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1 Department of Virology, University of Bremen, Leobener Straße/UFT, D-28359 Bremen, Germany
2 Children's Hospital, Department 1, University of Tübingen, Silcherstraße 7, D-72076 Tübingen, Germany
Correspondence
Andreas Dotzauer
dotzauer{at}uni-bremen.de
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ABSTRACT |
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B kinase 
(IKK). In consequence, interferon regulatory factor 3 (IRF-3) is not activated. As IRF-3 is necessary for IFN-β transcription, inhibition of this factor results in efficient suppression of IFN-β synthesis. This ability might be of vital importance for HAV, which is an exceptionally slow growing virus sensitive to IFN-β, as it allows the virus to establish infection and maintain virus replication for a longer period of time.
These authors contributed equally to this work. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Gust & Feinstone, 1988). The virus is eliminated from the organism mainly by the activity of HAV-specific cytotoxic T lymphocytes, resulting in massive hepatocyte destruction (Fleischer et al., 1990; Maier et al., 1988; Vallbracht et al., 1989). At the cellular level, HAV infections are not normally cytopathic (Brack et al., 1998), and in cell culture without adaptive immune responses, HAV replication results in a persistent, chronic infection (Dotzauer et al., 1994; Gauss-Müller & Deinhardt, 1984; Vallbracht et al., 1984). This indicates HAV mechanisms that evade the host cell-based, innate antiviral responses, and as persistent HAV infections in cell culture are eradicated by exogenously added type 1 interferons (IFNs) (Vallbracht & Flehmig, 1985a; Vallbracht et al., 1984), showing that HAV is not resistant to these IFNs, the virus should be able to interfere with type 1 IFN synthesis or secretion in infected cells. This is supported by experiments in which neither type 1 IFN nor interference with infection by other viruses could be detected in cells infected with HAV (Vallbracht et al., 1984, 1985b).
Recently, we presented data showing that HAV prevents beta interferon (IFN-β) synthesis (Brack et al., 2002) by blocking activation of interferon regulatory factor 3 (IRF-3) (Fensterl et al., 2005), which is necessary for IFN-β transcription. We demonstrated that HAV replication inhibits activation of IRF-3 through both the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA-5) pathways. RIG-I appears to be the major recognition receptor that senses single-stranded 5'-triphosphate RNA (Hornung et al., 2006; Kato et al., 2006; Pichlmair et al., 2006), whereas MDA-5 appears to be the major recognition receptor that senses poly(IC) and single-/double-stranded picornaviral RNA covalently linked to a viral protein (3B), respectively (Gitlin et al., 2006; Kato et al., 2006). Both RIG-I and MDA-5 regulate downstream activation of the TANK-binding kinase 1 (TBK1) and the inhibitor of NF-B kinase (IKK) (Fitzgerald et al., 2003; Hemmi et al., 2004; McWhirter et al., 2004; Sharma et al., 2003). These kinases phosphorylate IRF-3, which results in IRF-3 dimerization and cytoplasmic-to-nuclear translocation (Lin et al., 1998), where it induces IFN-β transcription as a central component of the enhanceosome complex by interaction with the positive-regulatory domain III-I (PRDIII-I) of the IFN-β enhancer (Thanos & Maniatis, 1995; Yang et al., 2004). After activation of the receptors RIG-I/MDA-5, HAV interferes within this pathway with a component or components involved in phosphorylation of IRF-3 by the IKK/TBK1 kinase complex (Fensterl et al., 2005). Furthermore, we found that HAV is also able to interfere with TRIF (TIR domain-containing adaptor inducing IFN-β), which serves as an adaptor connecting the RNA virus-sensing Toll-like receptor 3 with the IKK/TBK1 kinases (Oshiumi et al., 2003; Sato et al., 2003; Yamamoto et al., 2002). We suggested that the ability of HAV to inhibit transcription of IFN-β together with the ability to prevent dsRNA-induced apoptosis (Brack et al., 2002) helps the virus to establish an infection and preserves the sites of virus production for a longer period of time.
