at or before fusion. Notably, pretreatment with MβCD did not alter the binding of HCMV to cells (Fig. 3C). Together these data suggest that lipid rafts are essential for HCMV entry.

HCMV-induced IFN responses are dependent on cholesterol-rich microdomains

We next assessed whether disruption of lipid rafts by cholesterol depletion interferes with the induction of the IFN response to HCMV infection. Nuclear translocation of IRF3 induced by UV-HCMV infection was inhibited by pretreatment of cells with MβCD as compared with the infection of untreated cells (Fig. 4A, panels b and d). Activation of IRF3 was restored by the addition of water-soluble cholesterol (Fig. 4A, panel e). Neither MβCD nor cholesterol treatment alone activated IRF3 (Fig. 4A, panels c and f). Consistent with the lack of IRF3 nuclear translocation, the accumulation of representative ISG transcripts, IFN-β and ISG56, in response to UV-HCMV were inhibited by pretreatment with MβCD (Fig. 4B, lane 4). ISG transcript levels were restored when membrane cholesterol was replenished. MβCD and cholesterol treatments alone did not result in the accumulation of ISG mRNA. Consistent with our transcriptional studies, MβCD pretreatment diminished induction of the ISG56 protein (P56) by UV-HCMV. Densitometric analysis revealed a 5- to 6-fold decrease in P56 levels with MβCD pretreatment compared with P56 levels in untreated controls; P56 protein levels were restored by the addition of water-soluble cholesterol (Fig. 4C). Again, treatment with MβCD or water-soluble cholesterol alone did not induce P56 accumulation.


Figure 4
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  		FIGURE 4. Depletion of cellular cholesterol blocks HCMV-induced IFN responses. NHDF cells were pretreated with 8 mM MβCD to deplete membrane cholesterol. Water-soluble cholesterol was incubated with cells at indicated concentrations. Cells were washed with PBS and mock infected or infected with UV-HCMV (MOI = 1). A, Cells were fixed at 6 h postinfection, and IRF3 localization was determined by indirect immunofluorescence. In the merge panels, nuclei were stained with DAPI. B, RNA was isolated at 6 h postinfection, and IFN-β, ISG56, and GAPDH transcript levels were determined by RT-PCR. PCR products were analyzed on a 1% agarose gel and visualized by ethiduim bromide staining. C, Whole cell lysates were prepared at 7 h postinfection, and P56 protein and actin levels were determined by Western blot analysis. Cells were treated with recombinant type I IFNs (1000 U/ml) as a control. The levels of P56 protein were standardized to actin levels, and values indicated (*) represent the percentage of HCMV-infected cells under each condition. D, Cells were pretreated with MβCD and incubated with VSV (MOI = 150), Pam3CSK4 (40 µg/ml), or poly(I:C) (100 µg/ml). RNA was isolated at 6 h, and IFN-β, ISG56, IL-6, and GAPDH transcript levels were determined by RT-PCR. The resulting PCR products were analyzed on a 1% agarose gel and visualized by ethidium bromide staining.

 
To test the specificity of MβCD pretreatment on the HCMV-mediated IFN response, we also measured the accumulation of P56 following treatment with recombinant type I IFNs. No difference was observed upon IFN treatment (Fig. 4C). To further verify the specificity of MβCD pretreatment, RT-PCR analysis of IFN-β and IL-6 induction was examined in response to additional innate immune ligands. VSV, which enters cells through the classical clathrin-coated pit pathway in a lipid raft-independent manner was unaffected by MβCD pretreatment (Fig. 4D) (78). Similarly, treatment with poly(I:C), a TLR3 specific ligand, induced IFN-β in the presence and absence of MβCD pretreatment (Fig. 4D). Consistent with a previous report (65), the TLR2-specific ligand PamCSK did not cause the accumulation of IFN-β transcripts (Fig. 4D). Together these data suggest that virus entry and activation of the host IFN responses in fibroblasts are dependent on cholesterol-rich microdomains and are specific to HCMV.

HCMV activates IC secretion independent of the organization of cholesterol-rich microdomains

We next assessed the role of cholesterol-rich microdomains in the activation of NF-{kappa}B and the induction of ICs by UV-HCMV. NF-{kappa}B activation was determined by monitoring the degradation of I{kappa}B{alpha}, a protein that binds and retains the NF-{kappa}B dimer in an inactive state in the cytoplasm of cells (89). In response to stimuli such as cytokines or viruses, signal transduction cascades promote the phosphorylation and degradation of I{kappa}B{alpha} through ubiquitination, thereby releasing NF-{kappa}B to translocate to the nucleus where it drives target gene expression (16). We measured the degradation of the NF-{kappa}B inhibitory subunit, I{kappa}B{alpha}, as an indirect measure of NF-{kappa}B activation. In cells pretreated with MβCD and infected with gradient purified UV-HCMV, I{kappa}B{alpha} was degraded to a similar extent as UV-HCMV alone at both 1 and 3 h postinfection (7 and 12% of mock cells, respectively) (Fig. 5A and data not shown). I{kappa}B{alpha} degradation following UV-HCMV infection was slightly affected by treatment of cells with MβCD or 20 µg/ml water-soluble cholesterol (90% and 85% of mock cells, respectively) (Fig. 5A). Following I{kappa}B{alpha} degradation, NF-{kappa}B heterodimers composed of p50 and p65/RelA rapidly translocate from the cytosol to the nucleus (16). Using indirect immunofluorescence with Abs to RelA as well as the HCMV tegument protein pp65, we can simultaneously visualize HCMV-induced NF-{kappa}B activation and virus entry (Fig. 5B). At 2 h postinfection both RelA and pp65 can be visualized in the nucleus of cells infected with live HCMV. MβCD pretreatment blocks pp65 uptake but not RelA nuclear localization (Fig. 5B, panel b). Finally, IL-6 induction by UV-HCMV infection was unaffected following cholesterol depletion or replenishment as measured by ELISA (Fig. 5C). Treatment with MβCD or cholesterol alone did not affect IL-6 secretion. These results suggest that, unlike the IFN response, activation of NF-{kappa}B and subsequent production of ICs is independent of organized cholesterol-rich microdomains. These results also indicate that HCMV-induced IC activation is not dependent on virus entry into cells, as cholesterol depletion blocks virus entry (Fig. 3).


