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1 Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands
2 Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Dr Bohr Gasse 9/3, A-1030 Vienna, Austria
Correspondence
F. J. M. van Kuppeveld
f.vankuppeveld{at}ncmls.ru.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The genomic organization of all picornaviruses is essentially the same. The single-stranded viral genome is of positive polarity and contains a single, large open reading frame that encodes the viral polyprotein (reviewed by Bedard & Semler, 2004
). This polyprotein can be divided into the P1 region, comprising the structural capsid proteins, and the P2 and P3 regions, containing the non-structural proteins. The polyprotein is processed by virus-encoded proteases into the mature viral proteins. In enteroviruses and rhinoviruses, a primary cleavage is mediated by protease 2Apro (Sommergruber et al., 1989; Toyoda et al., 1986), which cleaves the polyprotein at the P1–P2 junction, separating the capsid proteins from the non-structural proteins. The capsid proteins are liberated proteolytically through cleavage by 3CDpro in trans. The P2 and P3 region proteins are liberated through the action of 3Cpro via a complex proteolytic cascade involving a number of subsequent cis-cleavage events (reviewed by Dougherty & Semler, 1993; Palmenberg, 1990). The cardiovirus and aphthovirus 2A proteins have no proteolytic activity. Instead, these proteins contain at their C terminus a unique amino acid sequence (DvExNPG/P motif) that is thought to prevent the formation of a peptide bond between the C-terminal glycine residue of 2A and the N-terminal proline residue of 2B, resulting in production of the L–P1–2A precursor protein (Donnelly et al., 2001; Palmenberg et al., 1992; Ryan et al., 1991). Translation then proceeds at the N-terminal proline residue of 2B, resulting in the production of the 2BC3ABCD precursor protein. Proteolytic processing of the capsid protein precursor is again carried out by 3CDpro in trans, whereas the 2BC3ABCD precursor is processed in cis by 3Cpro (Dougherty & Semler, 1993; Palmenberg, 1990).
Currently, few antiviral compounds are available against picornaviruses. The capsid-binding compound pleconaril (Pevear et al., 1999) and the 3Cpro inhibitor ruprintrivir are active against HRV and certain enteroviruses (Binford et al., 2005). Dipyridamole, a modified purine, was described recently as an effective inhibitor of cardiovirus growth in cell culture and seems to target reversibly an early step in both minus-strand and plus-strand RNA synthesis (Fata-Hartley & Palmenberg, 2005).
Pyrrolidine dithiocarbamate (PDTC) is a multifunctional compound. It is often used as an inhibitor of the transcription factor NF-
B (Schreck et al., 1992). Besides this, it can exert anti-apoptotic or pro-apoptotic properties, depending on the cellular system used (Erl et al., 2000). Moreover, PDTC was reported recently to hinder proteolytic protein degradation either by inhibiting E3 ubiquitin ligase (Hayakawa et al., 2003) or by inhibiting the proteasome directly (Kim et al., 2004). PDTC also exerts antiviral effects against influenza virus (Uchide et al., 2002). Previously, we showed that PDTC inhibits replication of HRV and poliovirus (Gaudernak et al., 2002; Krenn et al., 2005). The mechanistic basis of the antiviral function of PDTC is not yet resolved fully. We found that PDTC does not interfere with early steps of the HRV life cycle, such as receptor binding and internalization, but inhibits virus replication by interfering with proteolytic processing of the polyprotein (Krenn et al., 2005). Due to the lack of specific antibodies, however, the exact nature of the inhibition of proteolysis could not be clarified. In addition, we showed that the antiviral activity of PDTC is dependent on metal ions in the medium.
In this study, we tested the effects of PDTC on two other picornaviruses: coxsackievirus B3 (CVB3), a closely related enterovirus that is associated with myocarditis, and mengovirus, a more distantly related EMCV strain that causes severe problems in veterinary medicine, such as encephalitis and myocarditis in hoofed animals. We show that PDTC inhibits viral RNA replication of these two viruses by transporting zinc ions into cells and characterize the proteolytic-processing defects that are induced by this compound.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were provided by Drs A. Aminev and A. Palmenberg (University of Wisconsin-Madison, WI, USA).
Cells, viruses and replicons.
