Human cytomegalovirus (HCMV) is an opportunistic pathogen that is distributed worldwide with seroprevalences between 45% and 100% depending on location, age, gender, and socioeconomic status [1
]. As characteristic for other herpesviruses, HCMV persists life-long in a dormant state and can sporadically re-enter the lytic replication cycle. While lytic infection of healthy individuals is mostly asymptomatic, immunocompromised individuals (e.g., transplant recipients and HIV/AIDS patients) can develop life-threatening disseminating disease [2
]. Moreover, HCMV congenital infections are the underappreciated leading cause of congenital birth defects [3
]. The immediate recognition of invading pathogens is essential for the induction of a potent host immune response, leading to eventual control of the infection. In order to establish life-long infections, herpesviruses have evolved sophisticated strategies to modulate hosts’ immune responses [4
Cellular pattern recognition receptors (PRR) sense pathogen-associated molecular patterns (PAMP) such as nucleic acids and immediately activate the transcription of cytokine and antiviral factor genes [5
]. Cytosolic DNA can be detected by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) [6
] as well as other cytosolic DNA sensors. DNA binding to cGAS facilitates the generation of the second messenger cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) which activates the endoplasmic reticulum (ER) resident protein stimulator of interferon genes (STING) [7
]. STING undergoes dimerization and translocates to the Golgi compartment where it forms a platform for the activation of downstream effector proteins including TANK-binding kinase 1 (TBK1) and the transcription factor interferon regulatory factor 3 (IRF3) [8
]. TBK1 gets recruited, autophosphorylates itself and subsequently phosphorylates STING [9
]. STING phosphorylation enables IRF3 binding and TBK1-mediated IRF3 phosphorylation triggers IRF3 dimerization and its nuclear translocation, from where it facilitates IFNβ transcription. Similarly, RNA can be detected by cytosolic RIG-I-like receptors (RLR) such as retinoic acid inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5) [10
]. Following ligand binding, the adaptor protein mitochondrial antiviral-signaling protein (MAVS) multimerizes and forms a scaffold for TBK1 and IRF3, leading to the phosphorylation and nuclear translocation of IRF3 and subsequent IFNβ transcription [8
]. IFNβ protein produced following PRR-mediated activation is then secreted and binds to the type I IFN receptor (IFNAR) in an autocrine and paracrine manner. Activation of IFNAR signaling triggers the JAK-STAT pathway leading to the transcription of multiple interferon-stimulated genes (ISG), a hallmark of the antiviral state [12
Herpesviruses have developed numerous strategies to evade or impede the host immune response including PRR-signaling and downstream effectors [13
]. Recently, we identified the murine CMV (MCMV) M35 protein as a type I IFN antagonist that downmodulates type I IFN transcription downstream of multiple PRR [15
]. The M35 protein is present in the tegument of the MCMV virion and can therefore act immediately after viral entry into the host cell, without prior requirement for viral gene expression.
In this study, we investigated whether the HCMV homologue of M35, UL35, can likewise modulate PRR signaling. Although M35 and UL35 share only 25% amino acid identity, structure remodeling using the human herpesvirus 6B (HHV-6B) homologue U14 as the template [16
] predicted a high similarity for their three-dimensional protein structure. The UL35 open reading frame (ORF) codes for the 75 kDa ppUL35 phosphoprotein (herein referred to as UL35) which was identified as a minor tegument component of HCMV [17
]. In addition to UL35, the UL35 locus encodes a second protein, ppUL35a (herein referred to as UL35a), which is not present in the tegument [17
]. UL35a is identical to the C-terminus of UL35 as it is translated from an internal start codon leading to a 21 kDa protein. While tegument UL35 is expressed late, UL35a shows early and late expression kinetics during infection [17
]. It has been reported that deletion of the complete UL35 ORF led to a strong defect in virus replication, assembly, and particle formation, depending on the multiplicity of infection (MOI) [18
]. A recent study showed that loss of UL35a led to a comparable growth deficit as the complete deletion of the UL35 ORF [19
]. In contrast, when only UL35 expression was impaired but expression of UL35a was unaffected, a moderate defect in viral replication was observed [19
]. UL35 was shown to positively affect the activation of the viral major immediate-early promoter (MIEP) by interacting with the viral transactivator pp71, encoded by UL82 [20
Another example of the complex interplay between UL35, UL35a, and UL82 was reported by Salsman and colleagues in 2011 [21
]. Overexpression experiments revealed that UL35 forms ring-like nuclear bodies independent of the promyelocytic leukemia (PML) protein and co-localizes with PML and PML-associated proteins such as Daxx and SP100 [21
]. Additionally, the number of nuclear bodies formed by UL35 was enhanced by co-expressed UL82. When expressed individually, UL82 and UL35a localize to the nucleus, while they localize to the cytoplasm upon co-expression [21
]. These divergent functions of UL35 and UL35a underline the importance of their tight temporal control of their expression through the infection cycle. In 2012, Salsman et al. revealed multiple cellular interaction partners of transiently expressed UL35 and UL35a [22
]. DCAF1, a member of the Cul4DCAF1
E3 ubiquitin ligase complex, was identified as an interaction partner of UL35, but not UL35a. DCAF1 co-localizes with the ring-like UL35 nuclear bodies [22
Here, we identify the HCMV tegument protein UL35 as a novel type I IFN antagonist which acts downstream of the PRR cGAS and RIG-I, but upstream of the IFNAR signaling pathway. Upon HCMV infection, tegument UL35 antagonizes the proper activation of the STING-TBK1-IRF3 signaling platform, leading to impaired activation of IFNβ transcription and ISG expression.
