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Heinrich-Heine-Universität Düsseldorf, Institut für Virologie, 40225 Düsseldorf, Germany
Correspondence
Albert Zimmermann
albert.zimmermann{at}uni-duesseldorf.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work. 
A supplementary figure is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Immune control of mouse CMV (MCMV) infection is organized in a hierarchical and redundant manner by distinct components of innate and adaptive immunity (Polic et al., 1998). Interferons (IFNs) provide a direct defence against CMVs and connect innate and adaptive immunity (Lucin et al., 1992; Heise et al., 1998; Polic et al., 1998). Binding of IFN-
/β and IFN-
to their cognate transmembrane receptors initiates distinct Jak–signal transducer and activator of transcription (Jak-STAT)-dependent signalling pathways (Darnell, 1997; Stark et al., 1998). IFN receptors are composed of two subunits. Upon ligand binding, receptor-associated Jak kinases are activated and phosphorylate STAT proteins which subsequently dimerize, translocate to the nucleus and induce IFN-dependent gene expression. Experimental studies using MCMV indicate that IFN receptor-induced Jak–STAT signalling has an indispensable role in the control of CMV replication by the host immune system (Lucin et al., 1992, 1994; Heise et al., 1998; Presti et al., 1998). By replicating under the selective pressure of IFNs, CMVs have evolved effective mechanisms that counteract IFN induction (Le et al., 2008) and IFN-mediated antiviral defence mechanisms (Hengel et al., 2005; Zimmermann & Hengel, 2006).
CMVs are able to interfere with IFN-dependent signal transduction in infected cells. STAT1 tyrosine phosphorylation and nuclear translocation are blocked upon HCMV infection (Miller et al., 1998; Le Roy et al., 1999) by an HCMV-induced decrease in Jak1 levels (Miller et al., 1998) which also impedes IFN-/β-dependent signal transduction (Miller et al., 1999). Independently, a decrease in IRF-9/p48 expression contributes to the inhibition of ISGF3-mediated gene activation in HCMV-infected cells (Miller et al., 1999; Cebulla et al., 2002). The HCMV genes responsible for these functions have yet to be identified, but a post-translational mechanism involving proteasomal degradation of Jak1 has been proposed (Miller et al., 1998). Additionally, the HCMV IE1 protein pp72 exhibits an IFN-antagonistic function, forming a physical complex with STAT2 to inhibit STAT2-dependent gene expression (Paulus et al., 2006).
In contrast to the situation in HCMV, expression of STAT1 and IRF-9/p48 is unaffected by MCMV infection (Popkin et al., 2003; Zimmermann et al., 2005). However, we have recently identified that pM27 encoded by MCMV is an antagonist of STAT2 that mediates resistance to type I and type II IFNs. M27 acts by promoting proteasomal degradation of STAT2; deletion of the M27 ORF from the MCMV genome results in a strongly increased sensitivity to IFN- and IFN- in cell culture and in vivo (Abenes et al., 2001; Zimmermann et al., 2005).
In this study we demonstrate that STAT2 protein levels were consistently downregulated during the early phase of HCMV infection with the exception of HCMV laboratory strain Towne. This effect was sensitive to proteasome inhibitors, thus implicating a proteolytic mechanism of STAT2 degradation in infected cells. Even following restoration of STAT2 expression, tyrosine phosphorylation and activation by type I IFN was still impaired, thus revealing a second layer of HCMV interference with STAT2 functions. Remarkably, deletion of the MCMV M27 homologue, UL27, from the HCMV genome did not restore STAT2, suggesting that control over STAT2 was acquired independently by HCMV and MCMV.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Viruses, infection conditions and virus titration.