Meanwhile, an adaptor protein connecting RIG-I and MDA-5 with the kinases has been identified. Ligand binding to both RIG-I and MDA-5 results in complex formation with the adaptor molecule, variously called MAVS, IPS-1, VISA or Cardif (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). MAVS (mitochondrial antiviral signalling protein) is localized to the outer mitochondrial membrane and mediates the recruitment and activation of the IKK/TBK1 kinases (Seth et al., 2005). Based on our finding that HAV interferes with processes occurring between RIG-I/MDA-5 activation and IRF-3 phosphorylation, MAVS represents a possible target for HAV, and in a recent report, MAVS was suggested as the site of HAV intervention (Yang et al., 2007). These authors showed that MAVS is targeted for HAV protease 3C-mediated proteolysis by 3ABC, an intermediate product of HAV polyprotein processing targeted to mitochondria by 3A.
Our investigation aimed to clarify where HAV interferes with the RIG-I/MDA-5 pathway and which viral factor is involved. We also found that HAV interferes with the activity of MAVS, resulting in inhibition of IFN-β transcription. However, we observed that this ability correlated not only with ectopic expression of 3ABC but also with ectopic expression of HAV non-structural protein 2B, which in addition to its inhibitory effect on the function of MAVS also affects the functions of the IRF-3 kinases TBK1 and IKK.
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METHODS |
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) were maintained as continuous cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 % fetal calf serum (FCS). The cells were split at a ratio of 1 : 5 after detaching them from the tissue culture plate with trypsin supplemented with 0.2 g sodium EDTA l–1 and cultivated with DMEM plus 10 % FCS as growth medium. These cells, which are deficient in an IFN response (Brack et al., 2002), express RIG-I as well as MDA-5. Transient transfections were performed by jetPEI transfection of 5 µg plasmid for reporter assays (58 mm dishes) and of 1 µg plasmid for immunofluorescence analysis (chamber slides), as instructed by the manufacturer (Qbiogene).
Poly(IC) transfection was performed with serum-free DMEM supplemented with 20 µg poly(IC) ml–1 in the presence of 100 µg DEAE-dextran ml–1 for 2 h at 37 °C. After one wash with PBS, the cells were incubated for the times indicated in Results.
Viruses.
HAV/7 (Cohen et al., 1987) was propagated in FRhK-4 cells as described previously (Brack et al., 1998). Newcastle disease virus (NDV) strain R 05/93 (Fensterl et al., 2005) was passaged in FRhK-4 cells.
The 50 % tissue culture infective dose titres of HAV were determined by indirect immunofluorescence with the mouse anti-HAV IgG monoclonal antibody 7E7 (Mediagnost) and the NDV titre was determined by assessment of cytopathic effect. Titres were calculated using the method of Kärber (1931).
For reporter assays, HAV infections were carried out 10 days prior to analysis with an m.o.i. of 1. In order to guarantee infection of all cells, cells were split 1 day prior to experiments and infection of 100 % of the cells was shown by indirect immunofluorescence. For immunofluorescence experiments, HAV infections were carried out 3 days prior to analysis, with an m.o.i. of 0.1.
Chloramphenicol acetyltransferase (CAT) reporter assay.
Cell extracts were prepared by three freeze–thaw cycles, and expression of the CAT reporter gene was analysed by subjecting 100 µg protein to a CAT ELISA (Roche) as described by the manufacturer. The detection limit of the assay was 10 pg ml–1.
Luciferase (Luc) reporter assay.
Cell extracts were prepared by three freeze–thaw cycles and luc reporter gene expression was analysed by subjecting 20 µg protein to an assay system for the detection of firefly or Renilla Luc as described by the manufacturer (Promega) using a luminometer (Wallac MicroBeta).
Immunofluorescence analysis.