2. Materials and Methods
2.1. Cell Lines
Human embryonic kidney 293T/17 cells (HEK293T, CRL-11268™) were obtained from American Type Culture Collection, Manassas, VA, USA (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; high glucose, Sigma-Aldrich, Darmstadt, Germany) supplemented with 8% fetal calf serum (FCS) and 1× penicillin/streptomycin (P/S), Gibco (Sigma-Aldrich). Primary human foreskin fibroblasts, HFF-1, were obtained from ATCC (SCRC-1041™) and cultivated in DMEM high glucose supplemented with 10% FCS, 1 mM sodium pyruvate, and 1x P/S. Human lung fibroblasts MRC-5 were obtained from ATCC (CCL-171™) and maintained in DMEM, high glucose, supplemented with 10% FCS and 1× P/S. Cells were grown in a humidified incubator at 37 °C with 7 Vol% CO2. HFF-1 stably expressing empty vector (ev) or UL35-HA were generated by retroviral transduction using pMSCVpuro expression constructs and selection with 2 µg/mL puromycin.
2.2. Antibodies and Reagents
Rabbit anti-HA (#3724 clone C29F4), rabbit anti-IRF3 (#11904 clone D6I4C), rabbit anti-p65 (#6956 clone L8F6), rabbit anti-Fibrillarin (#2639 clone C13C3), mouse anti-myc (#2276 clone 9B11), rabbit anti-OGT (#24083 clone D1D8Q), mouse anti-O-GlcNAc (#9875 clone CTD110.6), rabbit anti-TBK1 (#3504 clone D1B4), rabbit anti-p-TBK1 (#5483 clone D52C2), rabbit anti-p-IRF3 Ser396 (#29047 clone D6O1M), rabbit anti-p-STING Ser366 (#85735), and rabbit anti-p-STAT1 Tyr701 (#7649 clone D4A7) were purchased from Cell Signaling Technology (Frankfurt am Main, Germany). Mouse anti-α-Tubulin (#T6199) and mouse anti-β Actin (A5441) were obtained from Sigma-Aldrich. Rat anti-HA-HRP (#12013819001 clone 3F10) was purchased from Roche (Mannheim, Germany). Mouse anti-pp65 (#ab6503 clone 3A12) was purchased from Abcam (Cambridge, UK) and mouse anti-UL44 ICP36 (#MBS530793) was obtained from MyBioSource (San Diego, CA, USA). Mouse anti-IE1 (clone 63-27), originally described in [23
], was a kind gift from Jens von Einem (Institute of Virology, Ulm University Medical Center, Ulm). Rabbit anti-UL82 (clone SA2932), originally described in [24
], was provided by Thomas Stamminger (Institute of Virology, Ulm University Medical Center, Ulm). HRP-conjugated and Alexa Fluor®
-conjugated secondary antibodies were purchased from Dianova (Hamburg, Germany) and Invitrogen (Life-Technologies, Darmstadt, Germany), respectively.
For production of mouse monoclonal anti-UL35 antibody, a section of UL35 corresponding to aa 465–641 of the UL35 ORF was subcloned into pET-28c (Novagen, Merck Millipore, Darmstadt, Germany). Recombinant protein was expressed in E. coli
strain BL21 (DE3) via IPTG induction. Immunization of mice and generation of hybridoma cultures was performed as reported previously [25
]. Specificity of antibodies was validated by ELISA on UL35 peptide used for immunization versus irrelevant His-tagged peptide. Antibodies were further tested on UL35 expressing cell lysates by immunoblotting, immunoprecipitation and immunofluorescence. Selected antibodies were purified from hybridoma supernatants using protein G columns (GE Healthcare, Chicago, IL, USA) on Äkta Prime Plus.
High molecular weight poly(I:C) was purchased from Invivogen (San Diego, CA, USA) (#tlrl-pic). Interferon-stimulatory DNA (ISD) was generated by the combination of complementary forward (ISD45 bp-for: 5′-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA) and reverse (ISD45 bp-rev: 5′-TGTAGATCATGTACAGATCAGTCATAGATCACTAGTAGATCTGTA) 45 bp oligonucleotides, heating to 70 °C for 10 min followed by annealing at room temperature. Protease inhibitors (4693116001) and phosphatase inhibitors (4906837001) were purchased from Roche. For transfections, Lipofectamine 2000, FuGENE HD, and polyethylenimine (PEI) were purchased from Life-Technologies, Promega (Walldorf, Germany), and Polysciences, Inc. (Warrington, PA, USA), respectively. JetPEI was obtained from Polyplus transfection (Illkirch, France) and OptiMEM was obtained from Thermo Fisher Scientific (Darmstadt, Germany). Recombinant human IFNβ (#300-02BC) was ordered from PeproTech (Hamburg, Germany).
