Adenovirus Sequesters Phosphorylated STAT1 at Viral Replication Centers and Inhibits STAT Dephosphorylation

Abstract

Tyrosine phosphorylation and nuclear translocation of STAT1 indicate activation of interferon (IFN) signal transduction pathways. Here, we demonstrate that tyrosine-phosphorylated STAT1 is targeted by a unique mechanism in adenovirus (Ad)-infected cells. Ad is known to suppress IFN-inducible gene expression; however, we observed that Ad infection prolongs the tyrosine phosphorylation of STAT1 induced by alpha IFN in infected cells. To understand this paradoxical effect, we examined the subcellular localization of STAT1 following Ad infection and found that nuclear, tyrosine-phosphorylated STAT1 accumulates at viral replication centers. This form of STAT1 colocalized with newly synthesized viral DNA. Viral DNA replication, but not viral late gene expression, is required for the regulation of STAT1 phosphorylation. Our results indicate that Ad infection regulates STAT1 dephosphorylation rather than STAT1 phosphorylation. Consistent with this idea, we show that Ad infection disrupts the interaction between STAT1 and its cognate protein tyrosine phosphatase, TC45. Our findings indicate that Ad sequesters phosphorylated STAT1 at viral replication centers and inhibits STAT dephosphorylation. This report suggests a strategy employed by Ad to counteract an active form of STAT1 in the nucleus of infected cells.

INTRODUCTION

Numerous studies have shown the molecular mechanisms employed by adenovirus (Ad) to counteract interferon (IFN)-mediated antiviral responses since virus-associated RNA I (VAI) and the E1A gene products were reported to be IFN antagonists a quarter century ago (2, 15). Ad infection inhibits both type I IFN (e.g., alpha/beta IFN [IFN-α/β]) and type II IFN (IFN-γ) signaling and thereby downregulates IFN-stimulated response element (ISRE)-driven) and IFN-γ-activating sequence (GAS)-driven transcription. Signal transducer and activator of transcription 1 (STAT1) is the only transcription factor shared by both IFN signaling pathways.

In response to type I IFNs, STAT1 and STAT2 undergo phosphorylation mediated by receptor-associated kinases, Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2), and form the heterotrimeric IFN-stimulated gene factor 3 (ISGF3) complex, together with IFN-regulatory factor 9 (IRF9), followed by nuclear translocation (reviewed in reference 27). The ISGF3 complex binds and activates the ISRE-containing promoters of IFN-inducible genes. After release from promoter regions, STAT1 is dephosphorylated by a nuclear protein tyrosine phosphatase, TC45, and exported to the cytoplasm (23). IFN signal transduction pathways are activated rapidly but persist only transiently in the absence of additional stimulation; aberrant regulation causes various immune disorders. Accurate regulation of STAT1 activity is crucial for the innate immune response, and it is modulated by various posttranslational modifications, such as tyrosine phosphorylation (38), serine phosphorylation (47), methylation (24), sumoylation (45), and ubiquitination (14). In addition, it was recently reported that acetylation of STAT1 causes recruitment of TC45 and subsequent dephosphorylation (16).

Several negative regulatory mechanisms for STAT1 activity have also been described. STAT1 can be dephosphorylated by specific phosphatases, such as TC45 (41) and Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) (48). Suppressor of cytokine signaling (SOCS) proteins block STAT1 tyrosine phosphorylation by inactivating kinases through proteasomal degradation (46) and also direct inhibition of tyrosine kinase activity (7). Protein inhibitor of activated STAT (PIAS) proteins prevent DNA binding of STAT1 (19). PIAS family members have been shown to promote STAT1 sumoylation; however, the functional consequences are controversial (32, 44, 45). These diverse STAT1 regulatory mechanisms can be exploited by viruses to impair IFN signal transduction pathways and, hence, evade host immune response (reviewed in reference 29).

STAT1 is a stable protein with a half-life of >24 h (17). Ad infection has not been reported to affect the basal protein level of STAT1 in unstimulated cells. In a previous study, E1A proteins were shown to interact with STAT1 and inhibit IFN-γ-inducible gene expression in primary human tracheobronchial epithelial (hTBE) cells (20). Using the same cells, these authors also showed that Ad blocks IFN-α-induced STAT1 tyrosine phosphorylation by downregulating JAK1 expression (37). The E1A proteins were also previously shown to inhibit ISGF3 DNA binding and IFN-stimulated gene expression (1, 2, 9, 13, 18, 31, 35). Ad and other viruses have evolved redundant mechanisms to counteract host antiviral responses. Thus, Ad may have evolved additional strategies to block early stages of an IFN signaling response, since this response is so detrimental to virus infection. This prompted us to investigate if other aspects of Ad infection inhibit IFN-induced antiviral activities.