Figure 5
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  		FIGURE 5. HCMV-induced IC signaling is unaffected by cholesterol depletion. NHDF cells were pretreated with 8 mM MβCD to deplete membrane cholesterol, and indicated wells were subsequently incubated with water-soluble cholesterol. Cells were washed with PBS and either mock-infected, infected with UV-HCMV (MOI = 1), or treated with IL-1β (100 pg/ml) or Pam3CSK4 (20 µg/ml). A, Gradient purified UV-HCMV was used, and whole cell lysates were prepared at 1 h postinfection. I{kappa}B{alpha} and actin protein levels were assessed by Western blot analysis. I{kappa}B{alpha} protein levels were measured by densitometry and standardized to actin levels. Values indicated (*) represent the percentage of mock-infected I{kappa}B{alpha} levels. B, Cells were fixed at 2 h postinfection and the nuclear localization of the NF-{kappa}B subunit, p65/RelA, and the tegument protein, pp65, was determined by indirect immunofluorescence. Nuclei were stained with DAPI. C, NHDF cells were pretreated as described for this experiment and stimulated with UV-HCMV (MOI = 0.1) or treated with IL-1β (100 pg/ml) or Pam3CSK4 (20 µg/ml). At 18 h postinfection, supernatant IL-6 levels were determined by ELISA.

 
HCMV entry inhibitors block HCMV-induced IFN signaling but not IC signaling

The observation that cholesterol depletion inhibits HCMV entry, as well as the induction of the IFN response (Figs. 3 and 4), suggests that 1) lipid rafts serve as a platform on which cellular machinery act to induce the IFN response, or 2) the process of HCMV entry is an important factor for activation of IFN signaling. To further probe the role of HCMV entry in HCMV-induced innate immune responses, we used two inhibitors of HCMV entry. The protein, gB-DLD, contains a highly conserved region of gB encompassing an integrin binding motif, the disintegrin-like domain. Protein gB-DLD acts as a ligand mimic and interacts with β1 integrin to potently block HCMV entry at a postattachment step (45 and A. L. Feire and T. Compton, manuscript in preparation). As a control, we used a protein termed gB-651 derived from a region of gB that contains no recognizable receptor binding motifs. Next, we used β-amino acid oligomers (β-peptide) designed to mimic the heptad repeat region of gB that block HCMV entry at a step before fusion. A β-peptide that does not block HCMV infection was used as a control. The inhibitory β-peptide displays a high degree of specificity for HCMV, as it does not block entry of mouse CMV or HSV-1 (57). Pretreatment of cells with gB-DLD resulted in a 92% decrease in virus infection as assessed by the HCMV-IE2 GFP reporter virus (Fig. 6A). The gB-651 protein did not block HCMV IE2-GFP infection (107% of control infection) (Fig. 6A). The β-peptide inhibitor, β-peptide (+), was preincubated with live HCMV before infection of NHDFs and caused a 89% decrease in HCMV IE2-GFP infection; whereas the negative control peptide, β-peptide (–), had little effect, resulting in a 12% reduction in virus infection (Fig. 6A).


Figure 6
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  		FIGURE 6. HCMV fusion inhibitors block HCMV-induced IFN signaling but not IC secretion. NHDF cells were pretreated with gB-DLD (250 µg/ml) or gB-651 (250 µg/ml) for 1 h at 37°C. Cells were washed and either mock-infected or infected with live HCMV (A) or UV-HCMV (B and C) (MOI = 1). HCMV virions (MOI = 1) were combined with 50 µM inhibitory β-peptide (+) or the control β-peptide (–) for 5 min and added to NHDF cells. A, At 18 h postinfection, the percentage of GFP-positive cells was determined by flow cytometry. No cellular toxicity was observed by propidium iodide staining (data not shown). These results are representative of three independent experiments. B, Whole cell lysates were prepared at 7 h postinfection, and P56 and actin levels were determined by Western blot analysis. P56 levels were normalized to actin. Values indicated (*) represent the percentage of infected cells. C, Whole cell lysates were prepared at 3 h postinfection, and I{kappa}B{alpha} and actin protein levels were assessed by Western blot analysis. I{kappa}B{alpha} protein levels were measured by densitometry and standardized to actin levels. Values shown (*) represent the percentage of mock-infected I{kappa}B{alpha} levels.

 
P56 protein accumulation was used as a measure of IFN activation following treatment with entry inhibitors. Preincubation of cells with gB-DLD prevented P56 induction after infection with UV-HCMV (33% of UV-HCMV alone). P56 levels were unaffected by the control gB-651 protein (Fig. 6B). Accumulation of P56 protein was not detected with gB-DLD or gB-651 pretreatments alone. Similarly, UV-HCMV-mediated induction of P56 was diminished when virus was treated with β-peptide (+), but not when treated with β-peptide (–) (60% and 114% of UV-HCMV alone, respectively) (Fig. 6B).