Expression constructs of HA-tagged and untagged UL35 were generated by subcloning PCR amplified ORF UL35 (GenBank accession# AAR31600.1) into pcDNA3.1+ (Invitrogen) via HindIII/NotI sites:
(HindIII-UL35_for 5′-GCATAAGCTTGCCACCATGGCCCAGGGATCGCGAGC-3′ and NotI-UL35-untagged_rev 5′-CCATGCGGCCGCtcaGAGATGCCGTAGGTTTTCGGC-3′ or NotI-UL35-HA_rev 5′-CCATGCGGCCGCctaTGCGTAGTCTGGTACGTCGTACGGATATGCGTAGTCTGGTACGTCGTACGGATAGAGATGCCGTAGGTTTTCG-3′). HA-tagged UL35 was subcloned into pMSCVpuro (Clontech) via blunt end cloning using HpaI/PmeI sites to generate pMSCVpuro UL35-HA. pEFBOS mCherry-mSTING (designated Cherry-STING) expressing N-terminal monomeric Cherry fused to murine STING and pIRESneo3 cGAS-GFP (GFP fused to the C-terminus of human cGAS) were kindly provided by Andrea Ablasser (Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Switzerland). The Renilla luciferase expression construct pRL-TK and pIRES2-GFP were purchased from Promega and Clontech, respectively. pGL3basic IFNβ-Luc (IFNβ-Luc) and pGL3basic ISG56-Luc (ISG56-Luc) were described previously [15
]. pcDNA3-FLAG-TBK1 was described previously by Søren Paludan, Aarhus University, Denmark [26
]. CMVBL IRF3-5D codes for human IRF3 containing five amino acid substitutions (S396D, S398D, S402D, S404D, S405D) which renders it constitutively active and was provided by John Hiscott (Institut Pasteur Cenci Bolognetti Foundation, Rome, Italy). pCAGGS Flag-RIG-I N, expressing a constitutively active truncation mutant of RIG-I, was kindly provided by Andreas Pichlmair (Technical University Munich, Germany). pFLAG-CMV2-MAVS was described previously [27
] and was kindly provided by Friedemann Weber (Justus-Liebig-Universität Giessen, Germany). pcDNA4/LacZ-myc/His was purchased from Invitrogen. C-terminally myc/His tagged M76 was subcloned from the Smith strain MCMV BAC into pcDNA4B myc/His (Invitrogen) using HindIII/XbaI sites. pcDNA4-M35-myc/His was previously described [15
]. Expression constructs for pcDNA3.1 M35-V5/His and M27-V5/His have been described previously [28
O-GlcNAcylation mutants of UL35 (all C-terminally HA tagged, pcDNA3.1+) were generated using the Q5 site-directed mutagenesis kit (New England Biolabs, Frankfurt am Main, Germany #E0554) according to the manufacturer’s instructions. For UL35 Ala529-553, serines and threonines within UL35 aa 529–553 were substituted for alanine. For UL35 Ala529-531, threonines at aa position 529–531 were substituted for alanines. For UL35 Ala534/537, serine 534 and threonine 537 were mutated to alanines. UL35 Ala550-553 was generated by mutating serines at position 550–553 to alanines. The expression construct of untagged OGT was subcloned from pOTB7-OGT (accession #BC014434, cDNA obtained from Dharmacon, Lafayette, CO, USA) into pcDNA3.1+ via BamHI/NotI sites.
Primer sequences as well as sequences of all constructs are available upon request.
2.4. BAC Mutagenesis
Manipulation of the UL35 open reading frame was carried out by en passant
] on the HCMV bacterial artificial chromosome (BAC) TB40/E-BAC4 (GenBank accession # EF999921.1) [30
]. To introduce a stop cassette (GGCTAGTAATAGCCT) at nucleotide position 228 within the UL35 ORF (nucleotide position 79145 of the HCMV genome), a linear PCR construct was generated using pori6K-RIT [31
] as template and the primers UL35stopEPfor: 5′-GGAGGCCCTGGTGGACTTCCAGGTGCGCAACGCTTTTATGGGCTAGTAATAGCCTAAGGTAAAGCCCGTGGCCCA
gacgcatcgtggccggatctc-3′ and UL35stopEPrev: 5′-TGCAGATACGGATAATCTCCTGGGCCACGGGCTTTACCTTAGGCTATTACTAGCCCATAAAAGCGTTGCGCACCT
gtgaccacgtcgtggaatgc-3′. HCMV specific sequences are underlined and the stop cassette is shown in bold. The PCR product was purified and transformed in GS1783 bacteria harboring TB40/E-BAC4 [30
]. Afterwards, the two-step recombination process was carried out essentially as described in [29
]. The resultant UL35stop HCMV BAC was fully sequenced by paired-end sequencing using the MiSeq system from Illumina.
2.5. HCMV Reconstitution
For virus reconstitution, endotoxin-free HCMV BAC DNA (TB40/E-BAC4 wildtype and UL35stop) was isolated from E. coli strain GS1783 using the NucleoBond® Xtra Midi EF kit (Macherey-Nagel, Düren, Germany). Isolated BAC DNA was transfected into MRC-5 cells using JetPEI (Polyplus transfection, Illkirch, France) and cells were cultivated until cytopathic effect was visible in the majority of cells. Infectious supernatant was used to infect HFF-1 seeded in T25 flasks. Approximately 10 days post infection, infectious HCMV containing supernatant was diluted with fresh HFF-1 medium and used to infect two T175 flasks containing 4 × 107 HFF-1 cells. Approximately 12 days later, supernatant and cells were removed and centrifuged at 2465× g to remove cell debris. Supernatant was further subjected to centrifugation at 26,000× g for 3 h at 4 °C to pellet virus. Subsequently, virus particles were purified on a Nycodenz cushion by centrifugation at 46,000× g for 3 h at 4 °C. Purified, pelleted virus was resuspended in VSB buffer (50 mM Tris-HCl, pH 7.8, 12 mM KCl, 5 mM Na2EDTA), aliquoted and stored at −70 °C.
