Mumps Virus V Protein Antagonizes Interferon witho
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病菌学杂志 2005年第6期
Department of Virology III, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo
Department of Microbiology, School of Medicine, Sapporo Medical University, Sapporo, Hokkaido, Japan
ABSTRACT
Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-? were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-?-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-?-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.
INTRODUCTION
The antiviral activity of interferon (IFN) is a major host defense mechanism generated during the early phase of viral infection. Antiviral activity is induced through an IFN signaling process called the Jak-STAT pathway. Briefly, the binding of IFN-/? to the cell surface type I IFN receptor activates the two receptor-bound kinases Jak1 and Tyk2, which subsequently phosphorylate the tyrosine residues (Y) of STAT1 and STAT2 at positions 701 and 689, respectively. The transcriptional activator, the ISGF3 complex, composed of Y701-phosphorylated STAT1 (pY701-STAT1), Y689-phosphorylated STAT2 (pY689-STAT2), and IRF9, is once formed and translocated to the nuclei. The ISGF3 complex then activates IFN-stimulated genes (ISGs). Typical ISG products such as 2',5'-oligoadenylate synthetase (2-5AS), RNA-dependent protein kinase (PKR), and Mx protein are known to exert antiviral activities (21).
However, it was previously reported that some viruses evolve to acquire the ability of antagonizing IFN functions through the suppression of the IFN signal transduction pathway (3, 14, 25-27, 49). Among these viruses, the members of the family Paramyxoviridae, which includes many important human and animal pathogens as well as emerging viruses such as Hendra virus and Nipah virus, have been found to antagonize IFNs (6, 7, 9, 15, 28, 31, 35, 36, 40, 46, 49). The individual paramyxoviruses have their own properties, which are obtained by coevolution with their respective hosts, but these viruses share common features regarding genome structure and function. They are enveloped viruses with a linear, nonsegmented negative-sense RNA genome of 15 to 18 kb. On the genome, basically six genes, the nucleocapsid protein, phospho (P) protein, matrix protein, fusion glycoprotein, attachment glycoprotein (HN, H, or G), and large (L) protein genes, are carried in that order from the 3' to 5' ends (20). The P and L proteins form RNA-dependent RNA polymerase. Among these genes, more than one protein is exceptionally produced from the P gene and at least one of these proteins is involved in the anti-IFN activities of the paramyxoviruses. The P gene gives rise to P and V mRNAs by a mechanism known as RNA editing. From these two mRNAs, P and V proteins are respectively translated in almost all paramyxoviruses, but in some paramyxoviruses, the C protein is additionally translated by use of an overlapping reading frame.
The carboxyl terminus of the V protein has seven cysteine residues and is highly conserved among the paramyxoviruses. Simian virus 5 (SV5), Simian virus 41 (SV41), Mumps virus (MuV), and Human parainfluenza virus type 2 (hPIV2) belonging to the Rubulavirus genus and Newcastle disease virus (NDV) belonging to the Avulavirus genus have the P and V proteins, but not the C protein in the P gene, and all of these viruses have been shown to antagonize IFNs by using the V protein (1, 12, 18, 28, 31, 33). The viruses of the Morbillivirus and Henipavirus genera have the P, V, and C proteins in the P gene and have also been shown to counteract IFNs by using V protein (33, 35, 36, 40, 49). Among the viruses in these genera, nonnegligible anti-IFN activity was also reported to be associated with the C protein of the Nipah and measles viruses (33, 37). Although the P gene of Sendai virus (SeV) of the Respirovirus genus codes P, V, and C proteins, the SeV C protein does counteract IFNs in the signaling process but V protein does not (15, 16).
The means by which such viral proteins inhibit the Jak-STAT pathway differ among the paramyxoviruses (9). For example, the V protein of MuV, SV5, SV41, and NDV induces the degradation of STAT1 (1, 12, 18, 28) and the V protein of hPIV2 induces the degradation of STAT2 (1, 28, 31). On the other hand, the V proteins of measles, Nipah, and Hendra viruses generate anti-IFN activity without STAT degradation (30, 35, 36, 40). In this latter case, instead of STAT degradation, IFN-induced phosphorylation and nuclear localization of STAT1 and STAT2 are inhibited.
The degradation of STATs found in members of the Rubulavirus and Avulavirus genera was originally demonstrated in persistently infected cells and by using a plasmid-based V expression system (6, 18, 28, 31). The importance of the V-unique carboxyl-terminal region for degradation was subsequently indicated by several V protein expression studies (12, 18, 28). Such observations have been confirmed in the context of viral replication by using recombinant hPIV2, SV5, and NDV lacking carboxyl-terminal V-unique regions (11, 12, 17). However, it is noteworthy that a spontaneous SV5 mutant with mutations in the P/V common domain showed no anti-IFN activity, indicating the contribution of the P/V common domain for generating anti-IFN activity (4, 45, 50). The degradation of these STAT proteins is thought to be the result of an ubiquitin-proteasome pathway because the amount of STAT mRNA does not change following viral infection; in addition, a proteasome inhibitor, MG132, recovers the STAT level, although the recoveries are partial (7, 47).
The interaction of V protein with cellular proteins was examined using glutathione S-transferase-V fusion protein and a yeast two-hybrid system. In those studies, p127 UV-damage-specific DNA binding protein 1 (DDB1) and receptor for activated C kinase 1 (RACK1) were identified as cellular counterparts of SV5 V and MuV V proteins, respectively (19, 22). The carboxyl-terminal V-unique region is necessary for both interactions. Copurification experiments revealed that the V protein of SV5 and hPIV2 forms a complex with STAT1, STAT2, DDB1, and Cul4A (31, 41). The good correlation between the binding of SV5 V to DDB1 and its ability to degrade STAT1 has been well demonstrated (2). As Cul4A is one of the components of cellular ubiquitin ligase, STAT1 degradation by the V protein was estimated to occur through an ubiquitin-proteasome pathway by using DDB1 and Cul4A. The ubiquitination of STAT1 has been demonstrated in MuV-infected cells (42, 47). On the other hand, the biological significance of RACK1 remains unclear. The V proteins of Rubulavirus have also been reported to bind to both STAT1 and STAT2 at their carboxyl termini, and these interactions are thought to be necessary for the ubiquitination and degradation of STATs (29, 32).
In this study, we demonstrated that the antiviral activity of IFN could be established in MuV-infected cells before the degradation of STAT1. Our observations therefore indicate that the complete degradation of STAT1 is not required for generating IFN antagonism of MuV.
MATERIALS AND METHODS
Cells, viruses, and IFN. Simian-kidney-derived CV1 and Vero cells were grown in Dulbecco's modified Eagle's medium and in Eagle's minimal essential medium in the presence of 10% fetal bovine serum and 1% penicillin-streptomycin solution (Invitrogen, Carlsbad, Calif.), respectively. The RW strain of MuV used throughout the present study was kindly supplied by J. S. Wolinsky, the Department of Neurology at Johns Hopkins University School of Medicine. The virus was grown in Vero cells and was titrated by plaque assay. Human IFN-? was purchased from Toray Industries, Inc. (Tokyo, Japan).
Plasmids and antibodies.
Four plasmids encoding MuV V protein of the Torii strain (pTM-V) and three mutant V proteins in which the cysteine residue at amino acid position 189, 207, or 214 was replaced by an alanine residue (pTM-Vc189a, pTM-Vc207a, or pTM-Vc214a, respectively) were used. These plasmids were constructed as described previously (19, 48). Polyclonal antibodies against human STAT1 p84/p91 (E-23), STAT2 p133 (C-20), and STAT1 phosphorylated at Y701 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Rabbit antibodies against Jak1, Tyk2, and STAT2 phosphorylated at Y689 were obtained from Upstate, Inc. (Charlottesville, Va.). Antibody to Jak1 phosphorylated at Y1022 and Y1023 [anti-Jak1pYpY(1022/1023) antibody] was obtained from BioSource International (Camarillo, Calif.). Antibody to Tyk2 phosphorylated at Y1054 and Y1055 [anti-Tyk2pYpY(1054/1055) antibody] was obtained from New England Biolabs, Inc. (Cambridge, Mass.). Monospecific polyclonal sera against MuV P, V (39), and HN, prepared at our institute, were also used. Labeled anti-mouse immunoglobulin G conjugates and anti-rabbit immunoglobulin G conjugates, used for the immunofluorescence experiments, were purchased from Molecular Probes, Inc. (Eugene, Oreg.).