To further understand the mechanisms by which Ad infection may modulate IFN signaling pathways, we examined tyrosine phosphorylation of STAT1 in Ad-infected HeLa and A549 cells, which are highly competent for virus replication and the induction of an IFN response. Unexpectedly, IFN-α-induced STAT1 tyrosine phosphorylation was prolonged longer in Ad-infected cells than in mock-infected cells, even though Ad infection per se was not able to promote STAT1 phosphorylation. In response to IFN stimulation, nuclear translocated STAT1 stayed longer within the nucleus following Ad infection and accumulated at viral replication centers. By separately blocking different aspects of the viral replication cycle, we found that Ad DNA synthesis, but not late gene expression, is required for prolonged STAT1 tyrosine phosphorylation. In addition, JAK inhibition did not interfere with this effect, indicating that Ad infection modulates STAT1 phosphorylation by inhibiting STAT1 dephosphorylation rather than enhancing phosphorylation. Finally, we observed that Ad infection inhibits the interaction between STAT1 and its cognate protein tyrosine phosphatase, TC45. Our results suggest that Ad sequesters tyrosine-phosphorylated STAT1 in the nucleus at viral replication centers to suppress an IFN-induced antiviral response.

MATERIALS AND METHODS

Cells and viruses.

HeLa human cervical carcinoma and A549 human lung carcinoma cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum. dl309 (phenotypically wild-type Ad type 5 [Ad5]) (12), dl312 (Ad5 E1A-deficient mutant) (12), and H5ts1 (Ad5 L4-100K temperature-sensitive mutant) (10) viruses were purified by CsCl equilibrium centrifugation.

Plasmids.

The expression vector for TC45 substrate-trapping mutant pMT2-TC45 (D182A/C216S) was kindly provided by Nicholas K. Tonks (Cold Spring Harbor Laboratory). pSTAT1-EGFP (enhanced green fluorescent protein) was a gift from Nancy C. Reich (Stony Brook University) and described previously (22).

Antibodies and reagents.

Anti-STAT1, anti-phospho-STAT1 (Tyr701), and anti-phospho-STAT3 (Tyr705) antibodies were purchased from Cell Signaling Technology; anti-STAT3 antibody was from Santa Cruz Biotechnology. The antibodies specific for T cell protein tyrosine phosphatase (TC-PTP [CF4]; Calbiochem), γ-tubulin (Sigma-Aldrich), and 5-bromo-2′-deoxyuridine (BrdU) (Roche) were purchased from the respective suppliers. The monoclonal antibody for adenovirus DNA binding protein (DBP) (B6-8) was provided by Arnold J. Levine (Princeton University). The rabbit polyclonal antipenton antibody was a gift from Carl W. Anderson (Brookhaven National Laboratory). Human IFN-α was purchased from PBL Interferon Source. JAK inhibitor I was from Calbiochem, BrdU was from Roche, and cytosine β-d-arabinofuranoside hydrochloride (AraC) and cycloheximide (CHX) were from Sigma-Aldrich.

Immunofluorescence.

HeLa cells seeded on coverslips in a 24-well plate were transfected with 0.2 μg pSTAT1-EGFP per well using polyethylenimine (PEI; Polysciences). At 24 h posttransfection, the cells were infected with 200 particles/cell of dl309 for 20 h and pulse treated with 500 U/ml IFN-α for 30 min. The cells were washed twice with phosphate-buffered saline and incubated further in fresh medium for the indicated periods of time. Then, HeLa monolayers on coverslips were fixed and permeabilized with methanol, blocked in 10% goat serum for 1 h, and incubated with mouse anti-DBP and rabbit anti-phospho-STAT1 (Tyr701) antibodies for 1 h. The Alexa 350-conjugated antimouse (Molecular Probes) and tetramethyl rhodamine isocyanate (TRITC)-conjugated antirabbit (Zymed) antibodies were used as secondary antibodies. Cell images were acquired on an Axiovert 200 M digital deconvolution microscope (Zeiss) and analyzed using Axiovision software. For BrdU labeling of Ad genomic DNA, 10 μM BrdU was added to the cells 30 min before fixation. Anti-BrdU and anti-phospho-STAT1 (Tyr701) primary antibodies and Alexa 350-conjugated antimouse and TRITC-conjugated antirabbit secondary antibodies were used for immunostaining.