The following HCMV strains were used: AD169 (Hengel et al., 1995), Towne (ATCC VR-977), the endotheliotropic strain TB40/E (Sinzger et al., 1999) reconstituted from its respective BAC clone TB40/E-BAC4 (Sinzger et al., 2008) and the low-passage clinical isolates UL1271 (Atalay et al., 2002) and UL1702 (a gift from T. Mertens, University of Ulm, Germany). Purified HCMV stocks were prepared on MRC-5 cells. The virus purification procedure was modified based on the method described by Stinski (1976). Briefly, infectious supernatants were harvested when all cells showed complete cytopathic effect. After centrifugation for 15 min at 5000 g at 10 °C, the virus-containing supernatant was centrifuged again at 20 000 g for 3 h at 10 °C. The virus pellet was resuspended in PBS, dounced and carefully placed on top of a 20 % sorbitol cushion. A further centrifugation step was performed at 65 000 g for 1 h at 10 °C and the virus pellet was homogenized in PBS before storing at –80 °C. Virus titres were determined by standard plaque assay; the titre of each virus stock was between approximately 1x108 and 2x109 p.f.u. ml–1. HCMV infection was enhanced by centrifugation at 800 g for 30 min. UV-inactivation of HCMV was performed for 20 min at 265 nm. Susceptibility to IFNs was assayed by virus growth in the presence of recombinant human IFN-,-β or - (PBL Biomedical Laboratories) after preincubation with IFN for 48 h prior to infection. Vaccinia virus (VACV) strain WR was used to generate recombinant VACVs.
Northern blot analysis of specific transcripts.
Total RNA was extracted from HCMV-infected MRC-5 fibroblasts using the RNeasy Mini kit (Qiagen). Total RNA was subjected to electrophoresis and transferred to nylon membranes using the TurboBlotter (Schleicher and Schuell). Probes were prepared by PCR with gene specific primers (STAT2 forw, 5'-GCTCATACTAGGGACGGGAAGTCG-3'; STAT2 rev, 5'-GGCTGAATGTCCCGGC AGA-3'; HCMV-IE2 forw, 5'-TCTGATAGCGAGGAAGAACAGGG-3'; HCMV-IE2 rev, 5'-CATACTGGGAATCGTGAAGGGC-3') and digoxigenin-labelled dUTP (Roche) for detection of STAT2 and HCMV-IE2 transcripts. Hybridization and detection were performed as described in the manufacturer's instructions.
BAC-based mutagenesis and construction of recombinant HCMV.
Recombinant HCMV were generated by PCR-directed mutagenesis according to the method described by Wagner et al. (2002) using BAC plasmid pTB40-4 (Sinzger et al., 2008). The PCR fragment containing a kanamycin-resistance gene (KanR) was generated from the template plasmid pSLFRTKn (Atalay et al., 2002) using primers 5'-delUL27 (5'-ACCCCACGGTGCTTATAACGCGCCGCCGCGGCCCAGTGAATTCGAGCTCGGTAC-3') and 3'-delUL27 (5'-TGTCTGGCTTTACCGCCGTCGCTCCGCGTCGCTTCGCCGCCACCTTCTTCTTCCTCTCAGCACCATGATTACGCCAAGCTCC-3') and inserted into pTB40-4 by homologous recombination. KanR was excised from the pTB40-4 by FLP-mediated recombination (Wagner et al., 2002), generating the HCMV BAC plasmid pTB40-
UL27. Correct mutagenesis was confirmed by restriction pattern and PCR analysis (data not shown). Recombinant HCMV was reconstituted from pTB40-UL27 by Superfect transfection as described by Borst et al. (1999).
Generation of vaccinia virus recombinants (rVV).
The UL27 ORF was amplified from HCMV strain AD169 using primers az-UL27-1 (5'-GCTCGGATCCATGAACCCCGTGGATCAGCC-3') and az-UL27-2 (5'-CTCGAGCTCTACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCTGTGGCGTGACCTCCGACCTCG-3'). Primer az-UL27-1 contains a BamHI site (italics); az-UL27-F2 contains a SacI site (italics) and the coding sequence for the V5 epitope (underlined). The PCR fragment was cloned into the VACV expression vector p7.5k resulting in p7.5K131-UL27, which was used to generate rVV-UL27 after homologous recombination into the thymidine kinase locus of VACV strain WR as described by Hengel et al. (1997). The recombinant VACVs (rVVs) were isolated by selection with bromodeoxyuridine using TK-143 cells.