Assay for antiviral activity.
Vero cells were plated 2 days prior to virus infection in 12-well or 48-well plates. Before the IFN treatment, monolayered Vero cells were inoculated with the RW strain of MuV at a multiplicity of infection (MOI) of 1 (Fig. 1) or that indicated in the text (Fig. 2). At 72 h (Fig. 1) or 24 h (Fig. 2) postinfection, the cells were treated with several concentrations of IFN-? as indicated in the text and were cultured for 24 h. Then, the cells were challenged with vesicular stomatitis virus (VSV) for 24 h and were fixed and stained with naftol blue black solution (0.1% naftol blue black, 0.1% sodium acetate, 9% acetate). Extracts from both attached and detached cells in parallel samples were prepared and analyzed by immunoblotting.
Immunoblotting.
MuV-infected and mock-infected cells were washed with phosphate-buffered saline (PBS) once just before harvesting, and the cells were lysed in a dish or plate on ice with cell lysis buffer (50 mM HEPES [pH 7.5], 4 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40) containing 1 mM phenylmethylsulfonyl fluoride, 2 mg of aprotinin/ml, and 0.1 mM Na3VO4. The cell lysates were centrifuged at 10,000 x g for 5 min, and the supernatants were used for the subsequent experiments.
For detection of the MuV proteins STAT1, pY701-STAT1, STAT2, pY689-STAT2, Jak1, and Tyk2, the soluble supernatants of cell lysates were subjected to NuPAGE (Invitrogen), electrotransferred onto nitrocellulose membranes, and immunoblotted with the specific antibody. For the detection of tyrosine-phosphorylated Jak1 at positions 1022 and 1023 and Tyk2 at positions 1054 and 1055, the soluble supernatants were first mixed with 5 μg of anti-Jak1 or anti-Tyk2 antibody and were incubated at 4°C for 1 h. Then, 20 μg of protein A-Sepharose 4B (Amersham Pharmacia Biotech, Inc., Uppsala, Sweden) was added to the cultures, which were then incubated at 4°C for 3 h. The respective proteins trapped by antibody were subjected to NuPAGE, electrotransferred, and then probed with anti-phospho Jak1 or anti-phospho Tyk2 antibodies.
Immunofluorescence.
An indirect-immunofluorescence assay was carried out to observe the protein localization in the cells. CV1 cells grown to 60% confluence on chamber slides (Nalge Nunc International, Naperville, Ill.) were inoculated with the RW strain of MuV at an MOI of 0.05. At 12 h postinfection, the cells were treated with IFN-? at 0 or 1,000 IU/ml for 30 min. The cells were then fixed with 10% formaldehyde in PBS for 10 min. After washing four times with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and were incubated with primary antibody against MuV NH and STAT1 for 1 h. The cells were then washed five times with PBS and were incubated with secondary antibody for 1 h. After being washed three times with PBS, immunofluorescence-stained cells were observed using a fluorescence microscope, Axiovert 135 (Carl Zeiss Japan, Tokyo, Japan).
Plasmid transfection.
CV1 and Vero cells grown on 24-well plates to approximately 70 to 80% confluence were transfected with pTM-V, pTM-Vc189a, pTM-Vc207a, or pTM-Vc214a by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Forty-eight hours later, the cells were then incubated with or without IFN-? at 1,000 IU/ml at the time indicated in Fig. 5 and 6, and the transiently expressed protein was analyzed by immunoblotting.
RESULTS MuV antagonizes the antiviral effects of IFN-?.
The RW strain of MuV is able to infect several cell lines, including human and monkey cell lines, without manifesting obvious cytopathic effects (23, 24). The final virus yield of the RW strain is almost the same as that of the other MuV strains that lead to the formation of giant cells by cell fusion. We used the nonapparent cytopathic character of the MuV RW strain to see how MuV infection inhibited the establishment of the antiviral state induced by IFNs. When the cells were incubated with IFN-? for 24 h at 0, 10, 100, or 1,000 IU/ml, MuV-preinfected cells were detached from the plate following the VSV challenge at any concentration of IFN used while the mock preinfected cells treated with IFN-? at 100 or 1,000 IU/ml attached to the plate even after the VSV challenge (Fig. 1A). The RW strain of MuV has thus been confirmed to antagonize the antiviral activity of IFN, as shown in the case of other MuV strains (18).
Until recently, the V protein of MuV has been reported to degrade STAT1, a key molecule of IFN-/? signal transduction, and to thereby antagonize the antiviral activity of IFN (18, 42). To observe the degradation of STAT1, we prepared whole-cell extracts of both attached and detached cells at 24 h post-VSV challenge following IFN-? treatment (Fig. 1B). The STAT1 levels in MuV-infected cells were certainly lower than those in uninfected cells, indicating the degradation of STAT1, but STAT1 was still detected and the degradation of STAT1 was also found to be incomplete (Fig. 1B). Viral P and V proteins in MuV-infected cells showed that MuV had actually grown in these cells.
These results suggested that anti-VSV activity induced by IFN was generated even in the presence of STAT1 in MuV-infected cells. However, it is also possible that the remaining STAT1 proteins were derived from cells that could not have been infected with MuV under these experimental conditions. To clarify the relationship between the MOI of the input MuV and the degradation of STAT1, we then investigated the correlation between the inhibition of IFN-?-induced antiviral activity and cellular STAT1 levels under conditions with a relatively high MOI. MuV-infected cells at different MOI doses were subsequently incubated with IFN-? at 0, 103, or 104 IU/ml for 24 h and were challenged with VSV (Fig. 2).
In MuV-uninfected cells preincubated with IFN-? at both 103 and 104 IU/ml induced resistance against VSV infection by the antiviral activity of IFN, as shown in Fig. 1, while in MuV-infected cells at an MOI of 1.56 or higher, no IFN-? antiviral activity was generated and those cells detached completely from the plate (Fig. 2A). However, the antiviral activity was generated in the MuV-infected cells at an MOI of 0.39 or lower, as was the case in MuV-uninfected cells, and those cells were attached to the bottom of the culture plate. These results indicated that MuV infection at an MOI of 1.0, in which almost all of the cells were expected to be infected by one virus, was a boundary point determining whether or not IFN-? led cells to an antiviral state, thus preventing VSV multiplication.
In these experimental conditions, the MuV P protein was detected in cells infected at an MOI of 1.56 or higher and the MuV V protein was detected at an MOI of 6.25 or higher (Fig. 2B). The discrepancy between the results obtained with these two antibodies was probably due to the differences in the intracellular viral protein levels and the antibody titers. The STAT1 level in the cells decreased in accordance with the increase in the MOI. There were no critical differences in the STAT1 levels in the cells at an MOI of 0.39 and an MOI of 1.56; the former resisted VSV infection, and the latter did not. Interestingly, even in the extract from cells that were infected with MuV at an MOI of 100, STAT1 was only incompletely degraded and was still present under these conditions. In addition, in the separate experiment designed as detailed above, the infected cells could be immunostained fluorescently with anti-MuV serum in proportion to the MOI by using fluorescence microscopy. The cells infected with an MOI of 1.56 or more were stained wholly (data not shown). These results clearly indicated that the remaining STAT1 was not preceded by cells that were not infected with MuV and that the inhibition of antiviral activity induced by IFNs occurred even in the presence of STAT1. The complete degradation of STAT1 is thus shown to be nonessential for antagonizing IFN.