Next, the effect of HCMV entry inhibitors on NF-{kappa}B activation was assessed. Pretreatment with gB-DLD or gB-651 resulted in similar levels of I{kappa}B{alpha} degradation as UV-HCMV alone (14% and 4% of the mock I{kappa}B{alpha} level, respectively, compared with 15% for HCMV alone) (Fig. 6C). The β-peptide (+) slightly inhibited I{kappa}B{alpha} degradation and the β peptide (–) did not affect I{kappa}B{alpha} degradation by UV-HCMV (15% and 35% of mock I{kappa}B{alpha} level, respectively) (Fig. 6C). Interestingly, pretreatment with the gB-DLD peptide alone resulted in I{kappa}B{alpha} degradation (11% of the mock I{kappa}B{alpha} level) (Fig. 6C). These data further support the hypothesis that UV-HCMV-induced innate immune responses are initiated through distinct mechanisms.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The goal of this study was to define the innate immune pathways elicited by HCMV and determine their relationship with virus entry. These experiments were performed in subconfluent, serum-starved secondary strains of human fibroblasts. Similar levels of IC and IFN induction are observed in the presence or absence of serum following HCMV infection (data not shown) (90). However, serum starvation reduces the basal level of some cytokines. In addition, serum starvation may increase the sensitivity of cells to ligands, such as IFN, because inhibitors have been found in serum (91). In addition, more robust innate immune signaling is observed following infection with UV-HCMV because replication-dependent counter-measures are not used (7). In this study we show that the IFN response to HCMV is independent of TLR2 because an antiviral state is intact in cells expressing a dominant negative construct of TLR2. We also did not find a role for intracellular localized TLRs, as inhibition of endosome acidification had no effect on HCMV-induced IFN responses. It has been reported that TLR3 can be found on both the cell surface and in intracellular compartments in fibroblast cells (92). However, a signaling defective, dominant negative construct of TLR3 also failed to block HCMV-induced IFN signaling (data not shown). In addition, we further define the mechanisms of activation for the HCMV-induced IC and IFN responses. Dissociating lipid rafts using cholesterol depletion greatly diminished IFN signaling, but the IC response was intact. Interestingly, depletion of cholesterol also inhibited HCMV entry suggesting that the process of IFN induction and virus entry are linked. These data also indicate that entry is not required for TLR2-dependent IC secretion. Finally, we used two HCMV-specific entry inhibitors to further explore the relationship between IC and IFN responses in regard to HCMV infection. Both entry inhibitors prevented IFN signaling, whereas the IC response was unaffected. Therefore we propose that the host cell has developed a multifaceted innate immune defense mechanism to recognize and battle infection at the earliest points in the virus entry process.

Lipid rafts are multifunctional assemblies involved in diverse processes including signal transduction, endocytosis, and cholesterol trafficking (87). These microdomains are enriched in cholesterol and glycosphingolipids, which facilitate protein-protein and lipid-protein interactions on the cell surface (93). The only other time that lipid rafts have been implicated in HCMV entry is in a report by Wang and colleagues (46). They found that lipid rafts are important for the coordination of signaling complexes within minutes of HCMV entry. In addition, two proposed HCMV coreceptors, {alpha}vβ3 and EGFR, localize to lipid rafts along with the signaling molecules, Src and the p85 subunit of PI3K (46). The exact mechanism by which lipid rafts initiate signal transduction has not been defined, nor is the role of lipid rafts in TLR signaling fully understood. TLR2 is reported to reside in lipid rafts on the apical surface of epithelial cells after bacterial infection (94), and clustering of TLR2 and GM1 ganglioside is detected in HEK293 cells expressing TLR2 and CD14 following treatment with lipoteichoic acid (95). Disruption of lipid rafts normally inhibits the function of raft-associated receptors, therefore we hypothesized that HCMV-induced IC responses would be diminished upon cholesterol depletion. In contrast, we found that the IC response remains intact when lipid rafts are disrupted. Under conditions in which HCMV entry is inhibited by cholesterol depletion, IC responses are unaffected suggesting that entry is not required for the initiation of IC secretion in response to HCMV. However, cholesterol depletion inhibits HCMV-mediated IFN signaling suggesting that it is a process that is independent of IC activation and reliant on the events that occur during the entry process.

Next, we tested two inhibitors of HCMV entry. β1 integrin, a HCMV cellular receptor, was blocked using a small protein fragment of gB that contains the disintegrin like domain integrin binding motif (A. L. Feire and T. Compton, manuscript in preparation). The gB-DLD effectively inhibited HCMV entry and IFN signaling but not IC activation. This result suggests that the interaction between β1 integrin and gB or subsequent steps after binding in the HCMV entry process are important determinants for IFN activation. Integrins have been shown to have immunomodulatory roles in promoting adaptive immune responses but to our knowledge there has been no link to innate immune activation. In addition, the gB-DLD protein appears to be functioning as a ligand mimic and is able to activate IC signaling alone (Fig. 6C), suggesting that gB-DLD protein may contain a PAMP from gB that is recognized by TLR2. The inhibitory β-peptide, designed to mimic the fusogenic heptad repeat region within gB was also used to impede HCMV entry (57). It is hypothesized that the β-peptide (+) forms a stable 12-helical conformation that physically interacts with gB of HCMV and blocks a step before fusion. The β peptides alone are not immunogenic (data not shown) (96, 97), likely due to their unnatural amino acid composition. The β peptide (+) effectively blocked both HCMV entry and IFN signaling. Activation of the IC response was observed with both β-peptide treatments and slight variations in the activation are likely due to alterations in the PAMP recognition of gB by TLR2.