2.6. HCMV Titration
HCMV titers were determined by standard plaque assay on HFF-1 cells. In short, HFF-1 seeded in 48-well plates were infected with serial dilutions of HCMV virus and overlaid with carboxymethylcellulose (CMC)-DMEM medium (DMEM supplemented with 11.64 g/L CMC, 5% FCS, 2 mM glutamine, 1× P/S, 0.37% NaHCO3 and 0.351% D-(+)-glucose). Plaque formation was monitored and counted approximately 14 days post infection. Titers were expressed as PFU/mL and used to calculate the MOI for infection experiments.
2.7. Immunolabeling of HCMV IE1 Antigen
IE1 immunolabeling was routinely performed to ensure equal input of HCMV WT and UL35stop in infection experiments and to determine HCMV titers from growth curve experiments. HFF-1 cells (5,000 cells per well of a 96-well plate) were infected with serial dilutions of HCMV virus stocks or supernatant from infected cells. 48 h post infection, cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 min. Fixed cells were permeabilized with 0.1% Triton X-100 for 5 min and blocked with PBS containing 5% FCS and 1% BSA for 30 min. Next, cells were immunolabeled with mouse anti-IE1 antibody, washed with PBS and subsequently labeled with secondary anti-mouse AF488 conjugated antibody. IE1 positive nuclei were counted using an IncuCyte S3 (Essen BioSciences, Sartorius, Göttingen, Germany) and resulting titers were calculated and expressed as IE1+ cells/mL.
2.8. Growth Curve
HFF-1 cells were seeded onto 48-well plates (35,000 cells per well) and infected with HCMV WT or UL35stop at an MOI of 0.1. At 0, 3, 5, 7, 9, 11, or 13 days post infection, supernatants were harvested and freshly seeded HFF-1 were infected with serial dilutions of the harvested supernatants to perform IE1 immunolabeling. 48 h post infection, cells were immunolabeled for IE1 as described above to calculate HCMV titers at the individual time points.
2.9. Luciferase-Based Reporter Assays
For all reporter assays, 25,000 HEK293T cells were seeded per well in 96-well plates.
cGAS-STING assay: HEK293T cells were transiently transfected with 120 ng plasmid of interest or empty vector (pcDNA3.1+), 60 ng of pEFBOS-cGAS (stimulated) or pIRES2-GFP (unstimulated), 60 ng pEFBOS-mCherry-STING, 100 ng pGL3basic IFNβ-Luc, 10 ng pRL-TK and 1.2 μL of Fugene HD (Promega) diluted in a total volume of 10 µL Opti-MEM (Thermo Fisher Scientific).
MAVS assay: HEK293T cells were transiently transfected with 5 ng pFLAG-CMV2-MAVS (stimulated) or pcDNA3.1+ (unstimulated) together with 100 ng pGL3basic IFNβ-Luc, 10 ng pRL-TK, and 50 ng plasmid of interest complexed with 0.6 μL FuGENE HD in 10 µL Opti-MEM.
RIG-I N: HEK293T cells were transiently transfected with 13 ng pCAGGS Flag-RIG-I N (stimulated) or pcDNA3.1+ (unstimulated) together with 50 ng pGL3basic IFNβ-Luc, 5 ng pRL-TK, and 130 ng plasmid of interest complexed with 0.66 μL FuGENE HD in 10 µL Opti-MEM.
TBK1 assay: HEK293T cells were transiently transfected with 100 ng pcDNA3-FLAG-TBK1 or 100 ng pIRES2-GFP (unstimulated), 100 ng pGL3basic IFNβ-Luc, 10 ng pRL-TK, 120 ng plasmid of interest, and 1 μL FuGENE HD diluted in 10 µL OptiMEM.
IRF3-5D: HEK293T cells were transiently transfected with 120 ng plasmid of interest, 60 ng of IRF3-5D (stimulated) or pIRES2-GFP (unstimulated), 100 ng pGL3basic-IFNβ-Luc, 10 ng pRL-TK and 1 μL of Fugene HD diluted in 10 µL Opti-MEM.
Cells from cGAS-STING, RIG-I N, MAVS, TBK1 and IRF3-5D reporter assays were lysed in 1 × passive lysis buffer (PLB) (Promega) 20 h post transfection.
IFNβ/ISG56 assay: HEK293T cells were transiently transfected with 120 ng plasmid of interest or pcDNA3.1+, 100 ng pGL3basic-ISG56-Luc, 10 ng pRL-TK and 0.8 μL of Fugene HD diluted in Opti-MEM. 24 h post transfection, cells were stimulated with 0.1 ng/mL recombinant human IFNβ (PeproTech, #300-02BC) or mock stimulated and lysed 16 h later in 1 × PLB.
Luciferase production was measured with the dual-luciferase reporter assay system (Promega) and the Tecan Infinite® 200 Pro microplate luminometer (Tecan, Männedorf, Switzerland). Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples.