Real-time reverse transcription-PCR.

Viral DNA was isolated using a genomic DNA extraction kit (Qiagen) according to the manufacturer's protocol. The following primers for quantitative PCR were designed using Primer3 software: Ad5 forward (5′-TAATGAGGGGGTGGAGTTTG-3′), Ad5 reverse (5′-GCGAAAATGGCCAAATCTTA-3′), GAPDH forward (5′-GTCAGTGGTGGACCTGACCT-3′), and GAPDH reverse (5′-TGACCAAGTGGTCGTTGAGG-3′). Quantitative PCR was performed on a LightCycler instrument (Roche) using a LightCycler-FastStart DNA master SYBR green I kit (Roche) according to the manufacturer's instruction.

Immunoprecipitation and Western blot analysis.

HeLa cells grown in 10-cm plates were transfected with 3 μg pSTAT1-EGFP and 3 μg pMT2-TC45 (D182A/C216S) per plate using PEI. At 24 h after transfection, cells were infected with 200 particles/cell dl309 and incubated for 24 h. Before harvesting, 500 U/ml IFN-α was added for 3 h. Cell pellets were resuspended in 300 μl lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, and 10 mM NaF) per plate, incubated on ice for 20 min, and further disrupted by passing them through a 21-gauge needle. After centrifugation at 12,000 × g for 10 min, the supernatant was precleared with protein A-Sepharose beads (Sigma-Aldrich) for 1 h and then incubated with monoclonal anti-TC-PTP antibody for overnight, followed by addition of protein A-Sepharose for 3 h. The beads were washed five times with lysis buffer and analyzed by 7.5% SDS-PAGE and Western blotting. Western blot analysis was performed using enhanced chemiluminescence according to the manufacturer's (Immobilon) instructions.

RESULTS

STAT1 tyrosine phosphorylation induced by IFN-α is prolonged in Ad-infected cells.

Although Ad is well-known to suppress IFN-inducible gene expression, Ad infection has not been observed to alter STAT1 protein levels. To further understand the mechanisms by which Ad infection may modulate IFN signaling pathways, we examined tyrosine phosphorylation at residue 701 of STAT1 in Ad5-infected HeLa cells, which are highly competent for virus replication and the induction of an IFN response. We hypothesized that Ad might have evolved a negative regulatory mechanism to inhibit IFN-activated STAT1 protein. We compared tyrosine phosphorylation levels of STAT1 in the presence and absence of Ad infection following a 30-min IFN-α pulse. Unexpectedly, we observed a higher level of tyrosine 701-phosphorylated STAT1 in Ad-infected HeLa cells than in mock-infected cells at 1 to 4 h after treatment with IFN-α (Fig. 1A). Since the phosphorylation and dephosphorylation kinetics of STAT1 are rapid, mock-infected HeLa cells showed a significant decrease in STAT1 tyrosine phosphorylation at 2 h and later after IFN-α treatment, while the tyrosine-phosphorylated status was maintained for more than 4 h after IFN-α induction in Ad-infected cells. A previous report demonstrated that an Ad vector for gene transfer stimulated STAT1 tyrosine phosphorylation in infected macrophages (26). Thus, we tested the possibility that Ad infection alone induces STAT1 phosphorylation. As shown in Fig. 1B, Ad infection alone did not increase STAT1 tyrosine phosphorylation in infected HeLa cells. In addition, an E1A-deleted, replication-defective Ad5 mutant, dl312, did not affect STAT1 tyrosine phosphorylation following IFN-α stimulation (Fig. 1C). We note that STAT1 migrated as a doublet in these immunoblots, which we believe represents the STAT1 alpha and beta isoforms.

Fig. 1.