These findings support a model in which virus binding promotes multiple receptor-ligand interactions between viral envelope glycoproteins and one or more cellular receptors. The IC response appears to be activated irrespective of events needed for a productive infection through outright sensing by TLR2. In contrast, the IFN response occurs by a postbinding or fusion-dependent mechanism. We hypothesize that the congregation of stable docking receptors promotes the activation of cellular IFN responses through one or more of the following events: 1) stable binding to cellular receptors; 2) the physical fusion event; or 3) delivery of virion contents into the cytoplasm. Our findings illustrate a clear distinction in the initiation of IFN and IC signal transduction cascades by UV-HCMV. RelA translocates to the nucleus in the absence of detectable pp65 localization, a measure of HCMV content delivery (Fig. 5B). However, type I IFN signaling is blocked in situations in which HCMV entry is impeded, suggesting a correlation between virus entry and IFN activation. The contribution of NF-{kappa}B to HCMV-induced IFN signaling has not been addressed in these experiments because it is activated before virus entry and IFN signaling. A recent report studying the contribution of NF-{kappa}B to type I IFN signaling following RNA virus infection found that the NF-{kappa}B subunits p50, RelA, and cRel play a minor role (98). Although the initiation of IFN signaling by RNA viruses and DNA viruses appear to be distinct, the RNA virus VSV was unaffected by cholesterol depletion (Fig. 4D). It is likely these viruses, at least partially, share downstream signaling pathways. An additional study by Wietek et al. (99) found that IRF3-mediated activation of the IFN stimulation response element by TLR4, but not TLR3, required the RelA subunit of NF-{kappa}B, suggesting that two parallel pathways may exist. Additional studies examining the degree of cross-talk between these two pathways following DNA virus infection are required. Because IL-8 levels are diminished but not eliminated in NHDF cells expressing the TLR2 dominant negative construct (Fig. 1A), we cannot rule out that other mechanisms of NF-{kappa}B activation coincide with later steps in the entry process. This model of innate immune activation is restricted to fibroblasts because HCMV entry can occur through alternate pathways in different cells types.

This report illustrates the existence of multiple mechanisms used by cells to detect HCMV and elicit distinct innate immune responses. In this study, we were able to dissect early events in the HCMV entry process and determine an order to the innate immune induction. It appears that the IC response is initiated via TLR2 before IFN activation and virus entry. The immediate activation of the IC response allows the cell to get a jump-start on the beneficial effects associated with NF-{kappa}B activation, such as the infiltration of professional immune cells. Interestingly, HCMV may also benefit from rapid IC induction. HCMV contains multiple NF-{kappa}B elements in its major immediate early promoter region, although the exact role of these elements in the initiation of viral gene expression remains controversial (100, 101). Postbinding or fusion-dependent steps trigger the IFN response, promoting an antiviral state to protect neighboring cells from the infection. The rapid recognition of HCMV by multiple pattern recognition receptors and the activation of both branches of the innate immune response are advantageous for the cellular defense mechanism. Increasing our understanding of HCMV-induced innate immune activation allows for a more comprehensive view of virus-host interactions and will aid in the identification of new targets for therapeutic intervention.


   Acknowledgments
 
We thank Ganes Sen (The Cleveland Clinic Foundation) for the P56 Ab, Deborah Spector (University of California, San Diego) for the HCMV AD169 IE2-GFP virus, John Yin (University of Wisconsin-Madison) for the VSV, and Rob Kalejta (University of Wisconsin-Madison) for the pCGN-HA-pp65 plasmid. We also thank Sam Gellman and Emily Payne-English (University of Wisconsin-Madison) for synthesizing the β peptides used in this study and the members of the T. Compton laboratory for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant R01 AI054915 (to T.C.) from the National Institutes of Health and National Institutes of Health Training Grant T32GM07215 (to K.W.B.). Back

2 Address correspondence and reprint requests to Dr. Teresa Compton, Novartis Institute for Biomedical Research, 500 Technology Square, Cambridge, MA 02139. E-mail address: teresa.compton{at}novartis.com Back

3 Abbreviations used in this paper: HCMV, human CMV; IC, inflammatory cytokine; ISG, IFN-stimulated gene; IRF, IFN regulatory factor; NHDF, normal human dermal fibroblast; VSV, vesicular stomatitis virus; MβCD, methyl-β-cyclodextrin; MOI, multiplicity of infection; gB, glycoprotein B; gH, glycoprotein H; PAMP, pathogen-associated molecular pattern. Back

