1 School of Biology, University of St Andrews, Fife KY16 9TS, UK
2 Department of Biochemistry and Immunology, St George's Hospital Medical School, University of London, London SW17 0RE, UK
Correspondence
R. E. Randall
rer{at}st-and.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Simian virus type 5 (SV5) and human parainfluenza virus type 2 (hPIV2) are classified within the genus Rubulavirus of the subfamily Paramyxovirinae of the family Paramyxoviridae (Lamb & Kolakofsky, 2001). It is now well-established that SV5 and hPIV2, and many other paramyxoviruses, at least partially circumvent the IFN response by blocking IFN signalling and IFN production (reviewed by Garcia-Sastre, 2004
; Horvath, 2004
; Nagai & Kato, 2004
). In human cells, SV5 and mumps virus block IFN signalling by targeting STAT1 for proteasome-mediated degradation, whilst human hPIV2 targets STAT2 for degradation (Andrejeva et al., 2002b
; Didcock et al., 1999a
; Nishio et al., 2001
; Parisien et al., 2001
; Yokosawa et al., 2002
; Young et al., 2000
). As a consequence, SV5 inhibits both IFN-
/
and IFN-
signalling, whilst, in human cells, hPIV2 only blocks IFN-
/
signalling. Intriguingly, it has recently been reported that mumps virus, but not SV5, can also target STAT3 for degradation, although the biological significance of this has yet to be established (Ulane et al., 2003
). The importance of IFN in controlling SV5 infections can be judged from studies in mice: SV5 fails to degrade STAT1 in murine cells and is non-pathogenic in normal and severe combined immunodeficient mice (which fail to make an adaptive immune response; Didcock et al., 1999b
; Randall & Young, 1991
), but is lethal in mice that lack STAT1 (He et al., 2002
). These results indicate that the relative lack of pathogenicity in normal mice can be at least partially attributed to the fact that SV5 fails to block IFN signalling in murine cells (Young et al., 2001
).
Much has been learned about the molecular mechanisms by which SV5, mumps virus and hPIV2 target STAT1/2 for degradation. Of the virus proteins, the V protein is necessary and sufficient to mediate degradation (Andrejeva et al., 2002b; Didcock et al., 1999a
; Parisien et al., 2002b
). Degradation is not dependent upon IFN signalling and SV5 can induce degradation of both phosphorylated and non-phosphorylated STAT1 (Andrejeva et al., 2002b
; Parisien et al., 2002b
). Nevertheless, there appears to be an interdependence of STAT1 and STAT2 for degradation; thus, hPIV2 failed to degrade STAT2 in STAT1-deficient (U3A) cells and SV5 failed to degrade STAT1 in STAT2-deficient (U6A) cells (Parisien et al., 2002b
). Furthermore, STAT2 was not degraded upon hPIV2 infection of human 2fTGH cells that constitutively expressed the V protein of SV5 (and thus lacked STAT1), whilst STAT1 was not degraded in 2fTGH cells expressing the hPIV2 V protein (which thus lacked STAT2) when infected by SV5 (Andrejeva et al., 2002b
). Also, the V protein of SV5 can target murine STAT1 for degradation in murine cells if human STAT2 is co-expressed (i.e. the V protein of SV5 is unable to utilize murine STAT2 in the degradation complex; Parisien et al., 2002a
).