Inhibition of IFN-?-induced STAT1 nuclear translocation in MuV-infected cells.
In MuV-infected cells, the antiviral effect of IFN-? could be blocked long before the complete degradation of STAT1. In virus-uninfected cells, IFN-? stimulation led to the nuclear translocation of STAT1, together with that of STAT2 and IRF9, thereby forming a multiprotein complex that functions as a transcriptional activator, ISGF3. To determine which step of IFN-/? signaling was inhibited, the IFN-?-induced nuclear translocation of STAT1 was examined by indirect immunofluorescence assay. As shown in Fig. 3, without IFN-? treatment, cellular STAT1 was located in the cytoplasms of uninfected cells. However, treatment with IFN-? for 30 min led to the accumulation of STAT1 in the nuclei. These nuclear translocations were also observed when anti-STAT2 antibody was used instead of anti-STAT1 antibody (data not shown). In the MuV-infected cells, no nuclear translocation of STAT1 (Fig. 3) and STAT2 (data not shown) was observed, even in the presence of IFN-?. These results indicated that the MuV infection inhibited IFN-? signaling, at least at the nuclear translocation step of STAT1 and STAT2 proteins, which is required for the activation of ISG transcription.
Effects of MuV infection on the IFN-?-induced phosphorylation of signaling molecules.
The binding of IFN-/? to the cell surface receptor triggers IFN signaling. The stimulation of IFN-? is transduced from the surface receptor to the nucleus by the sequential phosphorylation of signaling molecules such as the type I IFN receptors Jak1, Tyk2, STAT1, and STAT2 at a tyrosine residue. To determine why STAT1 and STAT2 were retained in the cytoplasms of MuV-infected cells regardless of IFN-/? treatment, the IFN-?-induced phosphorylation of STAT1 and STAT2 was initially examined in MuV-infected cells. MuV-infected and mock-infected cells were incubated with IFN-? for 30 min. Then, pY701-STAT1 levels, as well as total STAT1 levels, were respectively measured by immunoblot analysis using specific antibodies (Fig. 4A). In mock-infected cells, pY701-STAT1 was not detected without IFN-? but pY701-STAT1 was detected at 30 min following incubation with IFN-? (Fig. 4A). Within this short incubation period, the total STAT1 levels did not change significantly. On the other hand, in MuV-infected cells, the pY701-STAT1 level was significantly reduced after IFN-? treatment in comparison with that in mock-infected cells, thus indicating the inhibition of STAT1 phosphorylation by MuV. Likewise, the respective pY689-STAT2 levels were measured using the specific antibodies (Fig. 4B). Although the levels of STAT2 did not change significantly during this short period of incubation with IFN-?, pY689-STAT2 was detected in the mock-infected cells after 30 min of IFN-? incubation. pY689-STAT2 was not detected either before or after the IFN-? treatment in MuV-infected cells. This result was consistent with the previous finding obtained by using MuV-infected FL cells (47).
We then examined the IFN-?-induced phosphorylation of Jak1 and Tyk2, which are located upstream of STAT1 and STAT2 phosphorylation in the IFN signaling pathway. In the mock-infected cells, Jak1 and Tyk2 phosphorylation was detected after 15 min of IFN-? treatment (Fig. 4C and D). Under the same conditions, Jak1 and Tyk2 in MuV-infected cells were phosphorylated at levels comparable to those in the uninfected cells. These results indicated that MuV infection did not affect Jak1 and Tyk2 phosphorylation but that it did inhibit STAT1 and STAT2 phosphorylation.
The MuV V protein inhibits the IFN-?-induced phosphorylation of both Y701-STAT1 and Y689-STAT2.
It was previously reported that the MuV V protein inhibits host IFN signaling by degrading STAT1 protein (18, 42). We then investigated whether or not V protein was also involved in the inhibition of the IFN-?-induced phosphorylation of STAT1 on Y701. Plasmid DNA that expressed the V protein of MuV was transfected and was transiently expressed in these cells (Fig. 5). After 48 h of transfection, both nontransfected and V-expressing plasmid-transfected cells were treated with IFN-? for 0 or 30 min. The cells expressed the V protein in a manner that depended on the amount (0.01, 0.1, or 1 μg) of V-expressing plasmid used for the transfection (Fig. 5A). The total STAT1 levels were nearly equal in these cells, independent of the amount of transfected plasmid and also independent of the duration of IFN-? treatment. In 0.01 or 0.1 μg of plasmid-transfected cells, pY701-STAT1 was detected after 30 min of IFN-? treatment, as was observed in the case of the nontransfected cells (data not shown). However, in 1 μg of plasmid-transfected cells, IFN-?-induced STAT1 phosphorylation on Y701 was significantly inhibited.
To more clearly observe this type of inhibition, a time course study was carried out to examine IFN-?-induced STAT1 phosphorylation on Y701 in both V-expressing plasmid-transfected cells and nontransfected cells (Fig. 5B and C). In the nontransfected cells, the phosphorylation of STAT1 on Y701 was not seen at 0 min of incubation but was detected starting at 10 min. This trend was observed to increase linearly in Vero cells until 40 min had passed (Fig. 5B) and in CV1 cells until 50 min had passed (Fig. 5C) under the condition involving IFN-? treatment. In the V-expressing plasmid-transfected cells, although the phosphorylation of STAT1 on Y701 also increased linearly, the pY701-STAT1 level at each time point examined in these cells was significantly lower than that observed in the nontransfected cells (Fig. 5B and C). Since the total STAT1 levels were almost identical in the nontransfected cells and in the V-transfected cells during the incubation period, these results indicated that V protein is able to inhibit IFN-?-induced STAT1 phosphorylation on Y701. In the MuV-infected cells, phosphorylation of both STAT1 and STAT2 on tyrosines 701 and 689, respectively, was inhibited.
The effect of V protein expression on the tyrosine phosphorylation of STAT2 at 689 was then examined (Fig. 6). In the nontransfected cells, the phosphorylation of both STAT1 on 701 and STAT2 on 689 was not found in the IFN-?-untreated cells (0 min) but found in treated cells (30 min). The total STAT1 and STAT2 levels did not significantly change during the IFN-? incubation. In the same experimental condition but in the MuV V-protein-expressing cells, none of the pY701-STAT1 or pY689-STAT2 was found even after the incubation of IFN-?. Since the total STAT1 and STAT2 levels were not changed before and after the IFN-? treatment, the phosphorylation of STAT2 on Y689 was thus found to be inhibited by the V protein. These findings suggested that the MuV V protein alone can antagonize IFN-? by inhibiting the tyrosine phosphorylation of both STAT1 and STAT2.
Importance of the conserved C-terminal cysteine-rich region of MuV V protein for the inhibition of IFN-?-induced STAT1 and STAT2 phosphorylation.
The cysteine-rich region within the carboxyl terminus of the V protein has been shown to contribute to STAT1 degradation in MuV and SV5 (11, 18, 47), and this region also appears to contribute to STAT2 degradation in hPIV2 (17, 28) because artificially mutated V proteins lacking this region or those with a substitution at relevant cysteine were unable to degrade each STAT when they were expressed intracellularly (2, 18, 28, 47). To determine whether or not these cysteine residues are also involved in the inhibition of IFN-?-induced STAT1 and STAT2 phosphorylation, plasmids were transfected to cells that expressed mutated V proteins in which the cysteine residue at position 189, 207, or 214 was replaced with an alanine (Vc189a, Vc207a, or Vc214a, respectively) (Fig. 6). In the cells examined, the total STAT1 and STAT2 levels did not change significantly. In nontransfected cells, pY701-STAT1 and pY689-STAT2 were not detected before the incubation of IFN-? but were detected after the incubation (30 min) (Fig. 6).