Cells were cultured on Labtek chamber slides, fixed with 90 % acetone (for HAV immunofluorescence analysis) or 4 % paraformaldehyde and 0.2 % Triton X-100, and probed for HAV with mouse anti-HAV 7E7 (Mediagnost) and FITC- or Texas Red-conjugated goat anti-mouse (KPL), for Flag–MAVS with mouse anti-Flag M2 (Sigma) and Texas Red-conjugated goat anti-mouse, for MAVS with rabbit anti-MAVS (Abcam) and FITC-conjugated goat anti-rabbit (Santa Cruz) and for HAV 2B with rabbit anti-2B (see below) and FITC- or Texas Red-conjugated goat anti-rabbit (Santa Cruz). Nuclei were visualized by counterstaining with DAPI. Mitochondria were labelled with MitoTracker Red CMXRos (Invitrogen) before the cells were fixed.
Antibody against HAV 2B.
Rabbit polyclonal antiserum against HAV 2B was generated by peptide immunization (Genovac). The antiserum worked satisfactorily in immunofluorescence analysis, but insufficiently in immunoblot analysis.
Plasmids.
The cDNA sequences of HAV proteins and protein precursors were amplified by PCR from the plasmid pHAV/7 (Cohen et al., 1987) and the wild-type (wt) 2B-coding region by RT-PCR from different wt HAV stool suspensions and cloned into pI.18. Plasmid pCMV-HAV/7
1–351, which after transfection results in expression of viral proteins but not in virus replication, was obtained by deletion of nt 1–351 from HAV/7 and cloning into pcDNA3.1. All sequences were confirmed by sequence analysis.
Constructs (–110-IFN-β)–CAT, (PRDIII-I)3–CAT and (PRDIV)6–CAT were provided by T. Maniatis (Thanos & Maniatis, 1995), (PRDIII-I)4–Luc by S. Ludwig (Ehrhardt et al., 2004), pcDNA3/Flag-MAVS by Z. J. Chen (Seth et al., 2005), pcDNA3.1/IKK-myc by U. Siebenlist (Chariot et al., 2002), pcDNA3/Flag-TBK1 (pcDNA3/Flag-NAK) by M. Nakanishi (Tojima et al., 2000) and pI.18 by J. S. Robertson (National Institute for Biological Standards and Control, UK). Plasmid pI.18 contains the cytomegalovirus (CMV) promoter and the intron A sequence preceding the cloning site and results in high expression levels of cloned sequences, as verified by analysis of the expression of HAV VP1. Empty vectors were obtained by removal of the inserted sequences.
pHcRed-Tandem-N1 (Evrogen) was used as a transfection control. PiecasRenilla–Luc (CMV–Luc), which was provided by G. Keil (Friedrich-Löffler-Institut, Riems, Germany), was used as a control for CMV promoter-mediated protein expression in cells overexpressing HAV 2B.
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RESULTS |
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; Fensterl et al., 2005). Therefore, HAV inhibits induction of IFN-β mRNA transcription. In order to identify which protein or intermediate protein precursor of HAV might downregulate IFN-β transcription, we constructed vectors expressing the HAV proteins shown in Table 1. Processing of the HAV polyprotein, consisting of P1(VP4-VP2-VP3-VP1)-P2(2A-2B-2C)-P3(3A-3B-3C-3D), results in 11 distinct proteins and several intermediate precursors (Martin et al., 1999; Probst et al., 1998; Schultheiss et al., 1995). We tested the ability of HAV proteins from the cell culture-adapted HAV variant HAV/7 genome (Cohen et al., 1987) to inhibit dsRNA- or NDV-activated transcription from the IFN-β enhancer (IFN-β–CAT) by co-transfection of FRhK-4 cells with the reporter plasmid and the different HAV protein expression vectors. Empty expression vectors were used as controls. At 24 h post-transfection, the cells were infected with NDV at an m.o.i. of 0.1 or transfected with 20 µg poly(IC) ml–1 with DEAE-dextran, both strong inducers of IRF-3 activity and functionally equivalent in these experiments (Fensterl et al., 2005). At 18 h after this treatment, whole-cell extracts were prepared and analysed for IFN-β enhancer-mediated CAT expression.