Immunoblot analysis and electrophoretic mobility shift assay (EMSA).
For immunoblotting, equal amounts of EMSA cell lysate were suspended in Laemmli sample buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose filters. Immunoblot analyses were performed with the following antibodies: monoclonal anti-β-actin antibody produced in mouse (Sigma); mouse monoclonal antibody (mAb) against HCMV pp65 (3A12; AbCam); rabbit antiserum specific for STAT1 (E23; Santa Cruz), STAT2 (06-502; UpstateBiotechnology), STAT3 (C20; Santa Cruz), Jak1 (HR-785; Santa Cruz) or IRF-9/p48 (H-143; Santa Cruz); and mAb recognizing p-Tyr701 STAT1 (A2; Santa Cruz), p-Tyr689 STAT2 (Tyr 689; Upstate) or p-Tyr STAT3 (B7; Santa Cruz). Epitope-tagged proteins were detected by mAb against FLAG (M2; Sigma) or V5 (Invitrogen). Proteins were visualized using the ECL-Plus chemiluminescence system (Amersham).
EMSA analysis using a 32P-labelled M67 GAS probe (Haspel et al., 1996) was performed as described by Zimmermann et al. (2005).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This prompted us to compare the levels of STAT2 and STAT1 in HCMV-infected MRC-5 fibroblasts by Western blot analysis (Fig. 1). Testing of HCMV strain AD169-infected cells revealed that STAT2 levels were selectively downregulated 24 h post-infection (p.i.) and were almost undetectable 48 and 72 h p.i., whereas levels of STAT1 (Fig. 1a) and STAT3 (data not shown) remained unchanged. This finding was unexpected, as previous reports (Miller et al., 1999; Paulus et al., 2006) demonstrated that STAT2 protein levels remained unaltered during the course of infection of fibroblasts by HCMV strain Towne. Hence, we determined the STAT2 protein levels after infection with strain Towne and confirmed that the amounts of STAT2 remain relatively constant up to 72 h p.i. (Fig. 1a). The AD169 and Towne strains exhibited different capabilities for STAT2 regulation in infected cells.
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) and the clinical isolate UL1271. Both viruses downregulated STAT2 at 24 h p.i.; STAT2 was also efficiently suppressed at later time points of infection (Fig. 1b
). This result was reproduced with HCMV strain UL1702 (passage 3) which showed a strongly reduced STAT2 protein level 48 h p.i. (Fig. 1b). These data indicate that HCMV downregulation of STAT2 (an additional, as-yet unrecognized mechanism to control IFN signalling) is regularly found in naturally occurring HCMV isolates, whereas the Towne strain appears to have lost this function during extensive passage in fibroblast cultures.
HCMV controls STAT2 by proteasomal degradation
To gain insight into the mechanism by which HCMV downregulates STAT2, mRNA levels were measured by Northern blot analysis of HCMV strain AD169-infected cells (Fig. 2a). A probe that recognizes the 2.2 kb IE2 immediate early (IE) transcript and the 1.5 kb IE40 late transcript (Stenberg et al., 1989) was used to monitor the temporal course of HCMV infection. STAT2 mRNA was more abundant in infected cells compared with mock-infected MRC-5 cells; this was enhanced further during the course of HCMV replication. These data indicate that STAT2 expression might be downregulated at the post-transcriptional level. To test whether STAT2 was being targeted for proteasomal degradation, the selective inhibitor lactacystin was used. Lactacystin treatment of cells for 24 h partially rescued the STAT2 level in HCMV-infected cells when added 24 h p.i., but did not increase the levels in mock-infected fibroblasts (Fig. 2b). Notably, the STAT2 rescued in the presence of lactacystin was not tyrosine phosphorylated in response to IFN- stimulation (Fig. 2b), thus indicating that HCMV infection also affects STAT2 activation.