Buffalo green monkey (BGM) kidney cells and baby hamster kidney (BHK-21) cells were grown at 37 °C in minimal essential medium (MEM) (Gibco) supplemented with 10 % fetal bovine serum (FBS). CVB3 used in this study was derived from the p53CB3/T7 plasmid, which contains the cDNA of CVB3 strain Nancy behind a T7 RNA polymerase promoter (Wessels et al., 2005). The EMCV strain used in this study is the mengovirus strain, which was obtained upon transfection of in vitro-transcribed RNA from cDNA clone pM16.1 (Duke & Palmenberg, 1989). Virus yields were determined by end-point titration (Reed & Muench, 1938) and expressed as 50 % tissue culture infective dose (TCID50) values. The p53CB3-LUC replicon contains the CVB3 cDNA in which the P1 capsid coding region is replaced by the firefly luciferase gene (Wessels et al., 2005). The pEMCV-LUC replicons (Aminev et al., 2003b) were kindly provided by Drs A. Aminev and A. Palmenberg (University of Wisconsin-Madison, WI, USA).
Virus infection.
Confluent monolayers of BGM or BHK-21 cells were infected with virus for 30 min at 37 °C at an m.o.i. of 10 TCID50 unless otherwise indicated. Cells were then washed twice with PBS and cultured in MEM supplemented with 10 % FBS at 37 °C for the indicated periods of time. At the indicated times, the cells were disrupted by three cycles of freezing and thawing, and virus titres were determined by end-point titration.
Analysis of viral RNA synthesis.
Replicons were linearized with SalI, purified, transcribed in vitro by T7 RNA polymerase, transfected into BGM cells and tested for luciferase production as described previously (van Kuppeveld et al., 1995).
Fluorescence detection of metal ions.
Trypsinized HeLa cells were washed twice with buffer A containing 120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 20 mM HEPES, 15 mM glucose, 1.8 mM CaCl2, 10 mM NaOH (pH 7.4) and subsequently loaded in the same buffer with 3 µM mag-fura 2 at 37 °C for 30 min in the dark. Cells were washed twice with buffer A lacking Mg2+ or Ca2+. Intracellular zinc ion concentration was determined by measuring the fluorescence of the probe-loaded cells upon stimulation by using a spectrofluorimeter LS-55 (Perkin-Elmer) equipped with a fast filter accessory, alternating excitation wavelengths of 340 and 380 nm at 20 ms intervals and recording emission at 509 nm. All measurements were done at room temperature in 3 ml silica cuvettes containing 2 ml cell suspension, with stirring of the suspension.
Pulse-labelling and immunoprecipitation.
For pulse-labelling experiments, BGM monolayer cells were grown in 24-well plates to subconfluence and infected with virus at an m.o.i. of 50. After 5 h, the medium was replaced by 300 µl methionine-free MEM for 30 min at 37 °C. Proteins were pulse-labelled with [35S]methionine [20 µCi (740 kBq) per well] for 30 min at 37 °C and lysed at 6 h post-infection (p.i.) in lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 % Nonidet P-40, 0.05 % SDS]. During the starvation and labelling period, 125 µM PDTC or 10 µM TPEN was added. Laemmli sample buffer was added to the lysates, boiled for 5 min and analysed by SDS-PAGE.
For immunoprecipitation experiments, cells were labelled in the presence or absence of PDTC or TPEN and lysed at 6 h p.i. as described above. Antibodies (1 : 250) were added to the cell lysates and the mixtures were incubated at 4 °C for 5 h. Antibody–protein complexes were collected with protein A–Sepharose (Amersham Biosciences) for 16 h at 4 °C, washed once with dilution buffer [0.01 M Tris (pH 8.0), 0.14 M NaCl, 0.1 % BSA, 0.1 % Triton X-100], once with TSA [0.01 M Tris (pH 8.0), 0.14 M NaCl], once with 0.05 M Tris (pH 6.8) and then precipitated. The samples were resuspended in 20 µl Laemmli sample buffer and analysed as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Virus yields were determined by end-point titration at various times after infection. In the presence of PDTC, the multiplication of both viruses was suppressed strongly until 8 h p.i. Only after 24 h were increases in virus titre observed. Whether this is due to turnover of PDTC is unknown, because its half-life is unknown. PDTC prevented the production of progeny virus in a dose-dependent manner (Fig. 1c, d). Concentrations of 125 µM or higher resulted in the strongest inhibition of virus multiplication, as measured at 6 h p.i. Concentrations as low as 5 or 25 µM showed only a slight inhibitory effect. Similar results were obtained in HeLa cells for CVB3 and mengovirus, and in L929 as well as BHK-21 cells for mengovirus (data not shown). These data show that, although these two viruses are related only distantly, PDTC exerts a significant antiviral effect against both viruses in a variety of cell types.