Though in the V protein-expressing plasmid-transfected cells, pY701-STAT1 and pY689-STAT2 were not found, in three plasmid-transfected mutant cell lines expressing Vc189a, Vc207a, and Vc214a, pY701-STAT1 and pY689-STAT2 were again detected at the same levels observed in nontransfected cells. Since these three mutant V proteins and wild-type V protein were expressed equally in these transfected cells (Fig. 6), the normal appearance of pY701-STAT1 and pY689-STAT2 was not thought to be caused by indirect effects such as low expression of or instability of the mutated V protein but instead was attributed to the direct effects of amino acid substitution. Namely, these mutations rendered the V protein nonfunctional in terms of the inhibition of STAT1 and STAT2 phosphorylation on Y701 and Y689, respectively.
DISCUSSION Among natural immunity engaged during early periods of viral infection, the IFN system plays a principal role in the antiviral response that is dedicated to fighting viruses. However, infection by numerous viruses belonging to the subfamily Paramyxovirinae has acquired the ability to alter IFN susceptibility in host cells (6, 7, 9, 15, 28, 31, 33, 35, 36, 40, 46, 49). Among these IFN antagonisms observed in paramyxoviruses, viruses belonging to the genera Rubulavirus and Avulavirus have been reported to inhibit the establishment of the antiviral state via the degradation of STAT1 or STAT2 through an ubiquitin-proteasome pathway (7, 47). The viral factor concerning STAT degradation is the V protein, which is able to degrade STAT1 or STAT2 without the aid of other viral elements (6, 18, 28, 31). Expression of the carboxyl-terminal V region of MuV and NDV degrades STAT1, and that of hPIV2 degrades STAT2 (12, 18, 28). The V proteins of MeV, the genus Morbillivirus, the Nipah and Hendra viruses, and the genus Henipavirus also antagonize IFN-induced host antiviral effects (30, 35, 36, 38, 49). Even though the carboxyl terminus of the V protein is the most highly conserved region among the proteins of the paramyxoviruses, IFN antagonisms of these viral V proteins do not induce the degradation of any STAT proteins but do inhibit the signaling step leading to the ISGF3 complex formation. These findings revealed that the means of antagonizing IFN-induced antiviral activity are diverse, in spite of the structural conservation of viral V proteins.
As was demonstrated in the present study, the RW strain of MuV antagonizes IFN by its V protein, not only via STAT1 degradation, but also by the inhibition of STAT1 and STAT2 phosphorylation. These two antagonizing functions are not RW strain specific, though the RW strain of MuV leads to few cytopathic effects in infected cells compared to that of other strains. Because there is no amino acid difference in the carboxyl terminus of V protein which is important for degradation between the RW and Torii strains, a representative of other strains causes the degradation of STAT1. In addition, the fact that transfection of a plasmid expressing the V protein of the Torii strain resulted in a reduction of IFN-?-induced STAT1 and STAT2 phosphorylation (Fig. 5 and 6) can generalize the two anti-IFN functions of MuV V proteins, degradation of STAT1 and inhibition of STAT1 and STAT2 phosphorylation. The RW strain is thus not unique among MuV strains with respect to IFN antagonism.
Our present and previous studies (47) have demonstrated that cysteine residues in the carboxyl terminus of the MuV V protein are required for the inhibition of both STAT1 and STAT2 phosphorylation (Fig. 6) and the induction of STAT1 degradation. However, it remains unknown whether or not both abilities were generated via the mediation of the same host molecule that binds to the cysteine residues. For example, we previously found that host RACK1 binds to the carboxyl-terminal region of V protein (19). RACK1 has seven WD (tryptophan-aspartic acid) repeat motifs that function as a protein-protein interaction domain and are known to interact with many signaling molecules, such as STAT1, ?-chain of type I IFN receptor, Jak1, Jak2, and Tyk2 (5, 10, 44). It is of note that RACK1 interacts with STAT1 and mediates the binding of STAT1 to the IFN receptor because this binding regulates Jak-mediated phosphorylation of STAT1 in response to IFN (43). DDB1 is another host protein known to interact with V protein (22). However, the contribution of DDB1 protein to the inhibition of STAT1 phosphorylation remains unclear at present.
STAT degradation by the rubulavirus V proteins, including MuV, was demonstrated in present and previous studies. Whereas the interruption of IFN signaling must occur as early as possible in the viral infection process, the degradation of STAT protein does not appear to occur so quickly. SV5 infection led to a decrease in the STAT1 level from around 4 h postinfection and to a complete loss of STAT1 by 8 h postinfection in 2fTGH cells (7), and it resulted in the disappearance of STAT1 by at least 12 h postinfection in Vero cells (34). In addition, hPIV2 infection led to a nearly complete loss of STAT2 at 9 h postinfection in CV1 cells (31). In MuV-infected CV1 and Vero cells, STAT1 degradation progresses more slowly. For example, when the RW strain of MuV was used to infect CV1 cells at an MOI of 10, 64.3% of the STAT1 remained in the cells at 24 h postinfection compared to the level in uninfected cells (Fig. 4A). Taking into consideration the fact that IFN signaling can be triggered at a very early stage of infection, the complete degradation of STAT was found to take place too late to interrupt the generation of the antiviral activity of IFN. Thus, it is quite natural that rubulaviruses, and in particular MuV, have mechanisms other than protein degradation to achieve the inhibition of IFN signaling that functions prior to the activation of host IFN signaling.
The coauthors of this study, Yokosawa et al., previously described the complete degradation of STAT1 in MuV-Torii-infected FL cells (47). In our separate experiment, we observed that STAT1 disappears in the MuV-Torii-infected FL cells but not in the MuV-RW-infected Vero and CV1 cells. In addition, if the Torii strain was inoculated to the Vero and CV1 cells, the complete degradation of STAT1 did not occur within our experimental conditions. On the other hand, if the RW strain was inoculated to the HeLa cells, the degradation of STAT1 occurred earlier and more extensively (data not shown). By these results, it is possible to say that the difference in the degradation kinetics of STAT1 is due to the cell types rather than virus strains. Regarding the STAT degradation pathway driven by rubulavirus V proteins, the presence of STAT1, STAT2, and DDB1 was at least thought to be essential (2, 32, 41). Since the additional unknown factors may be involved in the degradation process, levels of these known and unknown factors in cells would affect the degradation level of STAT1 during the MuV infection.
In a previous study, the coauthors of this study, Yokosawa et al., demonstrated that treatment with the proteasome inhibitor MG132 partially recovers cellular STAT1 levels as well as sensitivity to IFN-, at least at certain levels (47). This previous finding was inconsistent with the notion that IFN signaling was inhibited independent of STAT1 degradation in the cells examined. However, since the observed increases in IFN--induced IRF1 and 2-5AS mRNA levels, as measured by reverse transcription-PCR, were significantly lower in MG132-treated MuV-infected cells than in the corresponding MuV-uninfected cells, the recovery of IFN sensitivity following MG132 treatment would not be sufficient to establish an antiviral state. Moreover, treatment with proteasome inhibitors such as MG132 and lactacystin has a side effect on several cellular regulatory mechanisms and alters the profiles of cellular proteins that are involved in signal transduction, transcription, and translation (13). It therefore remains difficult to precisely determine the cytokine effects resulting from MG132 treatment.
Our present results clearly indicated that MuV infection reduces IFN-?-induced STAT1 phosphorylation at Y701 and STAT2 phosphorylation at Y689 as well as STAT1 level for inhibiting the establishment of an antiviral state by using the carboxyl-terminal region of the viral V protein. The mechanism by which MuV V protein induces these two effects remains to be clarified in future studies.
ACKNOWLEDGMENTS
This work was partially supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
We thank Minoru Kidokoro, Naoko Miyajima, and Sakura Saito.