Received for publication January 30, 2008. Accepted for publication February 2, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Ljungman, P.. 1996. Cytomegalovirus infections in transplant patients. Scand J. Infect. Dis. Suppl. 100: 59-63. [Medline]
  2. Pass, R. F.. 2001. Cytomegalovirus. D. M. Knipe, and P. M. Howley, eds. In Fields Virology Vol. 2: 2675 Raven Press, New York.
  3. Ramsay, M. E., E. Miller, C. S. Peckham. 1991. Outcome of confirmed symptomatic congenital cytomegalovirus infection. Arch. Dis. Child. 66: 1068-1069. [Abstract/Free Full Text]
  4. Alford, C. A., W. J. Britt. 1993. Cytomegalovirus. B. Roizman, and R. J. Whitley, and C. Lopez, eds. The Human Herpesviruses 227-255. Raven Press, New York.
  5. Hendrix, M. G., M. M. Salimans, C. P. van Boven, C. A. Bruggeman. 1990. High prevalence of latently present cytomegalovirus in arterial walls of patients suffering from grade III atherosclerosis. Am. J. Pathol. 136: 23-28. [Abstract]
  6. Osame, K., Y. Takahashi, H. Takasawa, S. Watanabe, M. Kishimoto, K. Yasuda, Y. Kaburagi, K. Nakanishi, H. Kajio, M. Noda. 2007. Rapid-onset type 1 diabetes associated with cytomegalovirus infection and islet autoantibody synthesis. Intern Med. 46: 873-877. [Medline]
  7. Browne, E. P., B. Wing, D. Coleman, T. Shenk. 2001. Altered cellular mRNA levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mRNAs. J. Virol. 75: 12319-12330. [Abstract/Free Full Text]
  8. Simmen, K. A., J. Singh, B. G. Luukkonen, M. Lopper, A. Bittner, N. E. Miller, M. R. Jackson, T. Compton, K. Fruh. 2001. Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B. Proc. Natl. Acad. Sci. USA 98: 7140-7145. [Abstract/Free Full Text]
  9. Zhu, H., J. P. Cong, T. Shenk. 1997. Use of differential display analysis to assess the effect of human cytomegalovirus infection on the accumulation of cellular RNAs: induction of interferon-responsive RNAs. Proc. Natl. Acad. Sci. USA 94: 13985-13990. [Abstract/Free Full Text]
  10. Sen, G. C.. 2001. Viruses and interferons. Annu. Rev. Microbiol. 55: 255-281. [Medline]
  11. Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, R. D. Schreiber. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67: 227-264. [Medline]
  12. Laroux, F. S.. 2004. Mechanisms of inflammation: the good, the bad and the ugly. Front Biosci. 9: 3156-3162. [Medline]
  13. Perry, A. K., G. Chen, D. Zheng, H. Tang, G. Cheng. 2005. The host type I interferon response to viral and bacterial infections. Cell Res. 15: 407-422. [Medline]
  14. Theofilopoulos, A. N., R. Baccala, B. Beutler, D. H. Kono. 2005. Type I interferons ({alpha}/β) in immunity and autoimmunity. Annu. Rev. Immunol. 23: 307-336. [Medline]
  15. Hayden, M. S., A. P. West, S. Ghosh. 2006. NF-{kappa}B and the immune response. Oncogene 25: 6758-6780. [Medline]
  16. Baldwin, A. S., Jr. 1996. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu. Rev. Immunol. 14: 649-683. [Medline]
  17. Yurochko, A. D., E. S. Huang. 1999. Human cytomegalovirus binding to human monocytes induces immunoregulatory gene expression. J. Immunol. 162: 4806-4816. [Abstract/Free Full Text]
  18. Yurochko, A. D., T. F. Kowalik, S. M. Huong, E. S. Huang. 1995. Human cytomegalovirus upregulates NF-{kappa}B activity by transactivating the NF-{kappa}B p105/p50 and p65 promoters. J. Virol. 69: 5391-5400. [Abstract]
  19. Takeda, K., S. Akira. 2003. Toll receptors and pathogen resistance. Cell Microbiol. 5: 143-153. [Medline]
  20. Akira, S., S. Uematsu, O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124: 783-801. [Medline]
  21. Hornung, V., S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdörfer, T. Giese, S. Endres, G. Hartmann. 2002. Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168: 4531-4537. [Abstract/Free Full Text]
  22. Zarember, K. A., P. J. Godowski. 2002. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J. Immunol. 168: 554-561. [Abstract/Free Full Text]
  23. Janeway, C. A., Jr, R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197-216. [Medline]
  24. Kurt-Jones, E. A., L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, R. W. Finberg. 2000. Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat. Immunol. 1: 398-401. [Medline]
  25. Burzyn, D., J. C. Rassa, D. Kim, I. Nepomnaschy, S. R. Ross, I. Piazzon. 2004. Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus. J. Virol. 78: 576-584. [Abstract/Free Full Text]
  26. Compton, T., E. A. Kurt-Jones, K. W. Boehme, J. Belko, E. Latz, D. T. Golenbock, R. W. Finberg. 2003. Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2. J. Virol. 77: 4588-4596. [Abstract/Free Full Text]
  27. Boehme, K. W., M. Guerrero, T. Compton. 2006. Human cytomegalovirus envelope glycoproteins B and H are necessary for TLR2 activation in permissive cells. J. Immunol. 177: 7094-7102. [Abstract/Free Full Text]
  28. Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S. M. Liao, T. Maniatis. 2003. IKK{epsilon} and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4: 491-496. [Medline]