The SV5 V protein interacts strongly with the 127 kDa subunit (DDB1) of the UV-damaged DNA-binding protein (DDB; Lin et al., 1998) and a clear role for DDB1 in the targeted degradation of STAT1 by the V protein of SV5 has now been firmly established (Andrejeva et al., 2002a
; Leupin et al., 2003
; Ulane & Horvath, 2002
). In uninfected cells, DDB1 has been isolated as an interacting partner for several proteins, including Cullin 4a (Cul4a), which is associated with E3 ubiquitin ligase activity (Shiyanov et al., 1999
). Indeed, it has recently been shown that DDB1 and Cul4a form part of an E3 ligase complex that regulates c-Jun activity (Wertz et al., 2004
). Cullins function by serving as connectors between at least two groups of proteins. Firstly, they connect the ubiquitin E2 ligases to the E3 ligases and, secondly, they interact with substrate-recognition proteins to complete an E3 ligase complex that ubiquitinates the substrate protein specifically and hence targets it for proteasome-mediated degradation (for reviews on ubiquitination and proteasome-mediated degradation, see Glickman & Ciechanover, 2002
; Jackson et al., 2000
; Weissman, 2001
). Suggestive evidence for a direct role of Cul4a in the targeted degradation of STAT1 by SV5 comes from the observations that Cul4a was co-immunoprecipitated with V and DDB1, and that treatment of cells with small interfering RNA to Cul4a affected the degradation of STAT1 slightly (Ulane & Horvath, 2002
). Furthermore, preliminary evidence from in vitro ubiquitination assays suggests that SV5 V protein can be autoubiquitinated in the presence of E1 and E2 ubiquitin ligases, an activity that is a feature of some cellular E3 ubiquitin ligases (Ulane & Horvath, 2002
). It appears that the V proteins of SV5, hPIV2 and other rubulaviruses, together with STAT1, STAT2, DDB1 and probably Cul4a (or equivalent), form novel ubiquitin ligase complexes that target either STAT1 or STAT2 for ubiquitination and degradation.
To begin to define further the importance of these interactions, we have been developing in vitro assays to address questions concerning the nature of the targeted ubiquitination of STAT1 or STAT2 by the V proteins of SV5 and hPIV2. Here, we report on the ubiquitination and degradation of STAT1 and/or STAT2 in rabbit reticulocyte lysates by the addition of bacterially expressed V proteins of SV5 and hPIV2. Using this system, we have also shown that whilst the ubiquitination of STAT1 by the V protein of SV5 appeared to be highly specific, the V protein of hPIV2 could ubiquitinate both STAT1 and STAT2 proteins. On the basis of these observations, we re-examined the ability of SV5 and hPIV2 to target STAT proteins for degradation in a wide variety of cells. These studies revealed that whilst SV5 always targeted STAT1 for degradation, hPIV2 was more promiscuous and the apparent specificity of degradation of STAT1 or STAT2 by hPIV2 was dependent upon the species from which the cells originated.
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METHODS |
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Immunoblotting.
Immediately prior to harvesting, cells were washed twice with PBS and lysed into gel electrophoresis sample buffer. Cell lysates were then processed by sonication and heating at 100 °C for 5 min. Samples were subjected to SDS-PAGE, polypeptides were transferred to nitrocellulose membranes and STAT1 was detected by using a polyclonal anti-STAT1 antibody raised against the N-terminal 194 aa of the protein (cat. no. G16930; Transduction Laboratories). Proteinantibody interactions were visualized by enhanced chemiluminescence using horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences).
Plasmids.
Plasmids for T7 RNA polymerase-dependent in vitro translations were constructed by: (i) inserting a partial AflIII (5' end)BamHI fragment from pCEPp127 (a kind gift from Vesna Rapic-Otrin, University of Pittsburgh, USA) containing the entire ORF of DDB1 between the NcoI and BamHI sites of pGBKT7 (Clontech) to make pGBKT7.DDB1; (ii) inserting the entire ORF of human STAT2 flanked by NcoI (5' end) and EcoRI sites, obtained by RT-PCR, between the NcoI and EcoRI sites of pGBKT7 to make pGBKT7.hSTAT2. The plasmid pT7STAT1
has been described previously (King & Goodbourn, 1998
). The T7 polymerase-dependent Cul4a plasmid was a kind gift of Dr P. Zhou (Weill Medical College of Cornell University, NY, USA; Chen et al., 2001
).
Cloning and purification of glutathione S-transferase (GST)SV5 V fusion protein.
The SV5 V gene was amplified from an existing clone by PCR using the following primers: forward, 5'-GGATCCCCGAATTCCgaaaacctgtattttcagggcgccATGGATCCCACTG-3', and reverse, 5'-GCGGCCGCTCAAATTGCACTGCGGATGATTG-3', and ligated between the EcoRI and NotI restriction sites of pGEX-4T3 (Pharmacia). It should be noted that the forward primer contained coding sequences (lower-case letters) for the cleavage sequence of tobacco etch virus (TEV) protease. The plasmid was cloned into Escherichia coli B834 for optimum expression. GSTSV5 V protein was expressed by induction with 1 mM IPTG for 3 h at 30 °C. After centrifugation, the bacterial cells were resuspended in buffer A [50 mM Tris/HCl (pH 8·0), 200 mM NaCl, 2 mM dithiothreitol (DTT)], lysed by sonication and the lysate was clarified by centrifugation at 30 000 r.p.m. for 30 min in a Beckman 42.1 rotor. Expressed GSTSV5 V protein was bound to a glutathioneagarose column (Sigma), eluted with glutathione by using standard protocols and used without further purification.