2.10. siRNA Knockdown
Briefly, 15,000 HEK293T cells were combined with 50 nM siRNA complexed with 0.3 µL Lipofectamine 2000 and simultaneously seeded into 96-well plates. 48 h later, cells were transfected for luciferase reporter assays as described and luciferase activity was measured 20 h later. The following siRNAs were obtained from Dharmacon: ON-target plus non-targeting pool (#D-001810-10-05), SMARTpool siGENOME human OGT (#M-019111-00-0005), SMARTpool siGENOME human DCAF1 (#M-021119-01-0005), SMARTpool siGENOME human CUL4A (#M-012610-01-0005).
For the immunoblotting of luciferase samples, 20 µL of lysates (prepared in 1 × PLB) were boiled for 10 min at 95 °C in SDS sample buffer and subjected to SDS-PAGE. For immunoblotting of HCMV viral particles, 5 × 104 PFU from purified virus preparations were heated in SDS sample buffer and separated by SDS-PAGE. For analysis of HCMV protein expression levels, 100,000 HFF-1 (seeded in 24-well plates) were infected with either HCMV WT or UL35stop at an MOI of 0.1. Infection was enhanced by centrifugation at 800× g for 45 min at room temperature (RT). Afterwards, cells were washed with HFF-1 medium and incubated for the indicated time points until lysis. Whole cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors. For kinetic analysis of phosphorylated proteins, HFF-1 pMSCV or pMSCV UL35 were seeded into 24-well plates (100,000 cells per well) and were mock treated or infected with HCMV UL35stop at an MOI of 0.1 with 15 min enhancement via centrifugation (700× g, RT). Cells were further incubated for 30 min before the supernatant was exchanged with fresh HFF-1 medium. Cells were lysed at indicated time points in lysis buffer containing 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% IGEPAL CA-630, 1% sodium deoxycholate, 0.1% SDS, protease inhibitors and phosphatase inhibitors. For ISD stimulation, cells were seeded as described for UL35stop infection and either Lipofectamine 2000 containing OptiMEM was added (unstimulated control) or 10 µg/mL ISD complexed with Lipofectamine 2000 in OptiMEM. Cells were lysed at indicated time points.
Cell lysates were cleared by centrifugation at 17,000× g following separation by SDS-PAGE and transfer onto nitrocellulose or PVDF membranes (both GE Healthcare). Primary antibodies were added as indicated followed by incubation with secondary horseradish peroxidase (HRP) coupled antibodies. Membranes were developed with Lumi-Light (Roche Applied Science) or SuperSignal West Femto (Thermo Scientific) chemiluminescence substrates and imaged on a ChemoStar ECL Imager (INTAS, Göttingen, Germany).
For co-immunoprecipitation (coIP) experiments, 850,000 HEK293T cells were transiently transfected with 3 µg total DNA complexed with 12 µL PEI diluted in a total volume of 200 µL PBS. 24 h post transfection, cells were lysed in IP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630 (NP-40 replacement), 0.25% sodium deoxycholate, 0.1% SDS) freshly supplemented with protease inhibitors for 3 h at 4 °C on a rotator. 10% of the lysate was used as input control, and the remaining IP fraction was pre-cleared with protein A agarose beads (IPA300, Repligen) for 1 h. Cleared lysates were then incubated overnight with respective antibodies at 4 °C before addition of protein A agarose beads for 1 h. Beads were washed 7 times with IP lysis buffer and bound protein was eluted by heating samples in SDS sample buffer. Input lysates and IP samples were then analyzed by immunoblotting.
2.12. Subcellular Fractionation
HFF-1 were seeded at a density of 200,000 cells into a 12-well plate. The next day, cells were infected with HCMV WT or UL35stop at an MOI of 0.5 by centrifugal enhancement for 45 min at 800× g and RT. After centrifugation, cells were washed with HFF-1 medium and incubated until fractionation at 37 °C and 7% CO2. At the indicated time points, cells were collected in tubes containing 300 μL cold PBS and centrifuged at 10,416× g for 10 s at 4 °C. Pellets were lysed with 300 μL ice-cold 0.1% IGEPAL CA-630 in PBS. 45 μL of the lysate were removed and combined with 15 μL of 4× SDS sample buffer and designated as the whole cell lysate (WCL). The remaining lysate was centrifuged at 16,873× g for 10 s at RT and 45 μL of supernatant were added to 15 μL 4× SDS sample buffer and designated as the cytosolic fraction (C). The pellet was washed with 300 μL 0.1% IGEPAL CA-630 in PBS followed by centrifugation at 16,873× g for 10 s at 4 °C. The pellet was resuspended in 30 μL of 1× SDS sample buffer and designated as the nuclear fraction (N). WCL and N fractions were sonicated using a Bioraptor device at high power settings (30 sec on/30 sec off, 6 cycles). For immunoblotting, 10 μL of the WCL and C fractions and 5 μL of the N fraction were loaded per lane on SDS gel.