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1–351) resulted in a reduction in CAT expression to levels observed with cells infected with HAV (greater than 90 % reduction; Table 1
). As transfection of FRhK-4 cells with pCMV-HAV/71–351 did not result in virus replication (not shown), this result indicated that experimental expression of the HAV polyprotein blocked IFN-β transcription independently of virus replication. Separate co-expression of the P1-, P2- and P3-coding regions (P1+P2+P3) resulted in a weaker reduction in reporter expression (44 %), possibly reflecting less effective processing of the proteins. In summary, a reproducible reduction in reporter gene expression was observed in processing intermediates containing the 2B protein of HAV. Overexpression of 2B alone resulted in the most effective reduction in reporter synthesis (88 %), suggesting that the properties of 2B contribute to the ability of HAV to inhibit induction of IFN-β transcription. A weak but constant reduction in CAT expression was also observed with 2C (25 %) and 3ABCD (14 %). In order to prevent any possible influence on the function of the proteins, we did not fuse a Tag sequence to the HAV proteins for detection. We guaranteed high levels of protein expression by using the pI.18 vector, resulting in extraordinarily high expression levels as verified by analysis of the expression of HAV VP1 (not shown).
Confirmation that the ability of ectopically expressed HAV 2B correlated with the ability of HAV to suppress IFN-β synthesis by interfering with IRF-3 activation was obtained with a luc or cat reporter gene linked to the repeated individual enhancer element PRDIII-I [(PRDIII-I)3–CAT and (PRDIII-I)4–Luc, respectively], which represents the IRF-3-binding site. IRF-3 activity, which was strongly induced by poly(IC) transfection and NDV infection, respectively, was completely inhibited by HAV and almost completely suppressed by 2B (Fig. 1a). In addition, in cells not stimulated for IRF-3 activation (Fig. 1a, DD or mock), a reduction in basal Luc expression controlled by the IRF-3-specific PRDIII-I promoter occurred in the presence of HAV or 2B (see also Figs 1b, 2 and 4). This showed that, in unstimulated cells, a background of activated IRF-3 is present and is suppressed by HAV or 2B. Neither constitutive CMV promoter-controlled Renilla Luc expression, which was determined as an internal control for overall protein expression in cells co-transfected with the reporter plasmids (PRDIII-I)4–Luc (firefly) and CMV–Luc (Renilla) (Fig. 1a), nor constitutive ATF-2/c-Jun (PRDIV)-controlled CAT expression (not shown) was influenced by HAV or co-expression of 2B, showing that 2B did not decrease overall cellular transcription or translation.
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, left-hand panels) was completely inhibited in cells expressing 2B (Fig. 1c, 2B). HAV 2B expression was verified by immunofluorescence using antiserum against 2B. Co-expression of HcRed in cells overexpressing 2B confirmed that overexpressed 2B did not cause a general decrease in protein expression (see above) and nuclear staining with DAPI showed that no induction of apoptosis resulted from overexpressed 2B (Fig. 1d). Thus, the effect caused by HAV on RIG-I/MDA-5-mediated IRF-3 signalling was also caused by the non-structural 2B protein of HAV, indicating that 2B is a viral factor engaged in blocking IFN-β induction by HAV.
While this work was in progress, Yang et al. (2007) reported that ectopic expression of the HAV polyprotein processing intermediate 3ABC interferes with IRF-3 activation. We observed an inhibitory effect of 3ABCD on the transcriptional activity of the IFN-β enhancer-promoter (Table 1), presumably caused by 3ABC generated by autocatalytic cleavage of 3ABCD (Gosert et al., 1997; Probst et al., 1998). However, as this inhibitory effect was weak (14 %) compared with HAV protein intermediates that contained 2B, we focused on the effect of 2B. We next included 3ABC in our studies and directly compared the effects of 2B and 3ABC, each expressed from separate expression vectors, on IRF-3 activity. We observed that Luc expression controlled by the IRF-3-specific PRDIII-I enhancer domain was inhibited by 2B and 3ABC equivalently after induction by NDV (Fig. 1b). Co-expression of both 2B and 3ABC resulted in an increased suppression of Luc expression, compared with 2B or 3ABC alone (Fig. 1b). Nuclear translocation of GFP–IRF-3 induced by NDV was also inhibited equivalently by either 2B or 3ABC, as well as by co-expression of both (Fig. 1c). Transfection of cells with 3ABC was verified by co-transfection with pHcRed-Tandem-N1 (Fig. 1c, 3ABC) or pI.18/2B and proof of 2B expression by immunofluorescence (Fig. 1c, 2B+3ABC), respectively. Thus, either ectopically overexpressed HAV 2B or 3ABC resulted in equivalent inhibition of IRF-3 activation, whilst joint expression of both gave an increased cooperative inhibitory effect on IRF-3 activation.