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) on virus replication were compared. The compounds were added during infection at a concentration of 100 µM and virus titres were determined at 6 h p.i. by end-point titration. DDTC reduced viral growth to an extent similar to that with PDTC, whereas pyrrolidine was ineffective (Fig. 2b, c). This indicates that the dithiocarbamate moiety is essential for the inhibitory effect of PDTC. The nature of the counterion is not relevant for the antiviral property of dithiocarbamate derivatives; sodium, potassium or ammonium salts of PDTC were found to have comparable antiviral activities, whereas the respective counterions alone did not inhibit virus multiplication (data not shown). Similar results were obtained for HRV (data not shown).
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). To test whether the inhibitory effects of PDTC on CVB3 and mengovirus production are also due to defects in viral RNA replication, the effects on CVB3 and EMCV replicons were tested. These replicons contain the firefly luciferase gene in place of (parts of) the capsid coding region. The amount of luciferase activity produced by these replicons is a measure of viral RNA replication. BGM cells were transfected with RNA transcripts of p53CB3-LUC and pEMCV-LUC and then incubated in the presence or absence of PDTC for 8 h. Analysis of luciferase activity showed that PDTC had no effect on the initial translation of the transfected replicon RNA, but that replication of the replicon was inhibited strongly in the presence of PDTC (Fig. 3a). The amount of luciferase produced in the presence of PDTC was as low as that produced in the presence of guanidine/HCl, a well-known inhibitor of enterovirus replication (Rightsel et al., 1961). Replication of the EMCV replicon was also inhibited strongly by PDTC. The activity of luciferase produced was equal to that produced by a replicon containing an in-frame deletion in the polymerase 3Dpol (Fig. 3b). Thus, PDTC inhibits RNA replication of both picornaviruses.
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). FBS can be a significant source of metal ions, such as zinc, copper and iron, in cell culture that may be involved in the antiviral properties of PDTC. To test whether the antiviral effect of PDTC involves factors contained in the serum, BGM cells were infected with either CVB3 or mengovirus and then incubated in medium with or without serum. Analysis of the virus titre obtained at 8 h p.i. showed that the inhibitory effect of PDTC on both CVB3 and mengovirus replication was eliminated upon using serum-free medium (Fig. 4a). Notably, the dependence of the antiviral effect on serum was not observed with all batches of medium, implying that ion levels between different batches of medium are not consistent. Notwithstanding this, the availability of batches of medium in which PDTC had no inhibitory effect on virus replication in the absence of serum allowed us to investigate further which metal ion is relevant for the antiviral effect of PDTC. To this end, different concentrations, varying between 100 and 0.01 µM, of Zn2+, Cu2+ and Fe2+ ions were added to PDTC-containing serum-free medium during infection. The metal ions were added in the form of chloride salts. None of the ions used alone interfered with virus replication or showed signs of cellular toxicity in the absence of PDTC, except for 100 µM Cu2+, which was toxic to the cells and reduced CVB3 and mengovirus replication strongly. This condition was therefore not considered further. In the presence of PDTC, none of the concentrations of Fe2+ tested interfered with replication of CVB3 (Fig. 4b) or mengovirus (data not shown). Upon addition of 10 µM Cu2+ together with PDTC, a small (10-fold) reduction in virus titre was found for both CVB3 (Fig. 4c) and mengovirus (data not shown), but no inhibitory effects were observed at lower concentrations. The most severe effects on replication were observed upon addition of Zn2+ ions in combination with PDTC. Low concentrations of Zn2+ (0.1 µM) reduced CVB3 replication by more than 10-fold. Upon addition of 1 µM Zn2+, the CVB3 titre was reduced by almost four orders of magnitude (Fig. 4d). Similar results were obtained with mengovirus-infected BGM cells (data not shown) and also in CVB3- and mengovirus-infected HeLa cells (data not shown). These data indicate that the presence of metal ions, and particularly Zn2+ ions, is essential for the antiviral effect of PDTC.