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, upper panel); this is consistent with previous reports (Lee et al., 1997). In contrast, the half life of STAT2 was reduced to less than 6 h in AD169-infected cells (Fig. 2c, middle panel). This effect could be partially reversed by the proteasome inhibitor MG132 (Fig. 2c, lower panel). STAT2 stability was evaluated further by immunoprecipitation from lysates of 35S metabolically labelled cells. This revealed that protein levels and half-life were reduced in HCMV strain TB40/E-infected cells compared with mock-infected and UV-inactivated HCMV strain TB40/E-treated cells (see Supplementary Fig. S1, available in JGV Online).
Since both the downregulation of STAT2 by HCMV and the degradation of STAT2 in MCMV-infected cells by pM27 (Zimmermann et al., 2005) involved proteasomal degradation, we hypothesized that the HCMV homologue of pM27, pUL27, might be responsible for STAT2 proteolysis. To investigate this, we amplified the UL27 sequence from HCMV strain AD169 and constructed a recombinant vaccinia virus (rVV) expressing pUL27. As a positive control, MCMV M27 was expressed by rVV, which resulted in a complete degradation of STAT2 (Fig. 2d). In contrast, expression of UL27 by rVV did not affect the STAT2 level (Fig. 2d), suggesting that pUL27 is not sufficient to downregulate STAT2. To exclude UL27 from any role in the HCMV-mediated loss of STAT2, we constructed a BAC-derived deletion mutant lacking the complete UL27 ORF from HCMV strain TB40/E. When fibroblasts were infected with the TB40/E-UL27 mutant, an almost complete loss of STAT2 protein was observed (Fig. 2e). Since deletion of UL27 did not restore STAT2 protein levels in HCMV-infected cells, we concluded that the loss of STAT2 occurs independently of pUL27. In summary, these results revealed that HCMV induces a strong upregulation of STAT2 mRNA which is counteracted by a proteolytic mechanism for reducing STAT2 that at least partially involves the proteasomal degradation pathway, but is independent of pUL27.
STAT2 downregulation requires HCMV early gene expression
To identify the gene products leading to the loss of STAT2 in HCMV-infected cells, we tested components of the HCMV virion for their ability to regulate this effect. UV-inactivated HCMV TB40/E particles were not able to reduce STAT2 levels at any time point following fibroblast incubation, whereas STAT2 levels were rapidly affected in productively infected cells (Fig. 3a). MRC-5 cells treated with inactivated virus exhibited elevated STAT2 protein levels, which argues against a direct effect of particle components leading to STAT2 instability. To investigate the effect further, we preincubated the HCMV preparation with neutralizing antibodies before infection. Both the degradation of STAT2 associated with active infection and the elevation of STAT2 levels induced by UV-inactivated virus were abolished with neutralizing antibody (data not shown). These data rule out a substantial contribution of virion components to STAT2 degradation. Additionally, the data indicate that elevation of STAT2 mRNA does not require HCMV gene expression, underlining the impact of HCMV-mediated STAT2 downregulation in the presence of a simultaneous upregulation of STAT2 mRNA.
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), ruling out an aetiological requirement for HCMV late genes in this process.
The HCMV IE1 protein pp72 was reported to interact physically with STAT1 and STAT2 in HCMV strain Towne-infected cells (Paulus et al., 2006). To test whether STAT2 degradation might be mediated by IE1/pp72, we analysed the lifespan of STAT2 in MRC-5 cells infected with TB40/E under conditions of selective and enhanced IE/pp72 expression, achieved by addition of CHX and actinomycin D (Fig. 3c). Under these conditions, stability of STAT2 was not altered compared to uninfected cells, while in untreated TB40/E-infected cells entering the early phase of infection, STAT2 levels were downregulated (Fig. 3c). From these findings, we concluded that neither IE1/pp72 nor other HCMV IE genes are sufficient to downregulate STAT2. Collectively, these data indicate that downregulation of STAT2 is mediated by an HCMV early gene product.