2.13. Proteomic Analysis
For proteomic analysis, 1 × 106
HFF-1 stably expressing pMSCV or pMSCV UL35 were left untreated or stimulated with 10 µg/mL poly(I:C) complexed with Lipofectamine 2000. Four hours later, cells were washed with cold PBS and harvested. Cells were pelleted (300× g
, 5 min), shock frozen in liquid nitrogen and kept at –80 °C until analysis. All samples were generated in quadruplicates. Cell pellets were thawed on ice for 30 min and 60 µL 10× guanidinium choride buffer (6 M guanidinium chloride, 10 mM TCEP, 40 mM CAA and 0.1 M Tris-HCl pH 8) were added for an additional 30 min. Samples were heated at 99 °C for 15 min with gentle agitation (500 rpm) and sonicated for 5 min at 4 °C and high frequency (Bioruptor). After centrifugation at 15,000× g
for 30 min at 4 °C, the protein concentration in the supernatant fraction was measured on a Nanodrop™ 1000 spectrophotometer. Subsequently, 50 µg protein were used and resuspended in 20 µL of 0.1 M Tris-HCl pH 8. Proteins were digested into peptides with 1 µg LysC (Wako, Neuss, Germany #129-02541) for 3 h at 37 °C and 0.5 µg Trypsin (Promega # V5113) overnight at 30 °C. Peptides were purified and concentrated on stage tips with three C18 Empore filter discs (3M, Neuss, Germany) and analyzed by mass spectrometry as described before [32
]. Briefly, proteomes were measured via LC-MS/MS, using an EASY-nLC 1200 system (Thermo Fisher Scientific) coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Peptides were loaded on a 20 cm reverse-phase analytical column (75 μm column diameter; ReproSil-Pur C18-AQ 1.9 μm resin; Dr. Maisch, Ammerbuch, Germany) and separated using a 120 min acetonitrile gradient. Raw files were processed using MaxQuant version 188.8.131.52 with label-free quantification (LFQ) and Match between Runs options as described before [32
]. Using Perseus version 184.108.40.206., protein groups were filtered for reverse identification, modification site only identification, MaxQuant contaminants list and if not identified in three over four technical replicates in at least one condition. Significant protein expression changes between UL35 expressing cells and their corresponding empty vector control were determined by a two-sided Student’s t
= 0.1) and corrected for multiple hypothesis testing using permutation-based FDR statistics (FDR = 0.05, 250 permutations).
For all RT-qPCR experiments, 200,000 HFF-1 cells were seeded per well in 12-well plates the day before infection or stimulation.
ISD stimulation: HFF-1 cells stably expressing pMSCV or pMSCV UL35 were mock treated or transfected with OptiMEM containing 2.5 µg/mL ISD complexed with Lipofectamine 2000. Four hours post transfection, RNA was prepared using the innuPREP RNA mini kit 2.0 (Analytik Jena, Jena, Germany) according to the manufacturer’s instructions.
Sendai virus infection: HFF-1 cells stably expressing pMSCV or pMSCV UL35 were mock treated or infected with OptiMEM containing SeV for 1 h. Afterwards, medium was exchanged, and RNA was extracted 6 h post infection.
HCMV infection: Primary HFF-1 infected with either HCMV WT, HCMV UL35stop, or mock treated. To enhance infection efficiency, cells were centrifuged for 45 min at 800× g. Total RNA was extracted 6 h post infection. In parallel, aliquots of diluted HCMV WT and UL35stop virus used for infection were analyzed by IE1 immunofluorescence to control for equal inoculation doses of WT and UL35stop.
IFNβ stimulation: HFF-1 cells stably expressing pMSCV or pMSCV UL35 were mock treated or stimulated with 20 ng/mL human recombinant IFNβ for 6 h. Medium was removed, and RNA extracted from infected cells.
For all RT-qPCR experiments, genomic DNA was removed using the DNA-free kit (Ambion, Thermo Fisher) and 200 ng total RNA was used to generate cDNA (iScript cDNA synthesis kit-BioRad, Feldkirchen, Germany) according to the manufacturer’s instructions. Quantification of mRNA transcripts was performed using the GoTaq qPCR Master Mix 2× (Promega) on a LightCycler 96 instrument (Roche). qPCR primers were as follows: hHPRT1 for: 5′-GAACGTCTTGCTCGAGATGTG-3′, hHPRT1 rev: 5′-CCAGCAGGTCAGCAAAGAATT-3′, hIFNB1 for: 5′-TGTGGCAATTGAATGGGAGGCTTGA-3′, hIFNB1 rev: 5′-TCAATGCGGCGTCCTCCTTCTG-3′, hIFIT3 for: 5′-CCTACATAAAACACCTAGATGGT-3′, hIFIT3 rev: 5′-AAGTGATAGTAGACCCAGGCGT-3′, hIFIT1 for: 5′-CACCATTGGCTGCTGTTTAG-3′, hIFIT1 rev: 5′-CTCCTCTGAGATCTGGCTATTC-3′. Relative fold inductions were calculated using the 2−ΔΔCt method.
2.15. MSD Multiplex Assay
HFF-1 cells seeded into 12-well plates (200,000 cells per well) were infected with HCMV WT or UL35stop at an MOI of 0.5 by centrifugal enhancement (800× g, 45 min, RT). Cells were incubated for 2 h at 37 °C, 7 Vol% CO2 before medium was exchanged with fresh medium. Supernatants were harvested 24 h and 48 h post infection and used to measure secreted IFNβ using the U-Plex human interferon combo kit (Meso Scale Discovery, MSD, Rockville, MD, USA #K15094K) according to the manufacturer’s protocol. The plates were analyzed on a MESO™ Sector 600 using Discovery workbench 4.0.