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). The affinity of EDTA for different ions increases in the order Mg2+<Ca2+<Fe2+<Zn2+<Cu2+<Fe3+ (Perrin, 1979). As a control, we used the general metal ion-chelating agent EDTA, which was shown previously to alleviate the antiviral effect of PDTC (Krenn et al., 2005). BGM cells were infected with either CVB3 or mengovirus and incubated with 100 µM EDTA or one of the three specific metal ion–EDTA preparations during infection. Viral yields obtained at 8 h p.i. were determined. Neither EDTA nor any of the metal ion–EDTA complexes alone had an antiviral effect or showed signs of toxicity at the concentrations used (data not shown). The antiviral effect of PDTC on the replication of both viruses examined was abolished by Ca–EDTA and Mg–EDTA in a manner similar to that observed for free base EDTA (Fig. 5a, b). However, Zn–EDTA failed to eliminate the inhibitory effect of PDTC, again emphasizing that Zn2+ ions are essential for the antiviral activity of PDTC.
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; Parat et al., 1998). We measured the amount of virus produced in medium supplemented with serum and PDTC in the presence or absence of TPEN. TPEN alone had no effect on the multiplication of mengovirus, but it reduced the CVB3 titre by about 10-fold, probably because the activity of the CVB3 2A protease (partially) depends on the availability of Zn2+ (Glaser et al., 2003). Addition of PDTC resulted in a decrease in virus titres of both CVB3 and mengovirus by three to four orders of magnitude. However, when TPEN was added simultaneously, virus titres were restored to normal levels, indicating that TPEN abolishes the antiviral effect of PDTC (Fig. 5c, d
). Similar results were obtained upon using serum-free medium supplemented with 5 µM Zn2+ (Fig. 5c, d).
PDTC increases the intracellular level of labile Zn2+
PDTC binds various metal ions, leading to the formation of lipophilic dithiocarbamate–metal complexes that facilitate the transport of metal ions from the extracellular medium into the cell (Erl et al., 2000; Kim et al., 1999; Thorn & Ludwig, 1962; Verhaegh et al., 1997). To investigate the changes in the intracellular ion level induced by PDTC, we monitored the fluorescence of mag-fura 2 in a real-time mode in HeLa cells (Simons, 1993). The excitation wavelength of the fluorescent indicator mag-fura 2 is shifted from 380 to 340 nm when divalent cations, such as Mg2+ or Zn2+, are bound (Raju et al., 1989; Simons, 1993). HeLa cells were loaded with mag-fura 2 and transferred to a divalent cation-free buffer after intensive washing. Then, ZnCl2, PDTC, and EDTA or Zn–EDTA was added. Addition of 5 µM ZnCl2 alone had no effect on the fluorescence ratio (Fig. 6). Upon treatment with 125 µM PDTC, the signal increased gradually and reached a plateau approximately 150 s after treatment, indicating the influx of Zn2+ ions. Application of 10 µM EDTA, but not 10 µM Zn–EDTA, decreased the signal immediately to the basal level. These results show that, although mag-fura 2 is not specific for Zn2+ ions, the increased fluorescence ratio caused by combined application of 5 µM Zn2+ and 125 µM PDTC resulted solely from the influx of Zn2+ ions.
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). To investigate the effects of PDTC on CVB3 polyprotein processing, infected cells were labelled with [35S]methionine at 5.5 h p.i., a time point at which host-cell protein synthesis is shut off, due to the cleavage of eIF4G by 2Apro (Etchison et al., 1982), and predominantly viral proteins are synthesized. PDTC was added to the cell-culture medium during both the 30 min starvation period and the 30 min labelling period. The protein pattern of the newly synthesized and labelled proteins was analysed by SDS-PAGE and autoradiography (Fig. 7a). In untreated cells, the different viral proteins were clearly visible. Treatment with PDTC led to a dramatic reduction of the P1 region proteins VP0, VP1, VP2 and VP3. Production of the non-structural proteins was also affected, as indicated by the reduced amount of 2BC and 2C, but to a lesser extent. When TPEN was co-administered with PDTC, the protein pattern was similar to the untreated-control sample. PDTC had no effect on translation in non-infected cells (data not shown; Krenn et al., 2005).