HAV 2B interferes with the activities of MAVS and the TBK1/IKK kinases
Based on our previous finding that HAV did not inhibit the activities of overexpressed TBK1/IKK kinases, we assumed that the target site of inhibition of RIG-I/MDA-5-mediated IFN-β synthesis by HAV was located downstream of RIG-I/MDA-5 and upstream of the activator kinase complex for IRF-3 (Fensterl et al., 2005). As MAVS connects both sensors with the kinases (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005), we investigated the influence of HAV and 2B on MAVS-initiated signalling. FRhK-4 cells were co-transfected with a MAVS expression plasmid and CAT/Luc reporter constructs containing the complete IFN-β enhancer (IFN-β–CAT) or repeated IRF-3-binding sites [(PRDIII-I)4–Luc]. Overexpression of MAVS results in activation of these kinases without additional induction (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005), and we analysed reporter expression 42 h after transfection. As shown in Fig. 2(a) and (b), overexpression of MAVS resulted in significant CAT and Luc expression, respectively (Mock). However, in cells infected with HAV or transfected with 2B for overexpression, CAT expression controlled by the complete IFN-β enhancer as well as Luc expression controlled by PRDIII-I was significantly reduced (Fig. 2a). These experiments demonstrated the functional equivalence of 2B and replicating HAV in their ability to interfere with the transduction of RIG-I/MDA-5-signalling from MAVS to the kinases responsible for IRF-3 phosphorylation.
HAV 3ABC, which is suggested to target MAVS (Yang et al., 2007), also proved to be similar to 2B with regard to its inhibitory effect on MAVS-mediated IRF-3 activation (PRDIII-I controlled Luc expression), although significantly less effective than 2B (Fig. 2b). Co-expression of both 2B and 3ABC resulted in the most effective suppression of MAVS signalling, supporting their cooperative interference with the function of MAVS.
Compared with the reduction in reporter gene expression by HAV or 2B detected after induction of the signalling pathway by NDV infection and poly(IC), respectively (Fig. 1a), the ability of both HAV and 2B to inhibit MAVS activity obtained by overexpression of MAVS was less effective and only a decrease in overexpressed Flag–MAVS could be observed by immunoblotting, and no reduction in Flag–MAVS abundance could be identified by immunofluorescence in cells infected with HAV (not shown). This suggests that large amounts of MAVS compete with the inhibitory effect caused by HAV and 2B, and this partial restoration of MAVS function by overexpressed MAVS indicated that HAV as well as 2B influence MAVS. The same applied to 3ABC (Fig. 2b).
Interestingly, and in contrast to HAV or overexpressed 3ABC, which targets MAVS (Yang et al., 2007), no influence on the presence of endogenous MAVS could be detected in cells overexpressing 2B, either by immunofluorescence (Fig. 2c) or by immunoblotting (not shown), although 2B overexpression resulted in a reduction in MAVS function analogous to HAV (see above). Therefore, HAV 2B appeared to influence the function of MAVS without directly interfering with the antigenic structure of MAVS.