IFN-α-induced tyrosine phosphorylation of STAT1 is prolonged in Ad5-infected HeLa cells. (A) HeLa cells were mock infected (lanes −) or infected with 200 particles/cell using wild-type Ad5 dl309 (lanes +) for a total of 24 h. At the times indicated before cell harvest (0.5, 1, 2, and 4 h), cells were stimulated with 500 U/ml IFN-α for 30 min. For the 1-, 2-, and 4-h time points, fresh medium was added after the IFN-α pulse and the cells were further incubated for 30 min, 1.5 h, and 3.5 h, respectively. The experimental protocol is indicated in the schematic diagram shown below the immunoblot. Total STAT1 and tyrosine-phosphorylated STAT1 levels were measured by Western blotting using anti-STAT1 and anti-STAT1-phospho-Y701 antibodies. (B) A time course experiment was conducted to examine STAT1 tyrosine phosphorylation (pY) at different times after dl309 infection (8, 16, and 24 h postinfection [hpi]). Cells were left untreated (− IFN-α) or stimulated with 500 U/ml IFN-α for 30 min. Cells were then harvested immediately (30 min) or fresh medium was added after the IFN-α pulse for an additional 60 min prior to cell harvest (90 min). The experimental protocol is indicated in the schematic diagram shown below the immunoblot. (C) Using the same conditions described for panel A, the Ad5 E1A-deficient mutant virus dl312 (E1A⁻) was used in comparison to wild-type (WT) dl309. (D) Titer-dependent effects of Ad infection on STAT1 tyrosine phosphorylation. Using the same conditions described for panel A, different multiplicities of dl309 virus infection (10, 50, 200 particles/cell) were compared.

To confirm these unexpected results, we infected HeLa cells at different multiplicities of infection with Ad and for different periods of time and examined STAT1 tyrosine phosphorylation following IFN-α stimulation (Fig. 1B and D). These results showed that STAT1 tyrosine phosphorylation was prolonged by Ad infection by 16 h, but not 8 h, postinfection (Fig. 1B). This effect was augmented at higher multiplicities of infection (Fig. 1D), although it was observed even at 10 virus particles/cell, corresponding to ∼1 infectious unit/cell. A similar effect was seen in Ad-infected A549 cells compared to mock-infected cells (Fig. 2A and B). Taken together, these results demonstrate that IFN-α-induced tyrosine phosphorylation of STAT1 is sustained following Ad infection of HeLa and A549 cells, even though virus infection itself does not induce STAT1 phosphorylation.

Fig. 2.

IFN-α-induced tyrosine phosphorylation of STAT1 is prolonged in Ad5-infected A549 cells. (A and B) The experiments were conducted as described for Fig. 1A and C, respectively, using dl309-infected A549 cells.

Phosphorylated STAT1 is located at viral replication centers in Ad5-infected cells.

We examined if tyrosine-phosphorylated STAT1 translocated into the nucleus properly in response to IFN-α stimulation in Ad-infected cells. Since a previous report showed dynamic translocation of STAT1 using an EGFP fusion protein (22), we utilized this construct to monitor subcellular localization of STAT1. At 24 h posttransfection with a STAT1-EGFP expression plasmid, the cells were infected with Ad5 for 24 h. Cells were stimulated with IFN-α for 30 min and then incubated in fresh medium for different periods of time and analyzed by fluorescence microscopy for STAT1-EGFP and tyrosine-phosphorylated STAT1 subcellular localization (Fig. 3). Unstimulated cells displayed diffuse localization of STAT1-EGFP and undetectable tyrosine-phosphorylated STAT1 (Fig. 3A to C). At 30 min after IFN-α treatment, STAT1-EGFP translocated into the nucleus in both mock- and Ad-infected cells (Fig. 3H and K), but it remained nuclear at 90 min after IFN-α treatment only in Ad-infected cells (Fig. 3O and R). Interestingly, tyrosine-phosphorylated STAT1 colocalized with viral replication centers, which were visualized using anti-DBP antibody (Fig. 3L to N and S to U). We observed the same localization pattern of endogenous tyrosine-phosphorylated STAT1 in Ad-infected cells (Fig. 4A).

Fig. 3.

