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Institute of Medical Virology, University of Zürich, CH-8006 Zürich, Switzerland
Correspondence
Stefan A. Deuber
stefan.deuber{at}gmail.com
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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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). Semliki Forest virus (SFV) is transmitted by different species of mosquitoes from the genus Aedes. The first isolation of SFV from mosquitoes was reported by Smithburn & Haddow (1944) and subsequently several other SFV strains were isolated and propagated in mice or in cell culture. Virus strains are designated either virulent or avirulent according to their pathogenicity in adult wild-type (wt) mice (Bradish et al., 1971). Virulent strains of SFV enter the central nervous system (CNS) leading to the development of a fatal encephalitis (Fazakerley et al., 1993). Important determinants for neurovirulence of SFV and Sindbis virus have been found in the 5'-untranslated region, glycoprotein E2 and non-structural protein (nsP) 3 (Glasgow et al., 1994; Santagati et al., 1995; Tarbatt et al., 1997; Tucker et al., 1993; Tuittila & Hinkkanen, 2003; Tuittila et al., 2000).
Although many studies have focused on the characterization of neurovirulence determinants, little is known about the mechanisms involved in viral interference with the innate immune system. The type I interferon (IFN) system plays a central role in innate defence and is of great importance for protection against many viral infections. Mice lacking a functional IFN receptor (IFNAR–/–) are very sensitive to infection with various viruses, including SFV, that are avirulent for wt mice (Fazakerley et al., 2002; Muller et al., 1994).
The cytosolic replication of alphaviruses is initiated by translation of the replicase as a polyprotein from the positive-strand 42S genomic RNA (reviewed by Strauss & Strauss, 1994). The polyprotein is proteolytically cleaved, giving rise to an enzymic protein complex consisting of nsP1–4. This replicase regulates the synthesis of positive- and negative-strand genomic RNA, as well as the production of 26S subgenomic RNA encoding the structural proteins.
During co-evolution with their hosts, viruses have developed strategies to counteract the IFN-mediated antiviral response. Many viruses inhibit the induction of IFN by multiple mechanisms (reviewed by Weber et al., 2004). However, much less is known about viral interference with an established IFN-induced antiviral state.
The virulent L10 strain of SFV was derived from the original virus isolate by ten passages in mouse brain. In contrast, the avirulent strain, designated V42, is an earlier passage of the original virus isolate (Smithburn & Haddow, 1944). The attenuation of the SFV V42 strain in mice is strictly dependent on a functional IFN system, as shown previously, whereas the virulent L10 strain is lethal for wt mice (Fazakerley et al., 1993; Muller et al., 1994). In order to understand in more detail the IFN susceptibility of SFV, we investigated the distinct IFN sensitivities of these two closely related virus strains in cell culture.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were prepared by standard techniques. Normal growth medium consisted of Dulbecco's modified Eagle's medium supplemented with 10 % virus-free fetal calf serum (Chemie Brunschwig). All cell lines were maintained at 37 °C in 5 % CO2. Recombinant murine IFN-
A was purchased from PBL Biomedical Laboratories. Human IFN-2 was obtained from Roche. Exogenous IFN added to the cells for 16 h was washed away before virus infection in all cases. Cell survival was determined by staining the cells for 10 min in 1 % crystal violet, 20 % ethanol, 3.6 % formaldehyde, 1 % methanol. Quantification was performed by adding 80 % ethanol and reading the absorbance at 595 nm in a PowerWaveXS (BioTek) ELISA reader.
Detection of the SFV capsid protein was performed using a polyclonal rabbit antibody (Landis et al., 1998). Polyclonal rabbit nsP1 and nsP2 antisera were kindly provided by Tero Ahola, University of Helsinki, Finland. The expression of MxA was visualized using a monoclonal antibody raised against recombinant human MxA protein (Schnorr et al., 1993). A commercially available goat anti-human actin antibody (I-19; Santa Cruz) was used to demonstrate equal amounts of protein.
Virus strains.
SFV V42 (original strain) has been described previously (Muller et al., 1994). The L10 strain was kindly provided by John Fazakerley, University of Edinburgh, UK. Virus stocks of SFV were prepared and titrated on BHK-21 cells.
Isolation of total RNA and Northern blot analysis.
Total cellular RNA was extracted using TRI reagent (Sigma) according to the manufacturer's instructions. The integrity of the RNA was verified on a denaturing formaldehyde 1.2 % agarose gel.
Northern blot analysis was performed as described previously (Landis et al., 1998). A radioactively labelled DNA fragment was prepared using the RadPrime DNA Labelling System (Invitrogen). Membranes were exposed to a storage phosphor screen and bands were visualized by scanning with a Storm 840 PhosphorImager (Molecular Dynamics).
Immunofluorescence microscopy.