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Department of Microbiology, School of Medicine, Sapporo Medical University, Sapporo, Hokkaido, Japan
ABSTRACT
Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-? were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-?-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-?-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.
INTRODUCTION
The antiviral activity of interferon (IFN) is a major host defense mechanism generated during the early phase of viral infection. Antiviral activity is induced through an IFN signaling process called the Jak-STAT pathway. Briefly, the binding of IFN-/? to the cell surface type I IFN receptor activates the two receptor-bound kinases Jak1 and Tyk2, which subsequently phosphorylate the tyrosine residues (Y) of STAT1 and STAT2 at positions 701 and 689, respectively. The transcriptional activator, the ISGF3 complex, composed of Y701-phosphorylated STAT1 (pY701-STAT1), Y689-phosphorylated STAT2 (pY689-STAT2), and IRF9, is once formed and translocated to the nuclei. The ISGF3 complex then activates IFN-stimulated genes (ISGs). Typical ISG products such as 2',5'-oligoadenylate synthetase (2-5AS), RNA-dependent protein kinase (PKR), and Mx protein are known to exert antiviral activities (21).
However, it was previously reported that some viruses evolve to acquire the ability of antagonizing IFN functions through the suppression of the IFN signal transduction pathway (3, 14, 25-27, 49). Among these viruses, the members of the family Paramyxoviridae, which includes many important human and animal pathogens as well as emerging viruses such as Hendra virus and Nipah virus, have been found to antagonize IFNs (6, 7, 9, 15, 28, 31, 35, 36, 40, 46, 49). The individual paramyxoviruses have their own properties, which are obtained by coevolution with their respective hosts, but these viruses share common features regarding genome structure and function. They are enveloped viruses with a linear, nonsegmented negative-sense RNA genome of 15 to 18 kb. On the genome, basically six genes, the nucleocapsid protein, phospho (P) protein, matrix protein, fusion glycoprotein, attachment glycoprotein (HN, H, or G), and large (L) protein genes, are carried in that order from the 3' to 5' ends (20). The P and L proteins form RNA-dependent RNA polymerase. Among these genes, more than one protein is exceptionally produced from the P gene and at least one of these proteins is involved in the anti-IFN activities of the paramyxoviruses. The P gene gives rise to P and V mRNAs by a mechanism known as RNA editing. From these two mRNAs, P and V proteins are respectively translated in almost all paramyxoviruses, but in some paramyxoviruses, the C protein is additionally translated by use of an overlapping reading frame.
The carboxyl terminus of the V protein has seven cysteine residues and is highly conserved among the paramyxoviruses. Simian virus 5 (SV5), Simian virus 41 (SV41), Mumps virus (MuV), and Human parainfluenza virus type 2 (hPIV2) belonging to the Rubulavirus genus and Newcastle disease virus (NDV) belonging to the Avulavirus genus have the P and V proteins, but not the C protein in the P gene, and all of these viruses have been shown to antagonize IFNs by using the V protein (1, 12, 18, 28, 31, 33). The viruses of the Morbillivirus and Henipavirus genera have the P, V, and C proteins in the P gene and have also been shown to counteract IFNs by using V protein (33, 35, 36, 40, 49). Among the viruses in these genera, nonnegligible anti-IFN activity was also reported to be associated with the C protein of the Nipah and measles viruses (33, 37). Although the P gene of Sendai virus (SeV) of the Respirovirus genus codes P, V, and C proteins, the SeV C protein does counteract IFNs in the signaling process but V protein does not (15, 16).
The means by which such viral proteins inhibit the Jak-STAT pathway differ among the paramyxoviruses (9). For example, the V protein of MuV, SV5, SV41, and NDV induces the degradation of STAT1 (1, 12, 18, 28) and the V protein of hPIV2 induces the degradation of STAT2 (1, 28, 31). On the other hand, the V proteins of measles, Nipah, and Hendra viruses generate anti-IFN activity without STAT degradation (30, 35, 36, 40). In this latter case, instead of STAT degradation, IFN-induced phosphorylation and nuclear localization of STAT1 and STAT2 are inhibited.
The degradation of STATs found in members of the Rubulavirus and Avulavirus genera was originally demonstrated in persistently infected cells and by using a plasmid-based V expression system (6, 18, 28, 31). The importance of the V-unique carboxyl-terminal region for degradation was subsequently indicated by several V protein expression studies (12, 18, 28). Such observations have been confirmed in the context of viral replication by using recombinant hPIV2, SV5, and NDV lacking carboxyl-terminal V-unique regions (11, 12, 17). However, it is noteworthy that a spontaneous SV5 mutant with mutations in the P/V common domain showed no anti-IFN activity, indicating the contribution of the P/V common domain for generating anti-IFN activity (4, 45, 50). The degradation of these STAT proteins is thought to be the result of an ubiquitin-proteasome pathway because the amount of STAT mRNA does not change following viral infection; in addition, a proteasome inhibitor, MG132, recovers the STAT level, although the recoveries are partial (7, 47).
The interaction of V protein with cellular proteins was examined using glutathione S-transferase-V fusion protein and a yeast two-hybrid system. In those studies, p127 UV-damage-specific DNA binding protein 1 (DDB1) and receptor for activated C kinase 1 (RACK1) were identified as cellular counterparts of SV5 V and MuV V proteins, respectively (19, 22). The carboxyl-terminal V-unique region is necessary for both interactions. Copurification experiments revealed that the V protein of SV5 and hPIV2 forms a complex with STAT1, STAT2, DDB1, and Cul4A (31, 41). The good correlation between the binding of SV5 V to DDB1 and its ability to degrade STAT1 has been well demonstrated (2). As Cul4A is one of the components of cellular ubiquitin ligase, STAT1 degradation by the V protein was estimated to occur through an ubiquitin-proteasome pathway by using DDB1 and Cul4A. The ubiquitination of STAT1 has been demonstrated in MuV-infected cells (42, 47). On the other hand, the biological significance of RACK1 remains unclear. The V proteins of Rubulavirus have also been reported to bind to both STAT1 and STAT2 at their carboxyl termini, and these interactions are thought to be necessary for the ubiquitination and degradation of STATs (29, 32).
In this study, we demonstrated that the antiviral activity of IFN could be established in MuV-infected cells before the degradation of STAT1. Our observations therefore indicate that the complete degradation of STAT1 is not required for generating IFN antagonism of MuV.
MATERIALS AND METHODS
Cells, viruses, and IFN. Simian-kidney-derived CV1 and Vero cells were grown in Dulbecco's modified Eagle's medium and in Eagle's minimal essential medium in the presence of 10% fetal bovine serum and 1% penicillin-streptomycin solution (Invitrogen, Carlsbad, Calif.), respectively. The RW strain of MuV used throughout the present study was kindly supplied by J. S. Wolinsky, the Department of Neurology at Johns Hopkins University School of Medicine. The virus was grown in Vero cells and was titrated by plaque assay. Human IFN-? was purchased from Toray Industries, Inc. (Tokyo, Japan).
Plasmids and antibodies.
Four plasmids encoding MuV V protein of the Torii strain (pTM-V) and three mutant V proteins in which the cysteine residue at amino acid position 189, 207, or 214 was replaced by an alanine residue (pTM-Vc189a, pTM-Vc207a, or pTM-Vc214a, respectively) were used. These plasmids were constructed as described previously (19, 48). Polyclonal antibodies against human STAT1 p84/p91 (E-23), STAT2 p133 (C-20), and STAT1 phosphorylated at Y701 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Rabbit antibodies against Jak1, Tyk2, and STAT2 phosphorylated at Y689 were obtained from Upstate, Inc. (Charlottesville, Va.). Antibody to Jak1 phosphorylated at Y1022 and Y1023 [anti-Jak1pYpY(1022/1023) antibody] was obtained from BioSource International (Camarillo, Calif.). Antibody to Tyk2 phosphorylated at Y1054 and Y1055 [anti-Tyk2pYpY(1054/1055) antibody] was obtained from New England Biolabs, Inc. (Cambridge, Mass.). Monospecific polyclonal sera against MuV P, V (39), and HN, prepared at our institute, were also used. Labeled anti-mouse immunoglobulin G conjugates and anti-rabbit immunoglobulin G conjugates, used for the immunofluorescence experiments, were purchased from Molecular Probes, Inc. (Eugene, Oreg.).