Tyrosine-phosphorylated STAT1 colocalizes with viral replication centers. (A) HeLa cells were transfected with a STAT1-EGFP expression vector for a total of 24 h. The cells were then mock infected (Uninf.) (A to C, H to J, and O to Q) or infected with Ad5 (+Ad5) (D to G, K to N, and R to U) for a total of 24 h. Cells were left untreated (−; A to G) or treated with 500 U/ml IFN-α for 30 min and either harvested immediately (30 min; H to N) or incubated in fresh medium for an additional 60 min prior to fixation (90 min; panels O to U). The cells were immunostained for tyrosine-phosphorylated STAT1 using rabbit anti-STAT1-phospho-Y701 antibody and a TRITC-conjugated secondary antibody (pY-STAT1; B, E, I, L, P, and S) and for Ad DBP using a mouse anti-DBP antibody and an Alexa 350-conjugated antimouse secondary antibody (Ad DBP; F, M, and T). STAT1-EGFP was visualized using the EGFP tag (STAT1-EGFP; A, D, H, K, O, and R). Merged images are shown in the right column (Merge; C, G, J, N, Q, and U).

Fig. 4.

Tyrosine-phosphorylated STAT1 colocalizes with newly synthesized viral DNA. (A) Cells were prepared as described in the legend to Fig. 3, except that endogenous STAT1 instead of GFP-tagged STAT1 was visualized. Cells were immunostained for tyrosine-phosphorylated STAT1 (pY-STAT1; subpanels A, D, and G) and Ad DBP (DBP; subpanels B, E, and H). Merged images are shown in the right column (Merge; subpanels C, F, and I). (B) HeLa cells expressing STAT1-EGFP were infected with Ad5 for a total of 20 h. Cells were treated with IFN-α for 30 min and then incubated in fresh medium for 30 min. BrdU was the added to the culture medium for the final 30 min prior to fixation. STAT1-EGFP and tyrosine-phosphorylated STAT1 were detected as described for Fig. 3 for STAT1-EGFP (subpanel A) and pY-STAT1 (subpanel B). BrdU was detected using mouse anti-BrdU antibody and Alexa 350-conjugated secondary antibody (BrdU; subpanel C). A merged image is shown in the right column (Merge; subpanel D). The experimental protocol is indicated in the schematic diagram shown below the images.

A previous study suggested that tyrosine-phosphorylated STAT1 is dephosphorylated after disengagement from DNA (23). Thus, we labeled Ad DNA using BrdU to determine if tyrosine-phosphorylated STAT1 colocalizes with Ad DNA. Cellular DNA replication is inhibited by Ad infection during the late phase of infection; thus, Ad DNA may be selectively labeled and visualized by this approach. As shown in Fig. 4B, BrdU stained regions containing newly synthesized viral DNA colocalized with tyrosine-phosphorylated STAT1 in IFN-α-stimulated cells, suggesting that Ad DNA may be involved in prolonging tyrosine phosphorylation of STAT1. These findings suggest that nuclear, tyrosine-phosphorylated STAT1 may be trapped at viral replication centers in Ad-infected cells.

Viral DNA synthesis is required for sustained STAT1 tyrosine phosphorylation.

To assess the mechanism by which Ad infection modulates tyrosine phosphorylation of STAT1 in response to IFN-α treatment, we utilized traditional strategies to analyze if gene expression and viral DNA replication are required for this process. The time course experiment (Fig. 1B and 2B) already indicated that viral early gene expression was not sufficient to induce sustained STAT1 tyrosine phosphorylation. To analyze if viral DNA replication is required, Ad-infected cells were treated with the nucleoside analog AraC, which allows early gene expression but not viral DNA synthesis and late gene expression (8). AraC treatment significantly reduced the ability of Ad infection to sustain STAT1 tyrosine phosphorylation following IFN-α stimulation (Fig. 5A); a control experiment showed that AraC treatment reduced Ad DNA replication 16-fold under these conditions. Ad-infected cells were treated with the protein synthesis inhibitor CHX from 12 to 18 h postinfection to block late gene expression (11). The cells were then pulsed with IFN-α and incubated for different periods of time to examine the effect on STAT1 tyrosine phosphorylation (Fig. 5B). CHX treatment did not affect the ability of Ad infection to sustain STAT1 tyrosine phosphorylation following IFN-α stimulation, even though viral late gene expression (penton) was significantly reduced by CHX treatment. CHX treatment reduced viral DNA replication 3-fold. To confirm this result, HeLa cells were infected with wild-type Ad5 or with the Ad5 L4-100K temperature-sensitive mutant H5ts1 for 16 h at the nonpermissive temperature (40°C) or for 40 h at the permissive temperature (32°C). At the restrictive temperature, H5ts1 displays a significant reduction in viral late gene expression (10). Infection with the H5ts1 mutant virus resulted in sustained STAT1 tyrosine phosphorylation following IFN-α stimulation at the permissive and restrictive temperatures (Fig. 6A and B). We note that STAT1 phosphorylation was reduced at the 90-min time point at 40°C with both wild-type Ad5 and ts1; we suspect that this effect is due to increased cellular metabolism and turnover of STAT1 phosphorylation at the elevated temperature. We conclude from these results that viral DNA replication is required to sustain tyrosine phosphorylation of STAT1. The results indicate that Ad late protein expression is not required for this effect, although we cannot exclude the possibility that low levels of a late viral protein(s) are sufficient for this process to occur.