Based on this finding, it is possible that HAV 2B might interfere with the kinase complex, and although we did not observe an effect of HAV on the activity of overexpressed TBK1/IKK kinases in previous experiments (Fensterl et al., 2005), we investigated whether 2B was able to interfere with the activity of these kinases (Fig. 2d). FRhK-4 cells were co-transfected with a vector for 2B expression, a vector for luc as a reporter gene for IRF-3 activity linked to PRDIII-I and a plasmid for constitutive expression of IKK or TBK1. Reporter gene expression was analysed 42 h after transfection of the plasmids and empty pI.18 vector was used as a reference. Luc expression induced by overexpression of the kinases and without additional activation (Fensterl et al., 2005; Fitzgerald et al., 2003; Sharma et al., 2003) was significantly reduced by overexpressed 2B. In contrast to this finding, overexpression of 3ABC did not influence Luc expression induced by overexpression of the kinases. Co-expression of both 2B and 3ABC resulted in a reduction in Luc expression to the levels observed with expression of 2B alone, indicating that IRF-3 activity induced by overexpressed kinases was inhibited by overexpression of 2B. No cooperation between 2B and 3ABC, which does not interfere with the activities of the kinases, could be observed in these experiments.
HAV 2B partially co-localizes with MAVS
The C-terminal transmembrane domain of MAVS anchors this protein to the outer mitochondrial membrane (Seth et al., 2005). The C-terminal part of 2B contains a hydrophobic stretch, which can accommodate an amphipathic helix structure with a potential for membrane association (Gosert et al., 2000). It has been concluded that 2B is a peripheral membrane protein (Jecht et al., 1998) found in close proximity to the endoplasmic reticulum and to mitochondrial membranes (Gosert et al., 2000). Therefore, we examined the localization of MAVS and 2B after co-transfection of plasmids for expression of Flag–MAVS and 2B by immunofluorescence. We found that a certain amount of overexpressed 2B associated with mitochondria as analysed with MitoTracker dye and co-localized with mitochondrion-associated MAVS (Fig. 3).
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HAV 2B derived from wt viruses also inhibits RIG-I/MDA-5-mediated signalling
In the experiments described above, the cell-culture-adapted HAV/7 variant of HAV strain HM175 and ectopically expressed proteins derived from this variant were used. Adaptation of HAV to growth in cultivated cells seems to be achieved by varying sets of multiple interacting mutations in the viral genome, with adaptive mutations clustering in the 2B protein (Emerson et al., 1991, 1992, 1993). This may indicate functional differences in wt 2B compared with 2B of tissue-culture-adapted (tc) HAV variants. Therefore, we isolated the 2B-coding region of different wt viruses and investigated their ability to inhibit RIG-I/MDA-5-mediated signalling, which was demonstrated with tc 2B from HAV/7. FRhK-4 cells were co-transfected with the various 2B expression plasmids and the Luc reporter construct containing repeated IRF-3-binding sites [(PRDIII-I)4–Luc]. These cells were infected with NDV (m.o.i. of 1) 24 h after transfection or transfected with poly(IC) 48 h after transfection, respectively, and expression of the reporter was analysed 18 h after these treatments. NDV-induced (RIG-I-mediated) as well as poly(IC)-induced (MDA-5-mediated) IRF-3 activation were equally inhibited by ectopically expressed tc 2B and wt 2B (Fig. 4), indicating that HAV 2B of both tc and wt viruses is able to inhibit RIG-I/MDA-5 signalling independently of adaptive mutations of the 2B-coding region.
These experiments implied that wt HAV is also able to inhibit RIG-I/MDA-5-mediated IFN-β transcription caused by a function of the non-structural 2B protein, and that the inhibitory effect of tc variants of HAV on induction of IFN-β synthesis is not a quality acquired during adaptation to growth in cultivated cells by mutational changes in 2B, but is an inherent characteristic of HAV.
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) and that this is due to the ability of HAV to inhibit RIG-I/MDA-5-mediated IRF-3 activation (Fensterl et al., 2005).