  29. Sharma, S., B. R. tenOever, N. Grandvaux, G. P. Zhou, R. Lin, J. Hiscott. 2003. Triggering the interferon antiviral response through an IKK-related pathway. Science 300: 1148-1151. [Abstract/Free Full Text]
  30. Servant, M. J., B. tenOever, C. LePage, L. Conti, S. Gessani, I. Julkunen, R. Lin, J. Hiscott. 2001. Identification of distinct signaling pathways leading to the phosphorylation of interferon regulatory factor 3. J. Biol. Chem. 276: 355-363. [Abstract/Free Full Text]
  31. Taniguchi, T., A. Takaoka. 2002. The interferon-{alpha}/β system in antiviral responses: a multimodal machinery of gene regulation by the IRF family of transcription factors. Curr. Opin. Immunol. 14: 111-116. [Medline]
  32. Navarro, L., K. Mowen, S. Rodems, B. Weaver, N. Reich, D. Spector, M. David. 1998. Cytomegalovirus activates interferon immediate-early response gene expression and an interferon regulatory factor 3-containing interferon-stimulated response element-binding complex. Mol. Cell Biol. 18: 3796-3802. [Abstract/Free Full Text]
  33. Preston, C. M., A. N. Harman, M. J. Nicholl. 2001. Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus. J. Virol. 75: 8909-8916. [Abstract/Free Full Text]
  34. Boehme, K. W., J. Singh, S. T. Perry, T. Compton. 2004. Human cytomegalovirus elicits a coordinated cellular antiviral response via envelope glycoprotein B. J. Virol. 78: 1202-1211. [Abstract/Free Full Text]
  35. DeFilippis, V. R., B. Robinson, T. M. Keck, S. G. Hansen, J. A. Nelson, K. J. Fruh. 2006. Interferon regulatory factor 3 is necessary for induction of antiviral genes during human cytomegalovirus infection. J. Virol. 80: 1032-1037. [Abstract/Free Full Text]
  36. Netterwald, J. R., T. R. Jones, W. J. Britt, S. J. Yang, I. P. McCrone, H. Zhu. 2004. Postattachment events associated with viral entry are necessary for induction of interferon-stimulated genes by human cytomegalovirus. J. Virol. 78: 6688-6691. [Abstract/Free Full Text]
  37. Paulus, C., S. Krauss, M. Nevels. 2006. A human cytomegalovirus antagonist of type I IFN-dependent signal transducer and activator of transcription signaling. Proc. Natl. Acad. Sci. USA 103: 3840-3845. [Abstract/Free Full Text]
  38. Taylor, R. T., W. A. Bresnahan. 2006. Human cytomegalovirus IE86 attenuates virus- and tumor necrosis factor {alpha}-induced NF{kappa}B-dependent gene expression. J. Virol. 80: 10763-10771. [Abstract/Free Full Text]
  39. Penfold, M. E., D. J. Dairaghi, G. M. Duke, N. Saederup, E. S. Mocarski, G. W. Kemble, T. J. Schall. 1999. Cytomegalovirus encodes a potent {alpha} chemokine. Proc. Natl. Acad. Sci. USA 96: 9839-9844. [Abstract/Free Full Text]
  40. Wang, D., W. Bresnahan, T. Shenk. 2004. Human cytomegalovirus encodes a highly specific RANTES decoy receptor. Proc. Natl. Acad. Sci. USA 101: 16642-16647. [Abstract/Free Full Text]
  41. Stropes, M. P., W. E. Miller. 2004. Signaling and regulation of G-protein coupled receptors encoded by cytomegaloviruses. Biochem. Cell Biol. 82: 636-642. [Medline]
  42. Browne, H., G. Smith, S. Beck, T. Minson. 1990. A complex between the MHC class I homologue encoded by human cytomegalovirus and β2 microglobulin. Nature 347: 770-772. [Medline]
  43. Leong, C. C., T. L. Chapman, P. J. Bjorkman, D. Formankova, E. S. Mocarski, J. H. Phillips, L. L. Lanier. 1998. Modulation of natural killer cell cytotoxicity in human cytomegalovirus infection: the role of endogenous class I major histocompatibility complex and a viral class I homolog. J. Exp. Med. 187: 1681-1687. [Abstract/Free Full Text]
  44. Compton, T., D. M. Nowlin, N. R. Cooper. 1993. Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193: 834-841. [Medline]
  45. Feire, A. L., H. Koss, T. Compton. 2004. Cellular integrins function as entry receptors for human cytomegalovirus via a highly conserved disintegrin-like domain. Proc. Natl. Acad. Sci. USA 101: 15470-15475. [Abstract/Free Full Text]
  46. Wang, X., D. Y. Huang, S. M. Huong, E. S. Huang. 2005. Integrin {alpha}Vβ3 is a coreceptor for human cytomegalovirus. Nat. Med. 11: 515-521. [Medline]
  47. Wang, X., S. M. Huong, M. L. Chiu, N. Raab-Traub, E. S. Huang. 2003. Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus. Nature 424: 456-461. [Medline]
  48. Isaacson, M. K., A. L. Feire, T. Compton. 2007. Epidermal growth factor receptor is not required for human cytomegalovirus entry or signaling. J. Virol. 81: 6241-6247. [Abstract/Free Full Text]
  49. Cobbs, C. S., L. Soroceanu, S. Denham, W. Zhang, W. J. Britt, R. Pieper, M. H. Kraus. 2007. Human cytomegalovirus induces cellular tyrosine kinase signaling and promotes glioma cell invasiveness. J. Neurooncol. 85: 271-280. [Medline]
  50. Compton, T., R. R. Nepomuceno, D. M. Nowlin. 1992. Human cytomegalovirus penetrates host cells by pH-independent fusion at the cell surface. Virology 191: 387-395. [Medline]
  51. Ryckman, B. J., M. A. Jarvis, D. D. Drummond, J. A. Nelson, D. C. Johnson. 2006. Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion. J. Virol. 80: 710-722. [Abstract/Free Full Text]