Fig. 5.

Viral DNA synthesis is required to sustain STAT1 tyrosine phosphorylation in Ad-infected cells. (A) HeLa cells were infected with dl309 for a total of 24 h in the absence (−) or presence of 20 μg/ml AraC (+). Before harvest, the cells were stimulated with 500 U/ml IFN-α for 30 min and harvested immediately (30 min) or then incubated in fresh medium for an additional 1 h prior to harvest (90 min). Total and tyrosine-phosphorylated STAT1 levels were determined by Western blotting. (B) HeLa cells were infected with dl309 for a total of 18 h. Cells were untreated (−) or treated with 30 μg/ml of CHX from 12 to 18 h after infection (+ CHX). The cells were then stimulated with IFN-α and subsequently processed as described for panel A. Adenovirus penton protein levels were analyzed to monitor viral late gene expression. γ-Tubulin levels were analyzed as a loading control.

Fig. 6.

Viral late gene expression is not required to sustain STAT1 tyrosine phosphorylation in Ad-infected cells. HeLa cells were infected with 200 particles/cell of dl309 or H5ts1 and incubated at the permissive temperature (32°C) for a total of 42 h (A) or the nonpermissive temperature (40°C) for a total of 16 h (B) and then treated with IFN-α and processed as described in the legend to Fig. 1. Western blot analysis was performed using the indicated antibodies.

Ad infection negatively modulates STAT1 dephosphorylation.

On the basis of the result that Ad infection alone does not induce phosphorylation of STAT1 in HeLa cells, we examined if dephosphorylation of STAT1 may be inhibited during Ad infection. To test this, we examined the effect of a JAK inhibitor on STAT1 tyrosine phosphorylation following Ad infection (Fig. 7). Ad-infected cells were treated with a 30-min pulse of IFN-α and then incubated for the indicated periods of time with fresh medium alone (Fig. 7A) or with medium containing the JAK inhibitor (Fig. 7B). Our rationale was that enhanced STAT1 tyrosine phosphorylation following Ad infection would be inhibited by the JAK inhibitor if Ad infection promotes STAT1 tyrosine phosphorylation following IFN-α stimulation, whereas the inhibitor would have no effect if Ad infection interferes with STAT1 dephosphorylation following IFN-α stimulation. By treatment of cells with the JAK inhibitor prior to IFN-α stimulation, we confirmed that this inhibitor completely blocks JAK activity in HeLa cells (data not shown). In the presence of the JAK inhibitor, the duration of STAT1 tyrosine phosphorylation was still enhanced in Ad-infected cells, suggesting that Ad infection impairs the dephosphorylation of STAT1 rather than enhances STAT1 tyrosine phosphorylation.

Fig. 7.

Ad infection modulates STAT1 dephosphorylation. (A and B) HeLa cells were infected with dl309 for a total of 24 h. Cells were left untreated (A) or were treated with 1 μM JAK inhibitor (B) for the times indicated prior to cell harvest (15, 30, 45 min). Cells were treated for 30 min with 500 U/ml IFN-α and then harvested immediately (30 min), or fresh medium was added and the cells were harvested at the times indicated (15, 30, and 45 min after the IFN-α pulse, samples 45, 60, and 75 min, respectively). Total and tyrosine-phosphorylated STAT1 levels were analyzed by Western blotting. (C) Infected cells were treated and processed as described for panels A and B. Total STAT3 and tyrosine-phosphorylated STAT3α (86 kDa; upper panel) and β (79 kDa; lower panel) were analyzed by Western blotting. (D) Schematic diagram of experimental protocol.