STAT2 degradation only modestly affects IFN sensitivity of HCMV
The loss of STAT2 degradation resulting from deletion of M27 from MCMV reduced virus replication in the presence of IFN-/β and - (Zimmermann et al., 2005). For this reason, we assessed whether the ability of HCMV to reduce STAT2 may affect the replication efficiency in fibroblasts pretreated for 48 h with IFN-. After infection at an m.o.i. of 0.01, progeny virus was harvested from supernatants at the time points indicated in Fig. 4(a). To estimate IFN susceptibility, we calculated the ratio of progeny virus between untreated and IFN-treated cells at day 5 p.i. and defined the mean value from three independent experiments as the mean fold titre reduction. In the STAT2-degrading strain AD169 (Fig. 4a, left panel) and the non-degrading strain Towne (Fig. 4a, right panel) the mean titre reduction by IFN- (500 U ml–1) was 18- and 26-fold, respectively. Likewise, the presence of IFN-β (250 U ml–1) led to a 360-fold reduction of replication in strain AD169 compared with a 540-fold reduction in strain Towne (Fig. 4a). Since deletion of M27 strongly increased sensitivity of the M27 MCMV mutant to IFN- (Zimmermann et al., 2005), we analysed growth of these strains in the presence of 250 U IFN- ml–1 (Fig. 4b). AD169 (Fig. 4b, left panel) showed a median titre reduction of 150-fold, suggesting that it is slightly more resistant than Towne (Fig. 4b, right panel), which showed a 500-fold titre reduction. These data suggest that the ability of HCMV to degrade STAT2 only plays a minor role in overall IFN resistance, indicating that additional, functionally redundant mechanisms ensure IFN evasion, independent of STAT2 degradation.
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was strongly reduced at 24 h p.i. and eventually abolished (Fig. 5a), indicating that interference with STAT2 activation and downregulation of STAT2 are independent events in HCMV infection. Inhibition of STAT1 tyrosine phosphorylation proceeded with comparable kinetics in Towne-infected cells (Fig. 5a). The kinetic analysis also revealed a decrease in Jak1 level, as reported previously by Miller et al. (2000), that was temporally correlated with inhibition of STAT tyrosine phosphorylation. To test whether Jak1 represents a central target of HCMV interference that results in deficient STAT tyrosine phosphorylation, we determined the kinetics of Jak1-dependent IFN--mediated phosphorylation of STAT3 (Caldenhoven et al., 1999). Tyrosine activation of STAT3 was completely inhibited in HCMV strain Towne-infected cells (Fig. 5a). Moreover, IRF-9/p48 levels were decreased 48 h after Towne infection, consistent with previous data (Miller et al., 1999). To assess the DNA binding of phosphorylated STAT proteins, we performed EMSA analysis using a GAS probe. DNA binding to GAS recognition sites which bind STAT1–STAT1 homodimers could be detected early (6 h p.i.) but declined during the progression of HCMV infection, becoming undetectable at 48 h p.i. (Fig. 5a), which is also consistent with previous data (Miller et al., 2000). Taken together, these data indicate that HCMV is able to control the IFN-dependent tyrosine phosphorylation of STAT1, STAT2 and STAT3 within 24 h of infection. These data agree with the hypothesis that HCMV control of STAT-dependent gene expression includes targeting of Jak1 (Miller et al., 1998, 2000).