  52. Keay, S., B. Baldwin. 1991. Anti-idiotype antibodies that mimic gp86 of human cytomegalovirus inhibit viral fusion but not attachment. J. Virol. 65: 5124-5128. [Abstract/Free Full Text]
  53. Milne, R. S., D. A. Paterson, J. C. Booth. 1998. Human cytomegalovirus glycoprotein H/glycoprotein L complex modulates fusion-from-without. J. Gen. Virol. 79: (Pt. 4):855-865. [Abstract]
  54. Compton, T.. 1993. An immortalized human fibroblast cell line is permissive for human cytomegalovirus infection. J. Virol. 67: 3644-3648. [Abstract/Free Full Text]
  55. Sanchez, V., C. L. Clark, J. Y. Yen, R. Dwarakanath, D. H. Spector. 2002. Viable human cytomegalovirus recombinant virus with an internal deletion of the IE2 86 gene affects late stages of viral replication. J. Virol. 76: 2973-2989. [Abstract/Free Full Text]
  56. Guo, J., K. L. Peters, G. C. Sen. 2000. Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection. Virology 267: 209-219. [Medline]
  57. English, E. P., R. S. Chumanov, S. H. Gellman, T. Compton. 2006. Rational development of β-peptide inhibitors of human cytomegalovirus entry. J. Biol. Chem. 281: 2661-2667. [Abstract/Free Full Text]
  58. Highlander, S. L., S. L. Sutherland, P. J. Gage, D. C. Johnson, M. Levine, J. C. Glorioso. 1987. Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration. J. Virol. 61: 3356-3364. [Abstract/Free Full Text]
  59. Gault, E., Y. Michel, A. Dehée, C. Belabani, J. C. Nicolas, A. Garbarg-Chenon. 2001. Quantification of human cytomegalovirus DNA by real-time PCR. J. Clin. Microbiol. 39: 772-775. [Abstract/Free Full Text]
  60. Baldick, C. J., Jr, A. Marchini, C. E. Patterson, T. Shenk. 1997. Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle. J. Virol. 71: 4400-4408. [Abstract]
  61. Boyle, K. A., R. L. Pietropaolo, T. Compton. 1999. Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol. Cell Biol. 19: 3607-3613. [Abstract/Free Full Text]
  62. Nagai, K., H. C. Thogersen. 1987. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 153: 461-481. [Medline]
  63. Kurt-Jones, E. A., M. Chan, S. Zhou, J. Wang, G. Reed, R. Bronson, M. M. Arnold, D. M. Knipe, R. W. Finberg. 2004. Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis. Proc. Natl. Acad. Sci. USA 101: 1315-1320. [Abstract/Free Full Text]
  64. Wang, J. P., E. A. Kurt-Jones, O. S. Shin, M. D. Manchak, M. J. Levin, R. W. Finberg. 2005. Varicella-zoster virus activates inflammatory cytokines in human monocytes and macrophages via Toll-like receptor 2. J. Virol. 79: 12658-12666. [Abstract/Free Full Text]
  65. Toshchakov, V., B. W. Jones, P. Y. Perera, K. Thomas, M. J. Cody, S. Zhang, B. R. Williams, J. Major, T. A. Hamilton, M. J. Fenton, S. N. Vogel. 2002. TLR4, but not TLR2, mediates IFN-β-induced STAT1{alpha}/β-dependent gene expression in macrophages. Nat. Immunol. 3: 392-398. [Medline]
  66. Ahmad-Nejad, P., H. Häcker, M. Rutz, S. Bauer, R. M. Vabulas, H. Wagner. 2002. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32: 1958-1968. [Medline]
  67. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks, C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, D. T. Golenbock. 2004. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5: 190-198. [Medline]
  68. Nishiya, T., A. L. DeFranco. 2004. Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling properties of the toll-like receptors. J. Biol. Chem. 279: 19008-19017. [Abstract/Free Full Text]
  69. Diebold, S. S., T. Kaisho, H. Hemmi, S. Akira, E. S. C. Reis. 2004. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303: 1529-1531. [Abstract/Free Full Text]
  70. Häcker, H., H. Mischak, T. Miethke, S. Liptay, R. Schmid, T. Sparwasser, K. Heeg, G. B. Lipford, H. Wagner. 1998. CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. EMBO J. 17: 6230-6240. [Medline]
  71. de Bouteiller, O., E. Merck, U. A. Hasan, S. Hubac, B. Benguigui, G. Trinchieri, E. E. Bates, C. Caux. 2005. Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH. J. Biol. Chem. 280: 38133-38145. [Abstract/Free Full Text]
  72. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408: 740-745. [Medline]
  73. Krug, A., G. D. Luker, W. Barchet, D. A. Leib, S. Akira, M. Colonna. 2004. Herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9. Blood 103: 1433-1437. [Abstract/Free Full Text]
  74. Lund, J., A. Sato, S. Akira, R. Medzhitov, A. Iwasaki. 2003. Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J. Exp. Med. 198: 513-520. [Abstract/Free Full Text]
  75. Krug, A., A. R. French, W. Barchet, J. A. Fischer, A. Dzionek, J. T. Pingel, M. M. Orihuela, S. Akira, W. M. Yokoyama, M. Colonna. 2004. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21: 107-119. [Medline]
  76. Yoshimori, T., A. Yamamoto, Y. Moriyama, M. Futai, Y. Tashiro. 1991. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J. Biol. Chem. 266: 17707-17712. [Abstract/Free Full Text]
  77. tenOever, B. R., S. Sharma, W. Zou, Q. Sun, N. Grandvaux, I. Julkunen, H. Hemmi, M. Yamamoto, S. Akira, W. C. Yeh, R. Lin, J. Hiscott. 2004. Activation of TBK1 and IKKvar{epsilon} kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity. J. Virol. 78: 10636-10649. [Abstract/Free Full Text]