This result caused us to consider if the same effect on STAT1 tyrosine phosphorylation following Ad infection would be observed with other shared substrates of STAT1 tyrosine phosphatases. STAT3 has been reported to be a substrate of the tyrosine phosphatase TC45 following phosphorylation by IFN-α treatment (50). Thus, we measured the level of STAT3 phosphorylation at tyrosine 705 using a phospho-specific antibody. As shown in Fig. 7C, tyrosine phosphorylation of STAT3 was also sustained following Ad infection; the JAK inhibitor did not affect this process after 30 min of treatment, as was observed with STAT1, but did moderately reduce STAT3 phosphorylation after 45 min of treatment, although STAT3 phosphorylation was still readily apparent. We did not detect any significant decrease in TC45 protein levels in Ad-infected cells (data not shown), suggesting that Ad infection may impair TC45 activity without altering its expression level.

Ad infection disrupts the interaction between STAT1 and TC45.

To determine if Ad infection inhibits TC45 access to tyrosine-phosphorylated STAT1 in IFN-α-treated cells, we examined the interaction between STAT1 and TC45 using coimmunoprecipitation. Since the kinetics of STAT1 phosphorylation/dephosphorylation is rapid, it is difficult to detect transient binding of STAT1 and TC45 in vivo. Thus, we utilized a TC45 substrate-trapping mutant harboring the mutations D182A and C216S (49). This construct was coexpressed with STAT1-EGFP in HeLa cells, and transfected cells were then infected with Ad5 for 24 h with IFN-α stimulation for the final 3 h before the cells were harvested. The lysates were utilized for immunoprecipitation using a monoclonal anti-TC45 antibody, and the STAT1 interaction was analyzed by Western blotting (Fig. 8). In agreement with a previous report (16), the TC45 substrate-trapping mutant protein coimmunoprecipitated with STAT1 with mock-infected, IFN-α-stimulated cells. However, the interaction between TC45 and STAT1 was significantly reduced by Ad infection, indicating that Ad blocks the binding of tyrosine-phosphorylated STAT1 to its cognate protein tyrosine phosphatase.

Fig. 8.

Ad infection disrupts the interaction between STAT1 and TC45. Plasmids to express STAT1-EGFP and TC45 (D182A/C216S) were cotransfected into HeLa cells. At 24 h posttransfection, cells were mock infected (lanes −) or infected with dl309 for 21 h (lanes +), followed by IFN-α treatment for 3 h. Cell extracts were used for immunoprecipitation (IP) with an anti-TC45 antibody. Samples (IP: TC45, left) were analyzed by Western blotting using either anti-STAT1 or anti-TC45 antibody. Protein levels in cell lysates (Lysate, left) were monitored using 2% of the input sample. STAT1 and TC45 are indicated in the right; asterisks indicate the positions of IgG heavy chain.

DISCUSSION

Ad infection was previously shown to block STAT1 phosphorylation through downregulation of JAK1 (37). We confirmed these results when tyrosine-phosphorylated STAT1 levels were analyzed in Ad5-infected human diploid fibroblasts, IMR90 and HDF-TERT (unpublished data). However, we observed an unexpected STAT1 phosphorylation pattern in Ad5-infected HeLa and A549 cells. Here, Ad infection significantly prolonged the duration of STAT1 tyrosine phosphorylation (Fig. 1 and 2). The basis for this result was difficult to understand since Ad is well-known to repress ISRE-dependent transcription by inactivating the ISGF3 complex (1, 2, 9, 13, 18, 31, 35), and we also observed suppression of type I IFN-inducible genes in Ad-infected HeLa cells. Subcellular localization studies provided an important clue to understand this unexpected finding. During Ad infection, STAT1 was properly phosphorylated on tyrosine residue 701 and translocated to the nucleus in response to IFN-α treatment (Fig. 3 and 4). However, phospho-STAT1 remained phosphorylated for many hours after IFN-α stimulation, in contrast to mock-infected cells, where STAT1 phosphorylation turned over by 2 h after IFN-α treatment (Fig. 1 and 2). Tyrosine-phosphorylated STAT1 localized at viral replication centers in infected cell nuclei (Fig. 3 and 4), and we believe that STAT1 is trapped at these sites and not available for the normal cycle of dephosphorylation by TC45. The latter conclusion is supported by coimmunoprecipitation experiments, where Ad infection blocked STAT1-TC45 interaction following IFN-α stimulation (Fig. 8).

