E2F/Rb Family Proteins Mediate Interferon Induced Repression of Adenovirus Immediate Early Transcription to Promote Persistent Viral Infection

Interferons (IFNs) are cytokines that have pleiotropic effects and play important roles in innate and adaptive immunity. IFNs have broad antiviral properties and function by different mechanisms. IFNs fail to inhibit wild-type Adenovirus (Ad) replication in established cancer cell lines. In this study, we analyzed the effects of IFNs on Ad replication in normal human cells. Our data demonstrate that both IFNα and IFNγ blocked wild-type Ad5 replication in primary human bronchial epithelial cells (NHBEC) and TERT-immortalized normal human diploid fibroblasts (HDF-TERT). IFNs inhibited the replication of divergent adenoviruses. The inhibition of Ad5 replication by IFNα and IFNγ is the consequence of repression of transcription of the E1A immediate early gene product. Both IFNα and IFNγ impede the association of the transactivator GABP with the E1A enhancer region during the early phase of infection. The repression of E1A expression by IFNs requires a conserved E2F binding site in the E1A enhancer, and IFNs increased the enrichment of the E2F-associated pocket proteins, Rb and p107, at the E1A enhancer in vivo. PD0332991 (Pabociclib), a specific CDK4/6 inhibitor, dephosphoryles pocket proteins to promote their interaction with E2Fs and inhibited wild-type Ad5 replication dependent on the conserved E2F binding site. Consistent with this result, expression of the small E1A oncoprotein, which abrogates E2F/pocket protein interactions, rescued Ad replication in the presence of IFNα or IFNγ. Finally, we established a persistent Ad infection model in vitro and demonstrated that IFNγ suppresses productive Ad replication in a manner dependent on the E2F binding site in the E1A enhancer. This is the first study that probes the molecular basis of persistent adenovirus infection and reveals a novel mechanism by which adenoviruses utilize IFN signaling to suppress lytic virus replication and to promote persistent infection.

cells were seeded on glass coverslips and infected as described above. At appropriate time points, the cells were fixed with ice-cold methanol and processed for immunofluorescence as described [6]. Images were captured and analyzed using a Zeiss Axiovert 200M digital deconvolution microscope with AxioVision 4.8.2 SP3 software.

Electrophoretic mobility shift assays (EMSA)
HDF-TERT cells were incubated with IFNs or left untreated for 24 hr, and nuclear extracts were prepared as described previously [7]. EMSA binding reactions were performed in a total volume of 15 µl containing 20 mM HEPES, pH 7.9, 5 mM MgCl 2 , 50 mM KCl, 1 mM DTT, and 6% glycerol. 4 µg of each nuclear extract was incubated with 250 ng sonicated salmon sperm DNA, with or without 0.2 µg specific antibody, for 15 min at room temperature. 10 fmol (80,000 cpm) of 32 P-labeled probe was added to each reaction and incubated for an additional 30 min at room temperature. DNA-protein complexes were resolved in a native polyacrylamide gel. The gels were dried and autoradiographed. The E1A-ENH probe, 5'-GGTTCCATTTTCGCGGGAAAACTGCCGC-3', corresponds to Ad5 nt 271-288.
Replication-deficient retroviruses were generated by cotransfection of 293FT cells (Life Technologies) with helper virus and pVSV-G in combination with the respective pSIREN-RetroQ plasmids. Retrovirus-containing medium was repeatedly collected at 2 days, 3 days and 4 days post-transfection. For double and triple depletion of PML-NB components, lentiviral vectors, pLKO-shDP/shPS/shDPS, as well as a scrambled negative control, pLKO-shneg were used [10]. Lentivirus stocks were prepared as previously described [10]. To deplete endogenous IFI16 protein, scrambled shRNA in pLKO-shneg was replaced with shRNA against IFI16. shRNA target sequences against IFI16 were obtained from the RNAi consortium (TRC) shRNA library, siIFI16-1, GATCATTGCCATAGCAAATT; siIFI16-3, GGAAACTCTGAAGATTGATT. Low passage HDF-TERT cells were transduced with the retrovirus or lentivirus supernatants in the presence of 7.5 µg/ml polybrene (Sigma-Aldrich, Germany) overnight. To increase the transduction efficiency, HDF-TERT cells were reinfected with retroviruses or lentiviruses at day 2 and day 3. Stably-transduced pools of cell populations were selected using 2 µg/ml puromycin. At least two independent pools of cells with efficient knowdown of individual target genes were generated and analyzed.

Western blot analysis
Whole cell extracts were prepared by suspending cell pellets in SDS lysis buffer (50 mM Tris-HCl, pH 6.5, 4% SDS) and boiled for 10 min. The samples were clarified by centrifugation and the protein concentration was determined using a Bicinchoninic Acid (BCA) kit (Pierce). Equal amounts of whole cell extracts were resolved on SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with Tris-HClbuffered saline (TBS) buffer containing 3% BSA for 1 hr at room temperature, followed by incubation with primary antibodies (as indicated in the text and figure legends) at 4°C overnight. Membranes were washed with TBS buffer containing 0.1% Tween-20 (TBS-T) and then incubated with IRDye ® 800CW-conjugated goat anti-rabbit antibody (926-32211, Li-COR) and IRDye ® 680RD-conjugated goat anti-mouse antibody (925-068071, Li-COR) for 1 hr at room temperature. After three wash with TBS-T, images was captured using the ODYSSEY ® CLx infrared imaging system (Li-COR). Alternatively, HRP-conjugated secondary antibodies were used in conjunction with ECL western blotting (Millipore Immobilon).

Cell fractionation
For the preparation of isolated nuclei for qPCR, cells were washed twice with icecold PBS, harvested, resuspended in 1 ml ice-cold isotonic buffer containing NP-40 (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1.5 mM MgCl 2 , 0.6% NP-40), and incubated on ice for 10 min. Nuclei were pelleted by centrifugation at 2,000 x g for 10 min at 4 °C. Nuclear and cytoplasmic fractions for Western blot analysis were prepared using NE-PER TM nuclear and cytoplasmic extraction kit (Pierce).