Cells were fixed for 10 min in 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100 for 5 min. Nuclear counterstaining was performed with Hoechst 33258. Rhodamine-conjugated secondary antibodies were used for visualization by light microscopy. Pictures were taken with a DC350FX CCD camera mounted on a DM IRB inverted microscope (Leica).
Western blot analysis.
Separation of proteins was performed using a standard SDS-PAGE procedure (Laemmli, 1970). Proteins were transferred to nitrocellulose membranes and incubated overnight with the appropriate primary antibodies at 4 °C. Horseradish peroxidase-coupled secondary antibodies were used and protein bands were visualized using an enhanced chemiluminescence system. Quantification of the bands was performed using UTHSCSA IMAGETOOL (Wilcox et al., 2002).
Quantification of dose-dependent IFN sensitivity.
Vero cells were seeded into 96-well plates and stimulated overnight with a twofold dilution series of IFN ranging from 2048 to 0 U ml–1. IFN-treated cells were challenged with 104 p.f.u. of virus. Virus was left to multiply for 48 h and culture supernatant was replaced with medium containing 0.1 mg 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) ml–1 (Mosmann, 1983). Blue precipitates were dissolved in 100 µl 2-propanol and quantified by absorption at 560 nm in an ELISA reader.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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showed that SFV strain V42 is avirulent for wt but not IFNAR–/– mice, wt MEFs as well as IFNAR–/– MEFs were used. Untreated MEFs and MEFs treated with IFN- for 16 h were infected with V42 and L10 at an m.o.i. of 2. Virus yield in cell-culture supernatants was determined at 24 h post-infection (p.i.) and the development of a cytopathic effect (CPE) was monitored at 48 h p.i. In untreated wt or IFNAR–/– MEFs, both strains replicated to high titres (1x1010–4x1010 TCID50 ml–1) (Fig. 1a, b) and induced a CPE (Fig. 1c, d). Similarly, IFN-treated IFNAR–/– MEFs infected with either V42 or L10 produced large amounts of progeny virus (Fig. 1b) and induced a strong CPE (Fig. 1d). However, in IFN-treated wt MEFs, the virus titre of V42 was reduced by four orders of magnitude, whereas L10 was inhibited by two orders of magnitude only (Fig. 1a). In addition, IFN- treatment of wt MEFs prevented the development of a CPE by the V42 strain, whereas L10 still induced a complete CPE by 48 h p.i. (Fig. 1c).
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). In the IFN-treated wt MEFs infected with V42, 1 % of the cells at 8 h p.i. and 10 % of the cells at 24 h p.i. were positive for virus protein (Fig. 1e). In the IFN-treated wt MEFs infected with L10, 4 % of the cells at 8 h p.i. and >99.9 % of the cells at 24 h p.i. were virus protein positive (Fig. 1e). This result showed that the avirulent V42 strain propagated only poorly in IFN-treated wt MEFs in contrast to the virulent L10 strain. Hence, consistent with the previously described IFN-mediated inhibition of the avirulent V42 strain in vivo, our data demonstrated that V42 was much more sensitive than L10 to the action of IFN- in cell culture (Muller et al., 1994).
IFN reduces the synthesis of genomic and subgenomic RNA
We next analysed the effect of IFN on viral RNA synthesis. For this purpose, intracellular RNA was isolated from wt or IFNAR–/– MEFs that either were left untreated or were treated with IFN- for 16 h and subsequently infected with L10 or V42. In untreated wt MEFs, as well as in untreated or IFN-treated IFNAR–/– MEFs, both viruses showed similar kinetics of accumulation of viral RNA (Fig. 2a–d). Viral RNAs were first detected at 4 h p.i. (Fig. 2a, b, lanes 2 and 12). The decrease in viral RNA levels observed at later time points of infection (24 h p.i.) was due to the CPE. Quantification of the band intensities in untreated wt MEFs and untreated or IFN-treated IFNAR–/– MEFs revealed that the avirulent V42 strain synthesized about 20 % more genomic RNA than the virulent L10 strain throughout infection (Fig. 2c, d, f). Furthermore, the ratio of 42S to 26S RNA was around 1.0 for the virulent L10 strain but ranged between 1.5 and 2.0 for the avirulent V42 strain. In wt MEFs treated with IFN-, however, the kinetics of accumulation of viral RNA clearly differed between both strains. Although accumulation of viral RNA of both viruses was reduced in IFN-treated wt MEFs, L10 synthesized about tenfold more viral RNA than V42 throughout infection (Fig. 2a, lanes 6–10 and 16–20, and Fig. 2e). Taken together, both viruses grew to similar titres in untreated wt MEFs, but analysis of viral RNA synthesis revealed that the avirulent V42 strain produced more genomic but less subgenomic RNA than the virulent L10 strain.