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, PDTC reduced the levels of both 3Cpro and 3Dpol severely. Co-administration of TPEN again rescued the inhibitory effect by PDTC. These data indicate a role for metal ions, particularly Zn2+ ions, in disturbance of the proteolytic activity of CVB3 3CD pro.
We also tested the effects of PDTC on mengovirus polyprotein processing. Because the shut-off of host-cell protein synthesis in mengovirus-infected cells is mechanistically different from and not as prominent as in CVB3-infected cells (Mosenkis et al., 1985), we performed pulse-labelling experiments followed by immunoprecipitation. As we have shown that, in CVB3 infection, 3CDpro is targeted by PDTC, we first studied the fate of mengovirus 3CDpro, making use of a mAb directed against 3Dpol. Fig. 8(a) shows that the autocatalytic processing of 3CDpro was not affected in PDTC-treated cells. To examine the polyprotein processing further, we made use of two other antibodies, namely a mAb against the 2A protein and a polyclonal serum that was raised against virus particles and thus recognizes the viral capsid proteins. Fig. 8(b) shows that this latter antibody not only recognized the processed capsid proteins (VP0, VP1, VP2 and VP3), but also two larger P1-containing precursor proteins (right picture, control lane). These two proteins probably represent the P1–2A and P1 proteins, as the small L protein is cleaved quickly from the L–P1–2A precursor (Zoll et al., 1998). Indeed, the larger of these two proteins was also recognized by the 2A antibody (Fig. 8b, left picture, control lane). Unfortunately, the 2A protein itself could not be detected. PDTC did not abrogate the proteolytic production of the capsid proteins, but caused massive accumulation at the top of the gel of a high-molecular-mass protein that was recognized by both antibodies, indicating that this accumulating protein contains (at least) P1 and 2A coding sequences. Again, the processing defect was rescued by TPEN. These results show that PDTC also disturbs mengovirus polyprotein processing by transporting metal ions, particularly Zn2+ ions, into cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Due to the lack of specific HRV antibodies, the exact nature of the inhibition of proteolysis could not be clarified. Here, we showed that PDTC also inhibits RNA replication of two other picornaviruses: CVB3, a closely related enterovirus, and mengovirus, a more distantly related EMCV strain, and that this inhibition is due to the dithiocarbamate moiety of the compound. Furthermore, we characterized the proteolytic-processing defects that are induced by this compound. Finally, evidence is provided that PDTC transports zinc ions into cells and that these zinc ions play an important role in the antiviral activity mediated by PDTC.
Dithiocarbamates, such as PDTC, chelate various metal ions, leading to the formation of lipophilic dithiocarbamate–metal complexes (Thorn & Ludwig, 1962). Several studies have shown that PDTC can promote cell entry of metals (e.g. Erl et al., 2000; Kim et al., 1999), thereby altering the tightly regulated balance of ions in cells. Previously, we showed that the inhibitory effect of PDTC on HRV replication could be prevented by addition of metal chelators. Serum can be a major source of metal ions in cell culture. By replacing metal ions into serum-free medium (this study) or EDTA-containing medium (Krenn et al., 2005), we showed that replication of CVB3, mengovirus and HRV is extremely sensitive to Zn2+ ions in the presence of PDTC. Even low concentrations of Zn2+ (1.0–2.5 µM) inhibited replication to a significant extent. Previously, we found that Cu2+ ions could also inhibit HRV replication, but much higher concentrations (25 µM) of this ion were required for a strong antiviral effect (Krenn et al., 2005). It seems unlikely that Cu2+ ions contribute to the antiviral effect of PDTC, because this concentration is much higher than the concentration of Cu2+ that is reported for most commercial serum batches; most batches contain approximately 5 µM Cu2+, whereas they contain approximately 50 µM Zn2+ (Life Technologies). Thus, in cell cultures supplemented with 10 % serum, there is only 0.5 µM Cu2+, which is far below the inhibitory level that we observed in our studies. Direct evidence that PDTC indeed transports Zn2+ ions into cells is provided in this study by real-time measurements employing fluorescent ion indicators, showing a PDTC- and Zn2+-mediated increase in the fluorescence signal of the indicator mag-fura 2 that is sensitive to EDTA, but not to Zn–EDTA. The finding that PDTC increased mag-fura 2 fluorescence, furthermore, provides evidence that Zn2+ transported into cells can be separated from PDTC, and thus become available for other binding partners, such as cellular proteins. This finding, together with the previous observations that high concentrations of Zn2+ can affect picornavirus polyprotein processing (Butterworth & Korant, 1974; Korant & Butterworth, 1976; Korant et al., 1974; Nakai & Lucas-Lenard, 1976), suggests strongly that the inhibitory effects are mediated by Zn2+ ions, rather than by a complex of PDTC and Zn2+.