To purify SV5 V protein, GSTSV5 V was incubated overnight with recombinant TEV protease (Invitrogen), using a 50 : 1 ratio of protein : enzyme at room temperature with accompanying dialysis in buffer A. After removal of GST by a second glutathione column, the partially purified V protein was size-fractionated on a Superose S200 column (Pharmacia). Protein fractions were collected and analysed by SDS-PAGE. SV5 V protein-containing fractions were pooled and used in subsequent analyses.
GSTSV5 V capture of L-[35S]methionine-labelled, in vitro-synthesized proteins.
L-[35S]Methionine-labelled proteins were synthesized individually in the Quick prime reticulocyte lysate system (Promega) following the manufacturer's instructions. To identify those proteins that interacted with GSTSV5 V, 5 µl of each translation product was mixed with 10 µl glutathione beads saturated with either GST or GSTSV5 V and diluted to 1 ml in buffer A with the addition of 0·1 % Tween 20 (Sigma) and BSA (10 mg ml1). These samples were incubated at 4 °C for 30 min with end-over-end mixing, followed by three washes of 1 ml with buffer A/0·1 % Tween 20.
STAT degradation and ubiquitination assays.
To demonstrate the degradation of STAT1 or STAT2, individual protein syntheses of STAT1, STAT2, DDB1 or Cul4a were made in reticulocyte lysates as described above. Depending on the experimental conditions, either L-[35S]methionine or unlabelled methionine was added to the individual transcription/translation mixes. Five microlitres of the appropriate translation products were mixed together and to these was added 5 µl of a degradation buffer containing the following components: 240 mM Tris/HCl (pH 7·5), 30 mM MgCl2, 12 mM DTT, 3 mM ATP, 60 mM creatine phosphate, 20 U creatine phosphokinase ml1 and 1·2 mg ubiquitin ml1 (Sigma). Finally, 5 µl (10 µg) purified E. coli-expressed SV5 V protein in buffer A was also added where appropriate. The mixtures were made up to a total volume of 30 µl with additional reticulocyte lysate. To identify polyubiquitinated products, the protease inhibitor MG132 at a final concentration of 10 µM or the deubiquitination inhibitor ubiquitin aldehyde (UA) at a final concentration of 20 µg ml1 was added to the mixtures. The mixtures were incubated at 37 °C for 180 min and analysed by SDS-PAGE.
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RESULTS |
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Purified SV5 V protein expressed in E. coli causes the degradation of human STAT1 in vitro
The results of the precipitation experiments described above established that SV5 V protein expressed in E. coli was capable of interacting with at least some of the cellular components associated with STAT1 degradation and it was thus of interest to determine whether STAT1 degradation could take place in vitro. In our initial experiments, STAT2, DDB1 and Cul4a were translated individually and mixed in combinations with in vitro-translated and L-[35S]methionine-labelled STAT1. It can be seen from Fig. 2(a) that STAT1 was degraded in the presence of SV5 V protein and human STAT2. However, in vitro-translated DDB1 and Cul4a could be omitted, possibly because these proteins are present within the reticulocyte lysates. To confirm that in vitro-translated human STAT2 was necessary, reticulocyte lysates containing L-[35s]methionine-labelled stat1 were prepared for a degradation assay in the presence or absence of L-[35S]methionine-labelled STAT2 (Fig. 2b
). These results showed that the presence of human STAT2 was required in this system for degradation to occur.