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) and BHK-21 cells (Fig. 3b). Production of progeny virus of the virulent L10 strain was slightly faster during the first hours of infection. Nevertheless, at 24 h p.i. a similar high titre was found for both strains of about 1010 TCID50 ml–1 in NIH-3T3 cells and 5x1010 TCID50 ml–1 in BHK cells.
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by Western blot analysis. Indeed, larger amounts of nsP1 were measured at 8 h p.i. for L10 than for the V42 strain (Fig. 3c, e). Similarly, small amounts of nsP3 were observed as early as 4 h p.i. in L10-infected cells, whereas nsP3 synthesized by V42 was only detected at 6 h p.i. (Fig. 3d, f). Although less genomic RNA was detected in cells infected with L10, larger amounts of nsP1 and nsP3 were produced in these cells. This apparent inconsistency could be explained by a higher translation efficiency of the L10 genomic RNA than the V42 genomic RNA.
Replication of the V42 strain is blocked more strongly than L10 in Vero cells unable to produce IFN
Many viruses inhibit the induction of IFN to block the cellular antiviral response. To exclude the possibility that the lower sensitivity of L10 to IFN was due to reduced induction of IFN by this virus, we infected African green monkey (Vero) cells, which are unable to produce IFN but are still responsive to exogenous IFN (Emeny & Morgan, 1979). Untreated Vero cells infected with L10 and V42 developed a complete CPE by 48 h p.i. as revealed by crystal violet staining (Fig. 4a). In IFN-treated Vero cells, the virulent L10 strain still exerted an almost complete CPE in contrast to the avirulent V42 strain where no CPE was observed. Quantification of cell survival after virus infection showed that untreated Vero cells infected with either virus were killed efficiently, leaving fewer than 10 % of cells viable (Fig. 4b). Treatment with IFN- resulted in the survival of the culture infected with V42, whilst infection with the virulent L10 strain left fewer than 10 % of cells viable. This confirmed that the higher cytopathogenicity of L10 in the IFN-treated culture was due to a reduced susceptibility to the action of IFN and not due to a reduced IFN induction.
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). Infection of IFN-treated Vero cells with the avirulent V42 strain resulted in 105 fewer progeny virus ml–1, whilst the titre of the virulent L10 strain was reduced by only one order of magnitude.
In order to determine whether the IFN-mediated reduction of viral titres and of CPE were due to inhibition of virus replication or to reduced cell death, we analysed virus infection on a single-cell level. Vero cells were infected and stained for viral capsid protein at 8 and 24 h p.i. In untreated cultures infected with V42 or L10, virtually 100 % of the culture was positive for virus protein at 8 and 24 h p.i. (Fig. 4d). In the IFN-treated culture infected with V42, no virus-positive cells were found at 8 h p.i. and fewer than 0.1 % of the cells stained positive for virus protein at 24 h p.i. In contrast, about 0.3 % of the IFN-treated culture infected with L10 stained positive for virus protein at 8 h p.i. and >99.9 % of the cells were positive at 24 h p.i. These data showed that IFN treatment did not reduce the induction of cell death but blocked the production of viral proteins of the avirulent V42 strain but not of the virulent L10 strain in Vero cells.
The human MxA protein has been described as inhibiting SFV replication in cell culture and in vivo (Hefti et al., 1999; Landis et al., 1998). We therefore tested whether MxA was induced following IFN treatment of Vero cells. As a positive control, we used the lung carcinoma cell line A549, in which MxA is strongly induced upon IFN stimulation (Ronni et al., 1993). Vero and A549 cells were stimulated with IFN- for 16 h and analysed for MxA by immunofluorescence microscopy. As expected, IFN treatment of A549 cells resulted in strong induction of MxA, whereas no expression of MxA was detected in IFN-treated Vero cells (Fig. 4e). The inability to detect MxA in Vero cells was not due to lack of specificity of the antibody, as the anti-MxA antibody detected IFN-induced MxA in extracts of African green monkey-derived CV-1 cells (Fig. 4f). Hence, the MxA protein was not responsible for the stronger IFN-mediated inhibition of the avirulent V42 strain compared with the virulent L10 strain in Vero cells.
In order to quantify the distinct IFN- susceptibility of V42 and L10, we determined the viability of cells pre-treated with increasing IFN concentrations and infected with the L10 or V42 strain. An IFN concentration of 130 U ml–1 was required to protect 50 % of the culture infected with the virulent L10 strain (Fig. 4f). By contrast, as few as 16 U IFN- ml–1 were sufficient to protect 50 % of the culture infected with the avirulent V42 strain.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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susceptibilities of two closely related SFV strains in cell culture. In order to investigate the molecular events underlying the IFN response, we used an in vitro cell-culture system where the early effects of the type I IFN system could be analysed independently of the complex processes involved in vivo during the systemic infection and invasion of the CNS. The avirulent V42 strain, an early passage of the original virus isolate, is not pathogenic for wt mice. The low pathogenicity of this strain in mice and human cells is due to its sensitivity to IFN (Hefti et al., 1999; Landis et al., 1998; Muller et al., 1994). By contrast, the virulent L10 strain, which was derived from the original isolate by ten passages in mouse brain, induces the development of a fatal encephalitis in wt mice (Fazakerley et al., 1993). In the absence of a functional IFN system, however, the avirulent V42 strain replicates efficiently in the CNS and leads to the development of a fatal encephalitis (Hefti et al., 1999).