Assay for antiviral activity.
Vero cells were plated 2 days prior to virus infection in 12-well or 48-well plates. Before the IFN treatment, monolayered Vero cells were inoculated with the RW strain of MuV at a multiplicity of infection (MOI) of 1 (Fig. 1) or that indicated in the text (Fig. 2). At 72 h (Fig. 1) or 24 h (Fig. 2) postinfection, the cells were treated with several concentrations of IFN-? as indicated in the text and were cultured for 24 h. Then, the cells were challenged with vesicular stomatitis virus (VSV) for 24 h and were fixed and stained with naftol blue black solution (0.1% naftol blue black, 0.1% sodium acetate, 9% acetate). Extracts from both attached and detached cells in parallel samples were prepared and analyzed by immunoblotting.
Immunoblotting.
MuV-infected and mock-infected cells were washed with phosphate-buffered saline (PBS) once just before harvesting, and the cells were lysed in a dish or plate on ice with cell lysis buffer (50 mM HEPES [pH 7.5], 4 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40) containing 1 mM phenylmethylsulfonyl fluoride, 2 mg of aprotinin/ml, and 0.1 mM Na3VO4. The cell lysates were centrifuged at 10,000 x g for 5 min, and the supernatants were used for the subsequent experiments.
For detection of the MuV proteins STAT1, pY701-STAT1, STAT2, pY689-STAT2, Jak1, and Tyk2, the soluble supernatants of cell lysates were subjected to NuPAGE (Invitrogen), electrotransferred onto nitrocellulose membranes, and immunoblotted with the specific antibody. For the detection of tyrosine-phosphorylated Jak1 at positions 1022 and 1023 and Tyk2 at positions 1054 and 1055, the soluble supernatants were first mixed with 5 μg of anti-Jak1 or anti-Tyk2 antibody and were incubated at 4°C for 1 h. Then, 20 μg of protein A-Sepharose 4B (Amersham Pharmacia Biotech, Inc., Uppsala, Sweden) was added to the cultures, which were then incubated at 4°C for 3 h. The respective proteins trapped by antibody were subjected to NuPAGE, electrotransferred, and then probed with anti-phospho Jak1 or anti-phospho Tyk2 antibodies.
Immunofluorescence.
An indirect-immunofluorescence assay was carried out to observe the protein localization in the cells. CV1 cells grown to 60% confluence on chamber slides (Nalge Nunc International, Naperville, Ill.) were inoculated with the RW strain of MuV at an MOI of 0.05. At 12 h postinfection, the cells were treated with IFN-? at 0 or 1,000 IU/ml for 30 min. The cells were then fixed with 10% formaldehyde in PBS for 10 min. After washing four times with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min and were incubated with primary antibody against MuV NH and STAT1 for 1 h. The cells were then washed five times with PBS and were incubated with secondary antibody for 1 h. After being washed three times with PBS, immunofluorescence-stained cells were observed using a fluorescence microscope, Axiovert 135 (Carl Zeiss Japan, Tokyo, Japan).
Plasmid transfection.
CV1 and Vero cells grown on 24-well plates to approximately 70 to 80% confluence were transfected with pTM-V, pTM-Vc189a, pTM-Vc207a, or pTM-Vc214a by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Forty-eight hours later, the cells were then incubated with or without IFN-? at 1,000 IU/ml at the time indicated in Fig. 5 and 6, and the transiently expressed protein was analyzed by immunoblotting.
RESULTS MuV antagonizes the antiviral effects of IFN-?.
The RW strain of MuV is able to infect several cell lines, including human and monkey cell lines, without manifesting obvious cytopathic effects (23, 24). The final virus yield of the RW strain is almost the same as that of the other MuV strains that lead to the formation of giant cells by cell fusion. We used the nonapparent cytopathic character of the MuV RW strain to see how MuV infection inhibited the establishment of the antiviral state induced by IFNs. When the cells were incubated with IFN-? for 24 h at 0, 10, 100, or 1,000 IU/ml, MuV-preinfected cells were detached from the plate following the VSV challenge at any concentration of IFN used while the mock preinfected cells treated with IFN-? at 100 or 1,000 IU/ml attached to the plate even after the VSV challenge (Fig. 1A). The RW strain of MuV has thus been confirmed to antagonize the antiviral activity of IFN, as shown in the case of other MuV strains (18).
Until recently, the V protein of MuV has been reported to degrade STAT1, a key molecule of IFN-/? signal transduction, and to thereby antagonize the antiviral activity of IFN (18, 42). To observe the degradation of STAT1, we prepared whole-cell extracts of both attached and detached cells at 24 h post-VSV challenge following IFN-? treatment (Fig. 1B). The STAT1 levels in MuV-infected cells were certainly lower than those in uninfected cells, indicating the degradation of STAT1, but STAT1 was still detected and the degradation of STAT1 was also found to be incomplete (Fig. 1B). Viral P and V proteins in MuV-infected cells showed that MuV had actually grown in these cells.
These results suggested that anti-VSV activity induced by IFN was generated even in the presence of STAT1 in MuV-infected cells. However, it is also possible that the remaining STAT1 proteins were derived from cells that could not have been infected with MuV under these experimental conditions. To clarify the relationship between the MOI of the input MuV and the degradation of STAT1, we then investigated the correlation between the inhibition of IFN-?-induced antiviral activity and cellular STAT1 levels under conditions with a relatively high MOI. MuV-infected cells at different MOI doses were subsequently incubated with IFN-? at 0, 103, or 104 IU/ml for 24 h and were challenged with VSV (Fig. 2).
In MuV-uninfected cells preincubated with IFN-? at both 103 and 104 IU/ml induced resistance against VSV infection by the antiviral activity of IFN, as shown in Fig. 1, while in MuV-infected cells at an MOI of 1.56 or higher, no IFN-? antiviral activity was generated and those cells detached completely from the plate (Fig. 2A). However, the antiviral activity was generated in the MuV-infected cells at an MOI of 0.39 or lower, as was the case in MuV-uninfected cells, and those cells were attached to the bottom of the culture plate. These results indicated that MuV infection at an MOI of 1.0, in which almost all of the cells were expected to be infected by one virus, was a boundary point determining whether or not IFN-? led cells to an antiviral state, thus preventing VSV multiplication.
In these experimental conditions, the MuV P protein was detected in cells infected at an MOI of 1.56 or higher and the MuV V protein was detected at an MOI of 6.25 or higher (Fig. 2B). The discrepancy between the results obtained with these two antibodies was probably due to the differences in the intracellular viral protein levels and the antibody titers. The STAT1 level in the cells decreased in accordance with the increase in the MOI. There were no critical differences in the STAT1 levels in the cells at an MOI of 0.39 and an MOI of 1.56; the former resisted VSV infection, and the latter did not. Interestingly, even in the extract from cells that were infected with MuV at an MOI of 100, STAT1 was only incompletely degraded and was still present under these conditions. In addition, in the separate experiment designed as detailed above, the infected cells could be immunostained fluorescently with anti-MuV serum in proportion to the MOI by using fluorescence microscopy. The cells infected with an MOI of 1.56 or more were stained wholly (data not shown). These results clearly indicated that the remaining STAT1 was not preceded by cells that were not infected with MuV and that the inhibition of antiviral activity induced by IFNs occurred even in the presence of STAT1. The complete degradation of STAT1 is thus shown to be nonessential for antagonizing IFN.