HFF-1 stably expressing pMSCV or pMSCV UL35-HA were seeded on acid washed glass cover slips in 24-well plates (40,000 cells per well) one day prior immunolabeling. The next day, medium was removed, and cells were fixed with 4 % PFA in PBS for 20 min at RT. Fixed cells were washed with PBS and remaining PFA was inactivated with 50 mM NH4Cl for 10 min. Cells were washed with PBS and permeabilized with 0.4% Triton X-100 for 10 min. After washing with PBS, cells were blocked with 4% BSA for 1 h prior to incubation with primary antibody. For immunolabeling, rabbit anti-HA (C29F4) was added (1:1800) and incubated for 1 h at RT. Labeled cells were washed with PBS prior to addition of secondary anti-rabbit coupled to Alexa Fluor 488 (Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI). Secondary antibody was incubated for 1 h and finally cells were washed with PBS and subsequently embedded in Prolong Gold (Invitrogen).
For infection: 20,000 HFF-1 cells were seeded on µ-slide plastic chambers (ibidi # 80826, Gräfelfing, Germany) one day before infection. The next day, cells were infected with either HCMV WT or HCMV UL35stop at an MOI of 5 for 90 min. Infectious supernatant was removed, cells were washed with fresh HFF-1 medium and incubated for the remaining time at 37 °C, 7 Vol% CO2. At the indicated time points post infection, cells were fixed with PFA and immunolabeled as described above. Primary antibodies against mouse anti-UL35 (1:2 hybridoma supernatant) and rabbit anti-UL82 (1:500) were used for labeling. Secondary anti-mouse AF488 and anti-rabbit AF647 (both obtained from Invitrogen) were combined with DAPI.
Imaging was performed on a Nikon ECLIPSE Ti-E-inverted microscope equipped with a spinning disk device (Perkin Elmer Ultraview). For z-stacks, the integrated piezo drive was used in 5 µm steps. Images including 3D reconstruction were processed using Volocity software (version 6.2.1 and 6.5.1, Perkin Elmer, Hamburg, Germany).
Differences between two data sets were evaluated by Student’s t-test (unpaired, two-tailed) using GraphPad Prism (GraphPad Software, San Diego, CA). p values <0.05 were considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
The ability of CMV to establish a life-long infection in its host has been attributed to its large coding capacity, which expresses a diverse range of immune evasion proteins. Viral manipulation of and escape from PRR-mediated detection of infection leads to a dampened antiviral type I IFN response. CMV tegument proteins are rapidly delivered into infected cells and several have been characterized to immediately exert their immune evasion activity [15
Based on our previous finding that the MCMV tegument protein M35 inhibits the type I IFN response, we addressed whether its HCMV homologue UL35 may have a similar function during HCMV infection. Using quantitative luciferase reporter assays we could confirm that UL35 alone antagonizes IFNβ reporter activation upon PRR-stimulation. Similar to M35, UL35 inhibited cGAS-STING, RIG-I-MAVS, and TBK1-mediated activation of IFNβ transcription. In addition, we show that UL35, like M35, targets signaling upstream of the IFNAR. To our surprise though, UL35 did not inhibit activation of the IFNβ reporter following stimulation with the constitutively active IRF3-5D mutant. This is a clear difference to M35 and led us to conclude that UL35 and M35 follow different mechanisms to downmodulate PRR-mediated type I IFN induction.
By generating a UL35-deficient HCMV, UL35stop, and a mouse monoclonal antibody directed against the C-terminus of UL35, we were able to detect and verify expression and localization dynamics of endogenous UL35. When we followed the subcellular dynamics of UL35 during infection, we detected UL35 in the nucleus as early as 1.5 h upon infection. However, nuclear translocation was not complete as a smaller fraction of UL35 was still present in the cytoplasm throughout the infection. This observation raises the possibility that both the cytoplasm and the nucleus are potential sites of activity for UL35. When we compared the IFNβ response induced by HCMV WT and UL35stop in primary human fibroblasts, we could confirm the antagonistic function of UL35 in the context of infection.
To better understand the molecular mechanism of how UL35 modulates the type I IFN response, we first generated HFF-1 cells stably expressing UL35 and confirmed its antagonistic function upon PRR stimulation. To gain insights into the global proteome change upon PRR stimulation of UL35-expressing fibroblasts, we performed whole cell proteomics and could recover 2,194 proteins. We compared stimulated empty vector control cells to stimulated UL35-expressing cells and focused on proteins that are involved in IFN signaling. Several important proteins of the IFN signaling pathway were not recovered, most likely due to sensitivity limits. Nevertheless, we observed reduced expression of multiple proteins described to be induced by IFN signaling, such as IFI16, IFIT3 and IFIT2, in UL35 expressing cells. Interestingly, there was a significant reduction of the minichromosome maintenance (MCM) complex in UL35-expressing cells independent of poly(I:C) stimulation. The MCM complex is known to be an important factor for host DNA replication and modulates the cellular response to DNA double strand breaks [54
]. Several HCMV proteins including IE2 [56
] and pUL69 [57
] target the host DNA replication machinery to force viral DNA replication. In addition, Qian and colleagues reported that the HCMV protein pUL117 inhibits the accumulation of MCM proteins to reduce cellular DNA synthesis [58
]. Accordingly, our observation raises the question of whether UL35 contributes to the disruption of cellular DNA replication by downmodulating MCM proteins.