  78. Superti, F., L. Seganti, F. M. Ruggeri, A. Tinari, G. Donelli, N. Orsi. 1987. Entry pathway of vesicular stomatitis virus into different host cells. J. Gen. Virol. 68: (Pt. 2):387-399. [Abstract/Free Full Text]
  79. Matlin, K. S., H. Reggio, A. Helenius, K. Simons. 1982. Pathway of vesicular stomatitis virus entry leading to infection. J. Mol. Biol. 156: 609-631. [Medline]
  80. Simons, K., E. Ikonen. 1997. Functional rafts in cell membranes. Nature 387: 569-572. [Medline]
  81. Laude, A. J., I. A. Prior. 2004. Plasma membrane microdomains: organization, function and trafficking (Review). Mol. Membr. Biol. 21: 193-205. [Medline]
  82. Bender, F. C., J. C. Whitbeck, M. Ponce de Leon, H. Lou, R. J. Eisenberg, G. H. Cohen. 2003. Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry. J. Virol. 77: 9542-9552. [Abstract/Free Full Text]
  83. Chung, C. S., C. Y. Huang, W. Chang. 2005. Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts. J. Virol. 79: 1623-1634. [Abstract/Free Full Text]
  84. Choi, K. S., H. Aizaki, M. M. Lai. 2005. Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release. J. Virol. 79: 9862-9871. [Abstract/Free Full Text]
  85. Pietiäinen, V., V. Marjomäki, P. Upla, L. Pelkmans, A. Helenius, T. Hyypiä. 2004. Echovirus 1 endocytosis into caveosomes requires lipid rafts, dynamin II, and signaling events. Mol. Biol. Cell 15: 4911-4925. [Abstract/Free Full Text]
  86. Pelkmans, L., D. Puntener, A. Helenius. 2002. Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 296: 535-539. [Abstract/Free Full Text]
  87. Pike, L. J.. 2004. Lipid rafts: heterogeneity on the high seas. Biochem. J. 378: 281-292. [Medline]
  88. Rodal, S. K., G. Skretting, O. Garred, F. Vilhardt, B. van Deurs, K. Sandvig. 1999. Extraction of cholesterol with methyl-β-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol. Biol. Cell 10: 961-974. [Abstract/Free Full Text]
  89. Malek, S., Y. Chen, T. Huxford, G. Ghosh. 2001. I{kappa}Bβ, but not I{kappa}B{alpha}, functions as a classical cytoplasmic inhibitor of NF-{kappa}B dimers by masking both NF-{kappa}B nuclear localization sequences in resting cells. J. Biol. Chem. 276: 45225-45235. [Abstract/Free Full Text]
  90. Gealy, C., M. Denson, C. Humphreys, B. McSharry, G. Wilkinson, R. Caswell. 2005. Posttranscriptional suppression of interleukin-6 production by human cytomegalovirus. J. Virol. 79: 472-485. [Abstract/Free Full Text]
  91. Rossman, T. G., J. Vilcek. 1970. Blocking of interferon action by a component of normal serum. Arch. Gesamte Virusforsch. 31: 18-27. [Medline]
  92. Matsumoto, M., S. Kikkawa, M. Kohase, K. Miyake, T. Seya. 2002. Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochem. Biophys. Res. Commun. 293: 1364-1369. [Medline]
  93. Simons, K., D. Toomre. 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1: 31-39. [Medline]
  94. Soong, G., B. Reddy, S. Sokol, R. Adamo, A. Prince. 2004. TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells. J. Clin. Invest. 113: 1482-1489. [Medline]
  95. Triantafilou, M., M. Manukyan, A. Mackie, S. Morath, T. Hartung, H. Heine, K. Triantafilou. 2004. Lipoteichoic acid and toll-like receptor 2 internalization and targeting to the Golgi are lipid raft-dependent. J. Biol. Chem. 279: 40882-40889. [Abstract/Free Full Text]
  96. Arvidsson, P. I., N. S. Ryder, H. M. Weiss, G. Gross, O. Kretz, R. Woessner, D. Seebach. 2003. Antibiotic and hemolytic activity of a β2/β3 peptide capable of folding into a 12/10-helical secondary structure. Chembiochem. 4: 1345-1347. [Medline]
  97. Patch, J. A., A. E. Barron. 2002. Mimicry of bioactive peptides via non-natural, sequence-specific peptidomimetic oligomers. Curr. Opin. Chem Biol. 6: 872-877. [Medline]
  98. Wang, X., S. Hussain, E. J. Wang, X. Wang, M. O. Li, A. García-Sastre, A. A. Beg. 2007. Lack of essential role of NF-{kappa}B p50, RelA, and cRel subunits in virus-induced type 1 IFN expression. J. Immunol. 178: 6770-6776. [Abstract/Free Full Text]
  99. Wietek, C., S. M. Miggin, C. A. Jefferies, L. A. O’Neill. 2003. Interferon regulatory factor-3-mediated activation of the interferon-sensitive response element by Toll-like receptor (TLR) 4 but not TLR3 requires the p65 subunit of NF-{kappa}. J. Biol. Chem. 278: 50923-50931. [Abstract/Free Full Text]
  100. DeMeritt, I. B., L. E. Milford, A. D. Yurochko. 2004. Activation of the NF-{kappa}B pathway in human cytomegalovirus-infected cells is necessary for efficient transactivation of the major immediate-early promoter. J. Virol. 78: 4498-4507. [Abstract/Free Full Text]
  101. Gustems, M., E. Borst, C. A. Benedict, C. Pérez, M. Messerle, P. Ghazal, A. Angulo. 2006. Regulation of the transcription and replication cycle of human cytomegalovirus is insensitive to genetic elimination of the cognate NF-{kappa}B binding sites in the enhancer. J. Virol. 80: 9899-9904. [Abstract/Free Full Text]



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Immunobiology of Human Cytomegalovirus: from Bench to Bedside
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