Inhibition of IFN-?-induced STAT1 nuclear translocation in MuV-infected cells.
In MuV-infected cells, the antiviral effect of IFN-? could be blocked long before the complete degradation of STAT1. In virus-uninfected cells, IFN-? stimulation led to the nuclear translocation of STAT1, together with that of STAT2 and IRF9, thereby forming a multiprotein complex that functions as a transcriptional activator, ISGF3. To determine which step of IFN-/? signaling was inhibited, the IFN-?-induced nuclear translocation of STAT1 was examined by indirect immunofluorescence assay. As shown in Fig. 3, without IFN-? treatment, cellular STAT1 was located in the cytoplasms of uninfected cells. However, treatment with IFN-? for 30 min led to the accumulation of STAT1 in the nuclei. These nuclear translocations were also observed when anti-STAT2 antibody was used instead of anti-STAT1 antibody (data not shown). In the MuV-infected cells, no nuclear translocation of STAT1 (Fig. 3) and STAT2 (data not shown) was observed, even in the presence of IFN-?. These results indicated that the MuV infection inhibited IFN-? signaling, at least at the nuclear translocation step of STAT1 and STAT2 proteins, which is required for the activation of ISG transcription.
Effects of MuV infection on the IFN-?-induced phosphorylation of signaling molecules.
The binding of IFN-/? to the cell surface receptor triggers IFN signaling. The stimulation of IFN-? is transduced from the surface receptor to the nucleus by the sequential phosphorylation of signaling molecules such as the type I IFN receptors Jak1, Tyk2, STAT1, and STAT2 at a tyrosine residue. To determine why STAT1 and STAT2 were retained in the cytoplasms of MuV-infected cells regardless of IFN-/? treatment, the IFN-?-induced phosphorylation of STAT1 and STAT2 was initially examined in MuV-infected cells. MuV-infected and mock-infected cells were incubated with IFN-? for 30 min. Then, pY701-STAT1 levels, as well as total STAT1 levels, were respectively measured by immunoblot analysis using specific antibodies (Fig. 4A). In mock-infected cells, pY701-STAT1 was not detected without IFN-? but pY701-STAT1 was detected at 30 min following incubation with IFN-? (Fig. 4A). Within this short incubation period, the total STAT1 levels did not change significantly. On the other hand, in MuV-infected cells, the pY701-STAT1 level was significantly reduced after IFN-? treatment in comparison with that in mock-infected cells, thus indicating the inhibition of STAT1 phosphorylation by MuV. Likewise, the respective pY689-STAT2 levels were measured using the specific antibodies (Fig. 4B). Although the levels of STAT2 did not change significantly during this short period of incubation with IFN-?, pY689-STAT2 was detected in the mock-infected cells after 30 min of IFN-? incubation. pY689-STAT2 was not detected either before or after the IFN-? treatment in MuV-infected cells. This result was consistent with the previous finding obtained by using MuV-infected FL cells (47).
We then examined the IFN-?-induced phosphorylation of Jak1 and Tyk2, which are located upstream of STAT1 and STAT2 phosphorylation in the IFN signaling pathway. In the mock-infected cells, Jak1 and Tyk2 phosphorylation was detected after 15 min of IFN-? treatment (Fig. 4C and D). Under the same conditions, Jak1 and Tyk2 in MuV-infected cells were phosphorylated at levels comparable to those in the uninfected cells. These results indicated that MuV infection did not affect Jak1 and Tyk2 phosphorylation but that it did inhibit STAT1 and STAT2 phosphorylation.
The MuV V protein inhibits the IFN-?-induced phosphorylation of both Y701-STAT1 and Y689-STAT2.
It was previously reported that the MuV V protein inhibits host IFN signaling by degrading STAT1 protein (18, 42). We then investigated whether or not V protein was also involved in the inhibition of the IFN-?-induced phosphorylation of STAT1 on Y701. Plasmid DNA that expressed the V protein of MuV was transfected and was transiently expressed in these cells (Fig. 5). After 48 h of transfection, both nontransfected and V-expressing plasmid-transfected cells were treated with IFN-? for 0 or 30 min. The cells expressed the V protein in a manner that depended on the amount (0.01, 0.1, or 1 μg) of V-expressing plasmid used for the transfection (Fig. 5A). The total STAT1 levels were nearly equal in these cells, independent of the amount of transfected plasmid and also independent of the duration of IFN-? treatment. In 0.01 or 0.1 μg of plasmid-transfected cells, pY701-STAT1 was detected after 30 min of IFN-? treatment, as was observed in the case of the nontransfected cells (data not shown). However, in 1 μg of plasmid-transfected cells, IFN-?-induced STAT1 phosphorylation on Y701 was significantly inhibited.
To more clearly observe this type of inhibition, a time course study was carried out to examine IFN-?-induced STAT1 phosphorylation on Y701 in both V-expressing plasmid-transfected cells and nontransfected cells (Fig. 5B and C). In the nontransfected cells, the phosphorylation of STAT1 on Y701 was not seen at 0 min of incubation but was detected starting at 10 min. This trend was observed to increase linearly in Vero cells until 40 min had passed (Fig. 5B) and in CV1 cells until 50 min had passed (Fig. 5C) under the condition involving IFN-? treatment. In the V-expressing plasmid-transfected cells, although the phosphorylation of STAT1 on Y701 also increased linearly, the pY701-STAT1 level at each time point examined in these cells was significantly lower than that observed in the nontransfected cells (Fig. 5B and C). Since the total STAT1 levels were almost identical in the nontransfected cells and in the V-transfected cells during the incubation period, these results indicated that V protein is able to inhibit IFN-?-induced STAT1 phosphorylation on Y701. In the MuV-infected cells, phosphorylation of both STAT1 and STAT2 on tyrosines 701 and 689, respectively, was inhibited.
The effect of V protein expression on the tyrosine phosphorylation of STAT2 at 689 was then examined (Fig. 6). In the nontransfected cells, the phosphorylation of both STAT1 on 701 and STAT2 on 689 was not found in the IFN-?-untreated cells (0 min) but found in treated cells (30 min). The total STAT1 and STAT2 levels did not significantly change during the IFN-? incubation. In the same experimental condition but in the MuV V-protein-expressing cells, none of the pY701-STAT1 or pY689-STAT2 was found even after the incubation of IFN-?. Since the total STAT1 and STAT2 levels were not changed before and after the IFN-? treatment, the phosphorylation of STAT2 on Y689 was thus found to be inhibited by the V protein. These findings suggested that the MuV V protein alone can antagonize IFN-? by inhibiting the tyrosine phosphorylation of both STAT1 and STAT2.
Importance of the conserved C-terminal cysteine-rich region of MuV V protein for the inhibition of IFN-?-induced STAT1 and STAT2 phosphorylation.
The cysteine-rich region within the carboxyl terminus of the V protein has been shown to contribute to STAT1 degradation in MuV and SV5 (11, 18, 47), and this region also appears to contribute to STAT2 degradation in hPIV2 (17, 28) because artificially mutated V proteins lacking this region or those with a substitution at relevant cysteine were unable to degrade each STAT when they were expressed intracellularly (2, 18, 28, 47). To determine whether or not these cysteine residues are also involved in the inhibition of IFN-?-induced STAT1 and STAT2 phosphorylation, plasmids were transfected to cells that expressed mutated V proteins in which the cysteine residue at position 189, 207, or 214 was replaced with an alanine (Vc189a, Vc207a, or Vc214a, respectively) (Fig. 6). In the cells examined, the total STAT1 and STAT2 levels did not change significantly. In nontransfected cells, pY701-STAT1 and pY689-STAT2 were not detected before the incubation of IFN-? but were detected after the incubation (30 min) (Fig. 6).