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DISCUSSION |
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The results presented here on the interaction of GSTSV5 V with in vitro-translated cellular proteins thought to be involved in targeted degradation of STAT1 generally support those of Parisien et al. (2002b), which showed that GSTSV5 V bound DDB1, Cul4a, STAT2 and STAT1 from human cell extracts. Whilst this current study and that of Parisien et al. (2002b)
failed to determine whether or not these interactions were direct, in vitro-translated human STAT1 was not captured by GSTSV5 V. As STAT1 is the target molecule for degradation and was not polyubiquitinated or degraded in the absence of human STAT2, these results support the idea that STAT1 may be incorporated into the degradation complex by STAT2. The role of Cul4a in the system remains unclear. Clearly, Cul4a can interact with SV5 V protein, but it seems likely to do so via its association with DDB1. It has also been suggested that there may be some redundancy in the cullins that are involved in the STAT-degradation complex (Ulane & Horvath, 2002
); however, the in vitro results presented here indicate that at least Cul2 is not able to interact either directly or indirectly with SV5 V protein and thus is unlikely to play any role in STAT degradation. Additionally, evidence is increasing for the specificity of interactions of cullins with substrate-recognition proteins in E3 ligases (Pintard et al., 2004
), suggesting that it is perhaps unlikely that different cullins would be involved in the degradation of STATs. However, the presence of the rabbit proteins in the reticulocyte lysates, and our inability to substitute wheat-germ cell lysates in these assays, limit the amount of data that can be gained regarding cellular proteins involved in the degradation. To overcome this problem and to define the minimal requirements for ubiquitination of STAT proteins by rubulavirus V proteins, we are currently further developing in vitro assay systems based upon purified expressed proteins, rather than cell extracts.
Given the observation that hPIV2 V degraded STAT1 preferentially in rabbit reticulocyte lysates, even in the presence of human STAT2, it was decided to re-examine the degradation of STAT1 and STAT2 by hPIV2 in a variety of tissue-culture cells. At the same time, SV5 and mumps virus were included to look for evidence of whether both tissue and species specificity might, in some cases, be determined by the ability of these viruses to degrade STAT proteins and hence block IFN signalling. As previously reported, hPIV2 degrades STAT2 preferentially in human cells (Andrejeva et al., 2002b). However, in some human cell types (e.g. Hep2 and HD-MY-Z), as well as inducing the degradation of STAT2, infection with hPIV2 also led to a marked reduction in the amount of STAT1. Furthermore, in some animal cells (MDBK and NBL-6), infection with hPIV2 preferentially induced the degradation of STAT1. Additionally, in MDCK cells that expressed hPIV2 V protein constitutively, STAT1 was degraded fully but STAT2 was not. In contrast, SV5 and mumps virus were much more specific in that they always targeted STAT1 for degradation. The biological significance of these observations remains unclear. Nevertheless, the data suggest that the differences in specificity of STAT1 and STAT2 degradation shown by hPIV2 could be relatively minor and may reflect either the cellular background or even the relative concentrations of STAT1 and STAT2 within a cell (given that hPIV2 can induce the ubiquitination of both STAT1 and STAT2; once one STAT has been degraded, there will be no significant degradation of the other STAT, as both STAT1 and STAT2 are required in the degradation process). In this respect, it is relevant to note that complementation of STAT2-deficient human U6A cells with exogenous STAT2 led not only to the degradation of STAT2 by hPIV2, but also to a partial loss of endogenous STAT1, suggesting that the level of STAT2 within a cell may influence the apparent specificity of STAT degradation by hPIV2 (Parisien et al., 2002b
). Also, whilst mumps virus degraded STAT1 in rabbit cells, SV5 did not. The inability of SV5 to degrade STAT1 in rabbit cells can be explained on the basis of the in vitro assays, in that the V protein of SV5 failed to ubiquitinate or degrade STAT1 in rabbit reticulocyte lysates unless human STAT2 was also added to the extracts; this is a situation similar to that observed in murine cells, where human STAT2 is required for SV5 to target STAT1 for degradation (Parisien et al., 2002a
). Thus, although species specificity may be influenced by the ability of these viruses to target STAT1 or STAT2 for degradation, from these limited studies, there is no evidence that tissue specificity might be dictated in the same way, i.e. SV5, mumps virus and hPIV2 degraded either STAT1 or STAT2 in all the human cells tested, regardless of tissue of origin.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 7 May 2004;
accepted 30 September 2004.