In this study, we have presented data showing that HAV interferes with MAVS-dependent signal transduction towards IRF-3 phosphorylation, supplementing our previous results. Furthermore, we found that, analogous to HAV, ectopically expressed HAV 2B protein resulted in suppression of RIG-I/MDA-5-mediated IRF-3 activation and thus IFN-β transcription. In a recent report by Yang et al. (2007), HAV protease 3C was identified as interfering with MAVS-dependent signalling by proteolytic cleavage of MAVS after association with mitochondrial membranes, for which the 3ABC precursor of HAV polyprotein processing is necessary, and therefore we included 3ABC in our experiments, showing that 2B and 3ABC were functionally equivalent in suppressing IRF-3 activation and that they acted cooperatively in suppressing RIG-I/MDA-5 signalling towards IRF-3 phosphorylation. Both 2B and 3ABC individually prevented translocation of cytoplasmic IRF-3 to the nucleus and therefore IRF-3 activation.
We found that, similar to HAV, 2B suppressed the activity of overexpressed MAVS and cooperated with 3ABC in this context. However, in contrast to HAV and to overexpressed 3ABC, 2B did not result in proteolytic cleavage of endogenous MAVS, with which it partially co-localizes. Investigating the effect of overexpressed 2B on the activities of overexpressed TBK1 or IKK, we found that, in contrast to 3ABC or HAV (Fensterl et al., 2005), 2B interfered with the activities of these kinases.
These results allowed us to conclude that HAV 2B, but not 3ABC, interferes with IKK and TBK1, thus inhibiting kinase activity. The discrepancy between the effect of 2B and HAV on the activities of strongly overexpressed kinases might reflect a restricted inhibitory ability of only small amounts of 2B in cells infected with HAV compared with ectopically overexpressed 2B. The ability of HAV to act on MAVS was also impaired in the presence of strongly overexpressed MAVS (see above). In this case also, small amounts of viral non-structural proteins and their intermediates during infection might not be able to counteract the activity of larger amounts of MAVS efficiently. Although endogenous MAVS is not structurally influenced by 2B, a possible interference of 2B with MAVS, which is targeted by 3ABC, can also be assumed, as overexpressed MAVS was able to counteract the inhibitory effect of ectopically expressed 2B on IRF-3 activity, and 2B partially co-localized with mitochondrion-associated MAVS. As the activity of overexpressed RIG-I was completely suppressed by HAV (Fensterl et al., 2005), MAVS is thus the first component in the RIG-I/MDA-5 signalling pathway on which HAV and also 2B show an effect. HAV 2B could interfere simultaneously with MAVS and the kinases, as both components associate during signalling.
Under physiological conditions, HAV appears to inhibit induction of IFN-β transcription by interfering with the activities of MAVS and the TBK1/IKK kinases by cooperating synergistic effects of 2B and 3ABC, which seem to act in a supplementary way to inhibit signal transduction. This was supported by a dramatic loss of MAVS in HAV-infected cells, observed by both immunofluorescence and immunoblot analysis, and also described by Yang et al. (2007). The complete absence of MAVS, which is abundant in uninfected cells, is unlikely to be due to the action of a protein or processing intermediate present in low, inaccessible amounts in infected cells. Additional support for a cooperative effect of 2B and 3ABC comes from our observation that the most efficient inhibitory effect on IRF-3 activation was obtained by co-expression of 2B and 3ABC. This cooperative effect was particularly observed in experiments overexpressing MAVS, which was clearly able to counteract the separate inhibitory effects of 2B and, notably, 3ABC. A recent report showed that, in addition to NS3/4A protease-mediated cleavage of MAVS, the non-structural protein NS4B of hepatitis C virus (HCV) inhibited RIG-I signalling by an unknown mechanism (Tasaka et al., 2007). It can therefore be assumed that, similar to HCV infections, several viral proteins might also cooperate in suppression of IFN-β transcription in HAV-infected cells. In addition, further targets of 2B in the signalling pathway for induction of IFN-β transcription are possible.