Zn2+ is one of the most abundant trace metals found in eukaryotic organisms, second only to iron. Zn2+ is distributed non-uniformly throughout the cell, existing at nanomolar to picomolar concentrations in the cytosol and at up to millimolar concentrations within vesicles (Frederickson, 1989). It is an essential element for a great number of proteins, including enzymes involved in signalling processes and transcription factors needed in the regulation of gene expression, and has been shown to interfere with replication or maturation of various viruses (Geist et al., 1987; Haraguchi et al., 1999; Katz & Margalith, 1981). Increased levels of Zn2+ were reported many years ago to interfere with the replication of poliovirus, HRV and EMCV (Korant et al., 1974). Several studies suggested that Zn2+ ions affect polyprotein processing (Butterworth & Korant, 1974; Korant & Butterworth, 1976; Korant et al., 1974; Nakai & Lucas-Lenard, 1976), but the exact mechanism is not understood completely. In this study, we found that the PDTC-induced influx of Zn2+ interfered with the polyprotein processing of both CVB3 and mengovirus. In the case of CVB3, the proteolytic production of the capsid proteins seemed to be affected most severely, similar to what has been described previously for HRV (Krenn et al., 2005). Obviously, this cannot explain the defect in viral RNA replication, as the capsid proteins are dispensable for viral RNA replication in enteroviruses and HRV (Kaplan & Racaniello, 1988). Here, we showed that PDTC interfered with the autocatalytic processing of 3CDpro, indicative of an impaired activity of this protease. This finding provides a plausible explanation not only for the impaired trans-cleavage of the capsid protein-containing precursor, but also for the defect in viral RNA replication. 3CDpro is an important component of the ribonucleoprotein (RNP) complex involved in the initiation of minus- and plus-strand RNA synthesis (Andino et al., 1990, 1993). RNP complexes are formed at the cloverleaf in the 5' untranslated region (UTR) and the higher-order structures in the 3' UTR, by the binding of the viral proteins 3AB and 3CDpro together with host-cell factors (Andino et al., 1993; Harris et al., 1994). In this complex, 3CDpro is bound to specific elements in the UTR. Autocatalytic processing of 3CDpro, a process that is stimulated by 3AB, is required for the liberation of 3Dpol to become available for the synthesis of new RNA strands (Harris et al., 1994). Thus, by interfering with the autocatalytic processing of 3CDpro in cis, PDTC may inhibit viral RNA replication.
PDTC impaired the proteolytic processing of the CVB3 capsid proteins severely. This may be explained by a disturbed trans-cleavage activity of 3CDpro. The impaired production of the capsid proteins may also (partially) be due to direct interactions of Zn2+ ions with the capsid proteins. Korant & Butterworth (1976) showed that Zn2+ disturbed processing of the HRV1A polyprotein by interacting with protein precursors, particularly those containing capsid protein sequences, and altering them so that they cannot be cleaved. PDTC also affected the 3Cpro-dependent proteolytic processing of the non-structural proteins, which occurs mainly in cis (Dougherty & Semler, 1993; Palmenberg, 1990), but the adverse effects seemed to be less severe. PDTC reduced, but did not eliminate, the production of 2BC and 2C (Fig. 7a), and it also had relatively minor effects on the production of 3CDpro, which requires intramolecular cleavage by 3Cpro at its N terminus (Fig. 7b). The idea that Zn2+ ions can inhibit 3Cpro activity is supported by in vitro studies with recombinant HRV14 3Cpro (Cordingley et al., 1989). The reason that PDTC impaired the autocleavage of 3CDpro severely, whereas it had relatively mild effects on the 3Cpro-mediated cis-processing of the non-structural proteins, is unknown. In addition to the impaired autocleavage of 3CDpro, subtle alterations in the levels of the non-structural proteins may also contribute to the defect in viral RNA replication imposed by PDTC.
