Animals and primary cell cultures
C57BL/6J (BL6) mice were obtained from Jackson Laboratories (Bar Harbor, Maine). Mice were housed and bred in a specific-pathogen-free barrier facility in accordance with federal and institutional guidelines. All experimental manipulations of mice were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. Bone marrow was harvested from mice between 7 and 10 weeks of age. Primary bone marrow macrophages and murine embryonic fibroblasts were generated and infected as previously described (Tarakanova et al., 2007
Viral stock preparation
NIH 3T12 cells were infected with wild type MHV68 (WUMS clone, NCBI U97553) at MOI=0.05. At 7 days post infection cells and media were collected by freeze-thawing twice and centrifuged at 1000×g for 20m at 4°C to pellet cell debris. Supernatant was transferred to new tubes and centrifuged at 12,000×g for 2h at 4°C. Pellet was resuspended in buffer with 40 U DNase (Ambion, Austin, TX) and incubated at 37°C for 1h. DMEM containing 10% FBS was added following DNAse digestion and virus stock preparation centrifuged at 12,000×g for 2h. Pellet was resuspended in DMEM supplemented with 10% FBS, aliquoted, and stored at −80°C.
Virus infections and cidofovir treatment
Wildtype MHV68 was titered on NIH 3T12 cells as previously described (Weck et al., 1996
). Bone marrow-derived macrophages were infected at the indicated multiplicities of infection for 1 hr at 37°C and 5% CO2
to allow for adsorption and washed twice with PBS prior to replenishment of medium. Viral DNA synthesis was inhibited by maintaining macrophages in 1 μg/ml cidofovir (Gilead, Foster City, CA) immediately following adsorption.
Infected cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature, and cross linking was quenched with 125mM glycine. Cells were washed three times with cold PBS, collected in 3 mL PBS by scraping, and pelleted at 4°C. Cell pellet was resuspended in 2 mL lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with Halt protease cocktail (Thermo Scientific, Rockford, IL) and sonicated to produce approximately 500-bp fragments (Cell Disruptor 185, Branson Sonifier, Danbury, CT). Sonicated samples were cleared of debris by centrifugation and portion of supernatant was set aside for analysis of input DNA. Chromatin was precleared with 80 μL protein G sepharose beads (Invitrogen, Carlsbad, CA) for 1h at 4°C. Precleared chromatin was diluted 10-fold in the immunoprecipitation buffer (0.01% SDS, 1.1% Triton-10X, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, and 176 mM NaCl, 10 μL Halt protease inhibitor cocktail [Thermo Scientific, Waltham, MA]), immunoprecipitated with 2 μg anti-histone H3 (AbCam, Cambridge, MA) or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) overnight, and incubated with 60 μL protein G for 2h at 4°C. Immunoprecipitates were washed at 4°C with buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl pH 8.1) containing low salt (150 mM NaCl) and high salt (500 mM NaCl). The samples were subsequently washed with LiCl buffer (0.25% LiCl, 1% IGEPAL-CA630, 1% sodium deoxycholate, 2 mM EDTA, 10 mM Tris-HCl pH 8.1) and twice with Tris-EDTA buffer (2 mM EDTA, 10 mM Tris-HCl pH 8.1). Chromatin was eluted twice by incubating immunoprecipitates with elution buffer (100 mM NaHCO3, 1% SDS, 10 mM DTT) at room temperature for 15 minutes. Cross links of immunoprecipitated and input samples were reversed by treatment with NaCl overnight at 65°C. Proteins were cleared by proteinase K treatment at 45°C for 1h, and DNA was purified using GeneJET PCR purification kit (Fermentas, Glen Burnie, MD). Gene-specific DNA was amplified utilizing 280 nM primers directed against GAPDH (Forward: 5′-TGT-GAT-GGG-TGT-GAA-CCA-CGA-GAA-3′; Reverse: 5′-GAG-CCC-TTC-CAC-AAT-GCC-AAA-GTT-3′), core gene 50 promoter (Forward: 5′-TTT-AGC-ATC-TGC-CCG-ACC-TGA-GA-3′; Reverse: 5′-AAT-GGA-CCT-TGA-AAC-CCG-TGA-AGG-3′), distal gene 50 promoter (Forward: 5′-AGG-TGG-TGT-TGG-GTT-AGT-ACA-GCA-3′; Reverse: 5′-TAG-TGA-CAG-GTA-AAG-CAT-AGC-CTG-GG-3′), origin of lytic replication (Forward: 5′-GTG-TGG-CCT-TTG-TGT-GCC-TGT-AAA-3′; Reverse: 5′-AAA-TCG-GTT-TGC-GGT-TAG-ACC-AGG-3′), and orf57 promoter primers (Forward: 5′-AGA-ACA-GCT-TCG-TGC-TGA-CAA-ACC-3′; Reverse: 5′-TTT-GGT-AAG-CTG-GCC-ACA-GTC-TTG-3′). Amplification of DNA sequences was performed with Bulldog Taq polymerase (Portsmouth, NH) in the presence of SYBR Green (Invitrogen, Carlsbad, CA). PCR conditions used were 95°C for 30s followed by 40 cycles of 95°C for 10s, 57°C for 30s, and 72°C for 30s, performed using BioRad iQ5. Amplification efficiency was determined for all primer sequences using serially-diluted DNA purified from virally-infected cells. Enrichment was calculated using the ΔΔCT method.
Viral DNA quantitation
Input samples from sheared chromatin were probed with primers directed against the core gene 50 promoter and cellular GAPDH and analyzed by quantitative RT-PCR. Relative viral DNA was calculated using ΔCT values comparing GAPDH and core gene 50 promoter sequences, normalizing to 0 hpi () or 1 hpi () or normalizing the value obtained at 30 hpi or 48 hpi with cidofovir treatment to 1 ().
qRT-PCR quantitation of viral messages
Total RNA was harvested, DNAse treated, and reverse transcribed in the presence or absence of the enzyme as described in (Tarakanova et al., 2009
). cDNA and corresponding minus RT reactions were serially diluted (8-fold) and assessed, in triplicate, by real time PCR using iCycler (Bio-Rad, Hercules, CA). Gene 50 specific cDNA was amplified using primers decribed in (Tarakanova et al., 2009
). Delta Ct
method was used to quantify fold difference in the levels of RTA sequences between plus and minus RT reactions.