Little is known about the functions and abilities of HAV 2B, so we can only speculate on the mechanisms resulting in inhibition of RIG-I/MDA-5 signalling by 2B. Studies of chimeric viruses have implicated that mutations acquired in the 2B-coding region might contribute to the efficient growth of HAV in cell culture (Emerson et al., 1991, 1992, 1993), but nothing is known about the functional mechanisms of these mutations. In experiments demonstrating that HAV antagonizes the IFN response and that 2B contributes to this ability, tc HAV variants as well as 2B derived from tc HAV (HAV/7) were used, so it could be that this ability was a consequence of cell culture adaptation, allowing the virus to grow more efficiently in cultivated cells. We could demonstrate that 2B proteins derived from wt HAV and tc HAV were functionally equivalent in suppressing RIG-I/MDA-5 signalling. This implies that suppression of IFN-β synthesis has not evolved during adaptation of HAV to cell culture conditions, but is a general characteristic of HAV, and that the mutations acquired in 2B of tc HAV serve other functions.
So far, no distinct functional roles for 2B, which is essential for HAV replication and thus cannot be eliminated functionally, have been defined. HAV 2B is significantly larger than the respective proteins of other picornaviruses, which enhance membrane permeability, block the protein secretory traffic and may contribute to viral particle release (Doedens & Kirkegaard, 1995; Lama & Carrasco, 1992; van Kuppeveld et al., 1997). Studies by different laboratories with ectopically expressed HAV 2B imply that this protein is a peripheral membrane protein that increases membrane permeability and is involved in rearrangements of intracellular membranes of infected cells (Gosert et al., 2000; Jecht et al., 1998; Teterina et al., 1997). Besides its association with membranes of the endoplasmic reticulum, the protein was found in close proximity to the tubular interconnected network of mitochondrial membranes (Gosert et al., 2000). Association of 2B with mitochondria is supported by our finding that this viral protein co-localized with MAVS, which is located in the outer mitochondrial membrane, and that co-staining of 2B with MitoTracker occurred. We do not know by which mechanism 2B interferes with membranes. As overexpression of 3A, which is also targeted to mitochondrial membranes (Yang et al., 2007), does not interfere with the function of MAVS, it is not likely that, in the case of 2B, only production of relatively large amounts of a membrane protein normally not accessible in infected cells will induce this effect. HAV 2B, which did not influence overall protein synthesis and viability of cells ectopically expressing 2B, might interfere with the constantly occurring events of fusion and fission of mitochondria, which represent a highly dynamic membranous network (Chan, 2006; Chen & Chan, 2005), through its ability to induce membrane rearrangements resulting in an impairment of the functions of MAVS and possibly of the TBK1/IKK complex, which is associated with MAVS during activation. The direct association of 2B with MAVS and/or the kinases might be an alternative, but because of the insufficient performance of the 2B antiserum in co-precipitation studies, we could not clarify this.
As IRF-3 simultaneously activates innate immune responses by triggering IFN-β synthesis and suppresses metabolism in the liver (Chow et al., 2006), the ability of HAV to inhibit IRF-3 activation seems to be of considerable importance for HAV replication. HAV replicates exceptionally slowly and is sensitive to the antiviral activities of IFN-induced proteins (Vallbracht & Flehmig, 1985a; Vallbracht et al., 1984). Thus, this strategy may allow the virus to establish infection and spread to neighbouring cells. Simultaneously, the sites of virus production are preserved for a longer time, and without limiting amounts of cellular components needed for virus replication, as liver cell metabolism is not downregulated by IRF-3.
In summary, our investigation demonstrated that HAV interferes with MAVS-dependent IFN-β transcription induced by the RIG-I/MDA-5 pathway and that, in addition to HAV 3ABC, the non-structural HAV 2B protein contributes to this inhibition. It remains to be clarified whether and to what extent HAV interferes with the TBK1/IKK kinases under physiological conditions, and through which biochemical properties and mechanisms HAV 2B is able to interfere with MAVS/TBK1/IKK-dependent IRF-3 phosphorylation and possibly with further mechanisms that contribute to induction of IFN-β transcription. The concerted actions of 2B and 3ABC in this context also need to be investigated in detail.
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Received 16 October 2007;
accepted 11 March 2008.
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