PDTC also interfered with mengovirus polyprotein processing, but the underlying cause seems to differ from that of CVB3. PDTC had no effect on the autocleavage of 3CDpro or the 3CDpro-dependent proteolytic processing of the capsid coding region. Instead, it caused the accumulation of a high-molecular-mass protein that was recognized by antibodies against the capsid and the 2A protein (Fig. 8b). This high-molecular-mass protein is much larger than the P1- and 2A-containing precursor proteins (e.g. L–P1–2A and P1–2A) that are typically observed in mengovirus-infected cells. These precursor proteins are the result of the occurrence of a unique amino acid sequence (DvExNPG/P motif) at the C terminus of the 2A protein that is thought to prevent the formation of a peptide bond between the C-terminal glycine of 2A and the N-terminal proline of 2B (Donnelly et al., 2001). The accumulation in PDTC-treated cells of a P1- and 2A-containing high-molecular-mass protein is indicative of a normal peptide-bond formation at the 2A–2B junction, resulting in a large precursor protein that contains P2 and, possibly, P3 region proteins as well. This large precursor was not observed by using anti-3D antibodies (Fig. 8a), suggesting that it is unlikely to contain the polymerase. Experiments undertaken with antibodies against 2B and 3AB were not conclusive, due to the poor immunoprecipitation properties of these antibodies. The idea that the PDTC-induced influx of Zn2+ ions affects polyprotein processing by altering the configuration of the mengovirus polyprotein, rather than by inhibiting the activity of 3Cpro or 3CDpro, is in line with results of Nakai & Lucas-Lenard (1976). These authors showed that cleavage of a capsid-containg precursor was disturbed by high concentrations of Zn2+ only when these ions were present during polyprotein synthesis. When added after synthesis and folding of the precursor, Zn2+ ions had no effect on proteolytic processing. Collectively, these results lend support to the idea that the PDTC-induced influx of Zn2+ ions leads to an altered folding of the nascent polyprotein, thereby disturbing the translation termination and reinitiation mechanism at the 2A–2B junction. How this results in a defect in viral RNA replication is as yet unknown. It is tempting to speculate that this defect is due to reduced amounts of mature 2A and 2B but, clearly, more research is required to support this idea.
Although our data point strongly to an effect of PDTC on polyprotein processing, it should be considered that PDTC may also have an impact on other viral enzymes. Zn2+ ions are described to inhibit the activity of the HRV 3Dpol in vitro (Hung et al., 2002). Thus, the increased intracellular Zn2+ ion concentration caused by PDTC might also inhibit viral RNA replication by interfering with the polymerase function of 3Dpol.
While this work was in progress, Si et al. (2005) also reported that PDTC inhibits replication of CVB3. These authors, however, did not investigate the effect of PDTC on polyprotein processing. Instead, they showed that PDTC led to the accumulation of several short-lived proteins in infected cells (e.g. p53, MKP-1 and p21), which is consistent with other reports that PDTC inhibits ubiquitin–proteasome-mediated protein degradation (Chen et al., 2005; Hayakawa et al., 2003; Kim et al., 2004; Lovborg et al., 2006), and raised the suggestion that PDTC interferes with virus replication by preventing the degradation of host-cell proteins that limit virus replication. It remains to be established whether the inhibition of cellular protein turnover constitutes another antiviral mechanism of PDTC or whether it is linked to the mechanism proposed here. It can be hypothesized that the autocatalytic processing of 3CDpro that must take place to release 3Dpol for viral RNA synthesis is regulated negatively by a cellular RNP component. By preventing the proteasomal degradation of such a negative regulator, PDTC may interfere with the autocatalytic processing of 3CDpro.
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ACKNOWLEDGEMENTS |
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Received 10 October 2006;
accepted 23 December 2006.
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3D). At 8 h post-transfection, cells were collected and luciferase activity was determined. Luciferase levels of non-transfected cells were always below 50 RLU (relative light units).