Our results showed that the reduced susceptibility of L10 to the action of IFN was also observed in cell culture, suggesting that factors different from neurovirulence determinants define the lower susceptibility to IFN-. Virus infections were performed in cells pre-treated with IFN, suggesting that the distinct susceptibility to IFN was due to a distinct susceptibility to the IFN-induced antiviral state. IFN-treated Vero cells unable to produce IFN were also protected against infection with the avirulent V42 strain but not against the virulent L10 strain. Hence, we excluded the possibility that the observed phenotype was a consequence of reduced IFN induction. Although both viruses showed a dose-dependent, IFN-mediated inhibition of the CPE, the virulent L10 strain required ten times more IFN- for inhibition comparable to that of the avirulent V42 strain. We propose that part of the higher virulence of L10 compared with V42 is due to the reduced susceptibility to IFN.
Three IFN-regulated antiviral pathways, the Mx proteins, the dsRNA-dependent protein kinase (PKR) and the RNaseL system, have been investigated extensively (reviewed by Samuel, 2001). However, mice deficient in these pathways are still protected against infections by many viruses including alphaviruses, suggesting the existence of an alternative IFN-induced mechanism inhibiting alphavirus replication (Ryman et al., 2002; Zhou et al., 1999). Long dsRNA generated during virus replication is sensed by cellular pattern-recognition receptors, activating the IFN system (reviewed by Kawai & Akira, 2006). We detected reduced amounts of genomic RNA in cells infected with the virulent L10 strain. Different regions of viral genomes contain sequences with extensive secondary structures, required for replication and translation. Therefore, lower concentrations of genomic RNA might result in decreased levels of intracellular dsRNA. The reduced amounts of genomic RNA may lead to a weaker activation of antiviral proteins. Both PKR and the RNaseL system require binding to dsRNA for activation (Kerr & Brown, 1978). However, neither pathway is primarily responsible for the IFN-mediated inhibition of alphavirus replication (Ryman et al., 2005, 2002).
Translation of the genomic RNA represents a critical step in the life cycle of positive-strand RNA viruses. Although less genomic RNA was detected in cells infected with the virulent L10 strain, more nsPs were found, suggesting that the translation efficiency of the genome and therefore the expression of nsPs by the virulent L10 strain were higher than those of the avirulent V42 strain. Expression of nsPs in the absence of structural proteins is sufficient to induce the shut off of host protein synthesis (Frolov & Schlesinger, 1994; Wengler, 1975). Predominantly, mutations in the coding region of nsP2 have been found to reduce the shut off of host protein synthesis, allowing persistent infections without affecting virus viability (Frolov et al., 1999). The faster production of nsPs by the virulent L10 strain allowed the induction of a shut off of host protein synthesis even in the presence of IFN (data not shown) and may thereby lead to a more efficient interference with the cellular antiviral response, in particular when short-lived cellular proteins are involved.
The human MxA protein restricts the replication of SFV V42 strain in human cells and MxA-transgenic mice (Hefti et al., 1999; Landis et al., 1998). However, we were unable to detect MxA in Vero cells treated with IFN-. Therefore, we excluded MxA as the effector molecule responsible for the observed IFN-dependent inhibition of SFV replication in Vero cells. Based on the findings that small differences between V42 and L10 are potentiated in the presence of IFN, we propose the existence of a latent IFN-inducible protein that is constitutively expressed at low levels in the absence of IFN and that inhibits V42 more efficiently than L10. Results from recent studies suggest that several cellular proteins inhibit the translation of genomic alphavirus RNA (Berlanga et al., 2006; Bick et al., 2003). It would now be interesting to investigate the functions of these proteins in cells infected with the two SFV strains.
The characterization of the distinct IFN sensitivities of SFV V42 and L10 will be used as the basis for the identification of the viral sequence conferring IFN susceptibility. We are currently investigating recombinant viruses derived from both viruses by exchanging viral sequences between the two strains. Determination of the viral sequence may then help to identify the so far unknown cellular factor that inhibits the replication of SFV and potentially also other viruses.
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ACKNOWLEDGEMENTS |
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Received 2 June 2006;
accepted 16 March 2007.
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