Ad induces the formation of a number of unique nuclear structures, such as E4-ORF3 tracks, viral replication centers, and pIX inclusions, in virus-infected cells. During the early phase of infection, the E4-ORF3 protein organizes nuclear structures which have been implicated in the inhibition of host defense mechanisms: the redistribution of the Mrell-Rad50-Nbs1 (MRN) complex to prevent a DNA damage response and viral genome concatemerization (3, 6, 40) and promyelocytic leukemia (PML) nuclear bodies to impair the IFN-induced antiviral response (4, 5, 43). At the late phase of infection, Ad forms replication centers condensed with viral DNA, viral DNA replication proteins, and various cellular proteins to promote efficient viral DNA replication and late gene transcription (6, 21, 28). Another late-phase-specific nuclear structure is formed by viral intermediate protein pIX (33). Antiviral factors such as PML, double-stranded RNA-activated protein kinase R (PKR), and cellular protein kinase CK2 have been observed to relocate into this structure (34, 39). From our results, we suggest that viral replication centers may also promote the inhibition of a cellular, antiviral response by recruiting activated STAT1 into these structures, which blocks proper STAT1 regulation and may inhibit STAT1-mediated antiviral activity. It is possible that other cellular, antiviral effectors may be inhibited in the same manner.

STAT1 is known to expose phosphorylated tyrosine 701 to its cognate protein tyrosine phosphatase, TC45, when it is released from DNA (23). We visualized newly synthesized viral DNA by BrdU labeling and observed colocalization of Ad DNA with tyrosine-phosphorylated STAT1. Since Ad replication centers contain multiple viral products and recruit diverse cellular proteins, we do not know if STAT1 is trapped in viral replication centers by direct binding to viral DNA. We found that the inhibition of viral DNA replication, but not late gene expression, blocked the prolonged tyrosine phosphorylation of STAT1 seen in Ad-infected cells, suggesting that viral DNA synthesis, or perhaps the mere presence of abundant levels of viral DNA, is required to modulate STAT1. However, it is still unclear whether only viral DNA within the viral replication center retains STAT1 or if other factors are required. This phenomenon was observed in established cancer cell lines such as HeLa and A549 but was not observed in infected human diploid fibroblasts (data not shown). This could suggest that the association of STAT1 at viral replication centers occurs by binding to a cell type-specific cellular protein(s) found at these sites in some, but not all, cells. It would not appear that the viral DNA would be distinct in different infected cell types, although some aspect of how the DNA is organized or condensed could vary in different cells.

Several reports demonstrate ISRE-like sequences within viral promoter regions. Human immunodeficiency virus (HIV) type 1 is shown to have an ISRE-like element and exploit interferon regulatory factors for gene transcription and replication when viral transactivator levels are very low (36). An ISRE-like sequence in the hepatitis B virus (HBV) enhancer I region is also reported to bind IRF9 (25); however, the functional consequences are controversial (25, 30). We used software to search for transcription factor binding sites in the Ad5 genome (TFBIND; http://tfbind.ims.utokyo.ac.jp) (42) and identified over 50 candidate ISRE sites, although most of the highly scored sites are not in Ad promoter regions. If STAT1 indeed binds to the Ad genome, it is unclear whether STAT1 binds to specific ISRE sequences or random DNA sequences or even to single-stranded viral DNA, nor is it clear if Ad recruits STAT1 to block its function or to exploit STAT1 for viral gene transcription.

Since STAT1 plays a critical role in the IFN signal transduction pathway, a balance between phosphorylation and dephosphorylation is important for the innate immune system. For viruses to inactivate STAT1, blocking mechanisms upstream of IFN signaling pathways through targeting receptors or protein kinases would appear to be a more efficient and powerful defense strategy. However, similar to many viruses which block several stages of IFN signaling, our results suggest that under circumstances in which Ad cannot block upstream signals, Ad infection is still able to inhibit IFN signaling by sequestering the active form of STAT1 and, hence, suppress IFN-inducible gene expression.