Salsman et al. (2012) previously used a mass spectrometry approach to identify cellular interaction partners of transiently transfected UL35 in HEK293T cells and described a role for UL35 in the DNA damage response [22
]. DCAF1 was identified as a direct interaction partner of UL35 and shown to associate with UL35 nuclear bodies. In the same study, it was also reported that UL35 induces the accumulation of cells in the G2 cell cycle phase and activates the DNA damage and repair response in a DCAF1-dependent manner. To evaluate whether the interaction of UL35 with DCAF1 contributes to its inhibition of the IFNβ response, we performed an siRNA-mediated knockdown of DCAF1 and observed that UL35 could still inhibit IFNβ induction. In addition, we also depleted Cullin-4A (Cul4A), the scaffold protein of the Cul4ADCAF1
E3 ubiquitin ligase complex. Depletion of Cul4A also did not influence the inhibition of IFNβ transcription by UL35. Taken together, we conclude that the antagonistic function of UL35 on the IFN response is unrelated to its interaction with DCAF1.
We next assessed another reported interaction partner of UL35, the O-GlcNAc transferase (OGT), for its role in UL35-mediated IFNβ inhibition. OGT was identified as UL35 interaction partner by Salsman and colleagues [22
]. OGT is a ubiquitous protein involved in many cellular processes including transcription, translation and stress responses [59
]. Interestingly, the inhibition of OGT was reported to result in reduced HCMV titers [60
]. OGT reversibly modifies serine and threonine residues of proteins with O-GlcNAc which is akin to phosphorylation events [61
]. Recently, O-GlcNAcylation of MAVS was reported to be essential for the activation of type I IFN signaling in response to RNA viruses [41
]. In addition, Yang et al. showed that O-GlcNAcylation of the NF-κB subunit p65 decreased the binding to IκBα and increased the transcriptional activity of NF-κB [40
]. As OGT is progressively linked to host antiviral immunity, we investigated whether the interaction between UL35 and OGT was required to antagonize the IFNβ response. Knockdown of OGT resulted in a dramatic decrease of the type I IFN response in empty vector and UL35-expressing cells, confirming its crucial role for antiviral signaling, but prohibiting us from concluding that OGT is needed by UL35 to inhibit IFNβ activation.
We were, however, able to show that UL35 is GlcNAcylated between amino acid positions 534 to 553. To our knowledge, this is the second HCMV protein reported to be modified by OGT besides ppUL32 [63
]. Moreover, the interaction with OGT and its GlcNAcylation was abolished by alanine substitution of serine and threonine residues of UL35 between amino acid position 529 and 553. As all UL35 alanine substitution mutants showed comparable inhibition of the IFNβ luciferase reporter, we concluded that OGT is not required for the antagonistic function of UL35. So why does UL35 interact with OGT? A possible scenario may be that UL35 sequesters OGT to enhance viral replication, since several cellular transcription factors involved in the regulation of the HCMV major immediate early promoter (MIEP) are modified by OGT [40
]. Our mapping studies have opened up future paths to reveal the role of the UL35-OGT interaction during HCMV infection.
To this point, we have provided evidence that UL35 downmodulates the PRR-mediated IFNβ response downstream or at the level of TBK1 but upstream of the IFNAR, and we showed that DCAF1 and OGT do not contribute to its antagonistic effect. We further analyzed whole cell extracts of TBK1 transfected HEK293T cells and observed that in the presence of UL35, phosphorylation of TBK1 and IRF3 was remarkably reduced, mirroring the strong downmodulation in the TBK1-stimulated luciferase assay. Since IRF3-5D transfection stimulates IFNβ transcription independent of TBK1 activation or IRF3 phosphorylation, and HEK293T cells lack STING, these data suggested that UL35 may interact with TBK1 to interfere with IFNβ induction. Indeed, co-immunoprecipitation experiments showed that UL35 interacted with TBK1 in transiently transfected HEK293T cells. Subsequent experiments in fibroblasts stably expressing UL35 clearly showed that the presence of UL35 in HCMV-infected and ISD-stimulated fibroblasts led to a reduction of phosphorylated TBK1, STING, IRF3, and consequently, the downstream transcription factor STAT1.
Based on our findings we propose that the tegument protein UL35 is delivered into infected cells upon virion entry and binds to TBK1 in the cytoplasm, which acts downstream of both PRR cGAS and RIG-I, thereby interfering with activation of the STING-TBK1-IRF3 and MAVS-TBK1-IRF3 axis to antagonize IFNβ activation (Figure 8
). While described functions of nuclear UL35 include the activation of the DNA damage response [22
] or the activation of the HCMV MIEP [20
], we have uncovered a novel role for tegument-derived cytoplasmic UL35. Up to now, several herpesviral proteins have been described to antagonize PRR-mediated signaling at the level of TBK1 [13
]. For example, HSV-1-encoded UL46 (VP11/12) binds TBK1 and prevents TBK1 oligomerization [66
]. Furthermore, HSV-1 ICP27 has been shown to bind to the activated STING-TBK1 signalosome to prevent IRF3 phosphorylation [26
]. In addition, HSV-1 US11 binds to Hsp90 and promotes the proteasomal degradation of TBK1 [67
]. Through this study, we have added to the multifunctional role UL35 plays during HCMV infection by identifying it as the first HCMV protein that associates to TBK1, thereby enriching our understanding of the many mechanisms HCMV employs to evade the immune response to establish a successful infection in the host.