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(Fig. 5b). The amount of tyrosine-phosphorylated STAT1 (p-Tyr-STAT1) declined during the course of infection; the level was substantially reduced 10 h p.i. and was virtually undetectable 24 h p.i. To monitor the kinetics of STAT2 downregulation and tyrosine phosphorylation, we used Western blot analysis to compare the two events during HCMV strain AD169 infection (Fig. 5c). The inhibition of STAT2 phosphorylation occurred earlier than the onset of STAT2 degradation. Notably, the temporal progress of inhibition of STAT2 phosphorylation appeared to be very similar to the time-course observed with IFN--induced STAT1 tyrosine phosphorylation (Fig. 5b), consistent with the suggestion that a common mechanism is responsible for both events. Likewise, inhibition of STAT phosphorylation preceded STAT2 degradation in strain TB40/E and UL1702 (data not shown). These findings indicate that HCMV sequentially employs two independent mechanisms to target STAT2. This may reflect redundancy of HCMV gene functions but also leaves the possibility that HCMV-mediated STAT2 degradation targets other STAT1- and phosphorylation-independent functions of STAT2. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 1999, 2000; Paulus et al., 2006) reporting supposedly intact levels of STAT2 protein in HCMV infection.
The inherent need of HCMV to escape from the IFN response is underlined by the fact that HCMV utilizes at least four separate mechanisms to achieve this goal. Starting with the expression of the IE genes, the IE1/pp72 phosphoprotein was found to bind ISGF3 and prevent binding of this transcription factor to the promoters of IFN-responsive genes (Paulus et al., 2006). The loss of STAT tyrosine phosphorylation in HCMV-infected cells, which occurs independently of IE1/pp72, indicates a further viral factor that blocks Jak–STAT signalling. This effect begins after the onset of early gene expression at 24 h p.i. and is associated with declining levels of Jak1, leading to impaired and eventually abolished tyrosine phosphorylation of STAT1 (Miller et al., 2000), STAT3 and STAT2. Concurrently, the degradation of STAT2 is initiated soon after infection, resulting in an almost complete loss of STAT2 within 48 h p.i. Consistent with this, we have demonstrated that IE1/pp72 does not affect STAT2 protein stability. Finally, the downregulation of IRF-9/p48, the third component of ISGF3, has been documented at 72 h p.i. (Miller et al., 1999). In summary, subsequent and redundant disruption of IFN signalling by expression of multiple and independently operating HCMV gene products ensures an effective multi-layered escape from the IFN response during the complete replication cycle. Due to this redundancy of inhibitory mechanisms, the principal ability to counteract IFN responses, ensuring replication in the presence of IFNs, was conserved among all HCMV strains investigated. Most importantly, all HCMV strains including Towne exhibited a conserved capability to block tyrosine phosphorylation of STATs and thus subsequent ISGF3 and GAF formation (data not shown), irrespective of their ability to downregulate STAT2 during infection. The fact that tyrosine activation of STATs controls a vast majority of IFN receptor downstream signalling events implies that the unknown HCMV inhibitor of STAT tyrosine phosphorylation has a central role in the hierarchy of HCMV antagonists, taking a concerted action to block IFN responsiveness.
STAT2 as a target of HCMV interference with IFN signalling
The STAT2 protein is a prominent target for immune evasion by several viruses employing a variety of strategies to inactivate Jak–STAT signalling, including STAT degradation, STAT1 sequestration and inhibition of STAT tyrosine phosphorylation (Horvath, 2004). Among the herpesvirus family, STAT2 degradation has been reported for herpes simplex virus-1 (Chee & Roizman, 2004), MCMV (Zimmermann et al., 2005) and murine gammaherpesvirus-68 (Liang et al., 2004). At the molecular level, the paradigm for virus-induced STAT degradation is given by the simian virus 5 V protein that targets STATs to the proteasome by forming a DDB1–Cul4A degradation complex (Precious et al., 2005); the crystal structure of this has recently been resolved (Guma et al., 2006). The loss of STAT2 observed in HCMV-infected cells closely resembles the phenotypic changes of STAT2 in MCMV infection (Zimmermann et al., 2005). As in MCMV-infected cells, downregulation of STAT2 by HCMV depends on viral early gene expression, occurs independently of virion components, is sensitive to proteasome inhibition and is STAT-selective, i.e. it does not involve STAT1 and STAT3 (Zimmermann et al., 2005). Degradation of STAT2 by HCMV was not recognized in previous studies (Miller et al., 1998, 1999, 2000; Paulus et al., 2006) due to the fact that the Towne strain that was used exhibits a selective loss of STAT2 degradation capability. Detection of STAT2 decay requires the uniform infection of all cells, otherwise the concomitant upregulation of STAT2 mRNA by type I IFN compensates for the STAT2 degradation.