Though in the V protein-expressing plasmid-transfected cells, pY701-STAT1 and pY689-STAT2 were not found, in three plasmid-transfected mutant cell lines expressing Vc189a, Vc207a, and Vc214a, pY701-STAT1 and pY689-STAT2 were again detected at the same levels observed in nontransfected cells. Since these three mutant V proteins and wild-type V protein were expressed equally in these transfected cells (Fig. 6), the normal appearance of pY701-STAT1 and pY689-STAT2 was not thought to be caused by indirect effects such as low expression of or instability of the mutated V protein but instead was attributed to the direct effects of amino acid substitution. Namely, these mutations rendered the V protein nonfunctional in terms of the inhibition of STAT1 and STAT2 phosphorylation on Y701 and Y689, respectively.
DISCUSSION Among natural immunity engaged during early periods of viral infection, the IFN system plays a principal role in the antiviral response that is dedicated to fighting viruses. However, infection by numerous viruses belonging to the subfamily Paramyxovirinae has acquired the ability to alter IFN susceptibility in host cells (6, 7, 9, 15, 28, 31, 33, 35, 36, 40, 46, 49). Among these IFN antagonisms observed in paramyxoviruses, viruses belonging to the genera Rubulavirus and Avulavirus have been reported to inhibit the establishment of the antiviral state via the degradation of STAT1 or STAT2 through an ubiquitin-proteasome pathway (7, 47). The viral factor concerning STAT degradation is the V protein, which is able to degrade STAT1 or STAT2 without the aid of other viral elements (6, 18, 28, 31). Expression of the carboxyl-terminal V region of MuV and NDV degrades STAT1, and that of hPIV2 degrades STAT2 (12, 18, 28). The V proteins of MeV, the genus Morbillivirus, the Nipah and Hendra viruses, and the genus Henipavirus also antagonize IFN-induced host antiviral effects (30, 35, 36, 38, 49). Even though the carboxyl terminus of the V protein is the most highly conserved region among the proteins of the paramyxoviruses, IFN antagonisms of these viral V proteins do not induce the degradation of any STAT proteins but do inhibit the signaling step leading to the ISGF3 complex formation. These findings revealed that the means of antagonizing IFN-induced antiviral activity are diverse, in spite of the structural conservation of viral V proteins.
As was demonstrated in the present study, the RW strain of MuV antagonizes IFN by its V protein, not only via STAT1 degradation, but also by the inhibition of STAT1 and STAT2 phosphorylation. These two antagonizing functions are not RW strain specific, though the RW strain of MuV leads to few cytopathic effects in infected cells compared to that of other strains. Because there is no amino acid difference in the carboxyl terminus of V protein which is important for degradation between the RW and Torii strains, a representative of other strains causes the degradation of STAT1. In addition, the fact that transfection of a plasmid expressing the V protein of the Torii strain resulted in a reduction of IFN-?-induced STAT1 and STAT2 phosphorylation (Fig. 5 and 6) can generalize the two anti-IFN functions of MuV V proteins, degradation of STAT1 and inhibition of STAT1 and STAT2 phosphorylation. The RW strain is thus not unique among MuV strains with respect to IFN antagonism.
Our present and previous studies (47) have demonstrated that cysteine residues in the carboxyl terminus of the MuV V protein are required for the inhibition of both STAT1 and STAT2 phosphorylation (Fig. 6) and the induction of STAT1 degradation. However, it remains unknown whether or not both abilities were generated via the mediation of the same host molecule that binds to the cysteine residues. For example, we previously found that host RACK1 binds to the carboxyl-terminal region of V protein (19). RACK1 has seven WD (tryptophan-aspartic acid) repeat motifs that function as a protein-protein interaction domain and are known to interact with many signaling molecules, such as STAT1, ?-chain of type I IFN receptor, Jak1, Jak2, and Tyk2 (5, 10, 44). It is of note that RACK1 interacts with STAT1 and mediates the binding of STAT1 to the IFN receptor because this binding regulates Jak-mediated phosphorylation of STAT1 in response to IFN (43). DDB1 is another host protein known to interact with V protein (22). However, the contribution of DDB1 protein to the inhibition of STAT1 phosphorylation remains unclear at present.
STAT degradation by the rubulavirus V proteins, including MuV, was demonstrated in present and previous studies. Whereas the interruption of IFN signaling must occur as early as possible in the viral infection process, the degradation of STAT protein does not appear to occur so quickly. SV5 infection led to a decrease in the STAT1 level from around 4 h postinfection and to a complete loss of STAT1 by 8 h postinfection in 2fTGH cells (7), and it resulted in the disappearance of STAT1 by at least 12 h postinfection in Vero cells (34). In addition, hPIV2 infection led to a nearly complete loss of STAT2 at 9 h postinfection in CV1 cells (31). In MuV-infected CV1 and Vero cells, STAT1 degradation progresses more slowly. For example, when the RW strain of MuV was used to infect CV1 cells at an MOI of 10, 64.3% of the STAT1 remained in the cells at 24 h postinfection compared to the level in uninfected cells (Fig. 4A). Taking into consideration the fact that IFN signaling can be triggered at a very early stage of infection, the complete degradation of STAT was found to take place too late to interrupt the generation of the antiviral activity of IFN. Thus, it is quite natural that rubulaviruses, and in particular MuV, have mechanisms other than protein degradation to achieve the inhibition of IFN signaling that functions prior to the activation of host IFN signaling.
The coauthors of this study, Yokosawa et al., previously described the complete degradation of STAT1 in MuV-Torii-infected FL cells (47). In our separate experiment, we observed that STAT1 disappears in the MuV-Torii-infected FL cells but not in the MuV-RW-infected Vero and CV1 cells. In addition, if the Torii strain was inoculated to the Vero and CV1 cells, the complete degradation of STAT1 did not occur within our experimental conditions. On the other hand, if the RW strain was inoculated to the HeLa cells, the degradation of STAT1 occurred earlier and more extensively (data not shown). By these results, it is possible to say that the difference in the degradation kinetics of STAT1 is due to the cell types rather than virus strains. Regarding the STAT degradation pathway driven by rubulavirus V proteins, the presence of STAT1, STAT2, and DDB1 was at least thought to be essential (2, 32, 41). Since the additional unknown factors may be involved in the degradation process, levels of these known and unknown factors in cells would affect the degradation level of STAT1 during the MuV infection.
In a previous study, the coauthors of this study, Yokosawa et al., demonstrated that treatment with the proteasome inhibitor MG132 partially recovers cellular STAT1 levels as well as sensitivity to IFN-, at least at certain levels (47). This previous finding was inconsistent with the notion that IFN signaling was inhibited independent of STAT1 degradation in the cells examined. However, since the observed increases in IFN--induced IRF1 and 2-5AS mRNA levels, as measured by reverse transcription-PCR, were significantly lower in MG132-treated MuV-infected cells than in the corresponding MuV-uninfected cells, the recovery of IFN sensitivity following MG132 treatment would not be sufficient to establish an antiviral state. Moreover, treatment with proteasome inhibitors such as MG132 and lactacystin has a side effect on several cellular regulatory mechanisms and alters the profiles of cellular proteins that are involved in signal transduction, transcription, and translation (13). It therefore remains difficult to precisely determine the cytokine effects resulting from MG132 treatment.
Our present results clearly indicated that MuV infection reduces IFN-?-induced STAT1 phosphorylation at Y701 and STAT2 phosphorylation at Y689 as well as STAT1 level for inhibiting the establishment of an antiviral state by using the carboxyl-terminal region of the viral V protein. The mechanism by which MuV V protein induces these two effects remains to be clarified in future studies.
ACKNOWLEDGMENTS
This work was partially supported by research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
We thank Minoru Kidokoro, Naoko Miyajima, and Sakura Saito.
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