An obvious candidate for the HCMV gene product that specifically targets STAT2 was the homologue of the MCMV M27 gene, the UL27 gene product. However, our data clearly indicate that the HCMV UL27 and MCMV M27 gene products do not share a conserved STAT2-related function. Hence, the genetic and molecular basis of the HCMV-mediated STAT2 degradation remains to be elucidated. Despite their collinear genomes, almost no homologous genes of MCMV and HCMV with conserved immuno-evasive functions have been identified to date. This suggests that, during co-evolution of CMVs with their natural host, originally conserved genes like M27/UL27 were selected by divergently evolving immune systems and thus adapted to different functions. Remarkably, STAT1 tyrosine phosphorylation, which is shown to be strongly inhibited in HCMV-infected cells, remains fully intact after MCMV infection (Zimmermann et al., 2005) and is balanced only by an additional downstream mechanism which escapes IFN--mediated responses by blocking the assembly of STAT1-driven promoters (Popkin et al., 2003).
Potential consequences of STAT2 degradation
The ability of HCMV to degrade STAT2 independent of its inhibition of tyrosine activation may simply reflect the redundancy of molecular mechanisms mediating immune escape from IFN responses. However, this finding could also mean that STAT2 degradation fulfils a biological function distinct from IFN interference. STAT2 has been reported to interact physically with the HCMV IE1/pp72 protein (Paulus et al., 2006), raising the possibility that STAT2 degradation contributes to the regulation of IE1/pp72 either by degradation of the IE1/pp72–STAT2 complex or by concomitant degradation of IE1/pp72. In fact, pp72 is the subject of active regulation by HCMV (Grey et al., 2007) and the relative abundance of pp72 decreases with the progress of HCMV replication (Benz et al., 2001; White & Spector, 2005). Another explanation could be that STAT2 is an independent target of immune evasion due to its STAT1-independent functions. There is growing evidence for additional roles of STAT2 beyond the accepted Jak–STAT signalling pathway. IFNs exert STAT1-independent actions on mononuclear phagocytes by regulating the expression of many genes during CMV infection (Gil et al., 2001). Moreover, a recent study demonstrated the existence of a collection of IFN-inducible GAS-regulated target genes that are expressed independently of ISGF3 but dependent on the STAT2 DNA-binding domain (Brierley et al., 2006). It is therefore tempting to speculate that HCMV disrupts this STAT1-independent IFN pathway when degrading STAT2. Furthermore, STAT2 is critically involved in the IFN-dependent activation of STAT4, which is STAT1- and STAT2-Y690-phosphorylation-independent (Farrar et al., 2000a, b), a pathway that is thought to be essential for IFN- induction during viral infection (Nguyen et al., 2002). The fact that IFN-dependent induction of STAT4 represents an antiviral pathway in cell types that represent primary targets for HCMV infection, i.e. endothelial cells (Torpey et al., 2004), macrophages and dendritic cells (Fukao et al., 2001), raises the intriguing possibility that HCMV affects STAT4-dependent functions by attacking STAT2 protein stability.
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ACKNOWLEDGEMENTS |
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Received 28 February 2008;
accepted 2 June 2008.
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