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1 Department of Microbiology, Kanazawa Medical University, Ishikawa 920-0293, Japan
2 Division of Pathology, Sendai City Hospital, Miyagi 984-0075, Japan
Correspondence
Yoshiro Ohara
ohara{at}kanazawa-med.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of NeuroBiology and Therapeutics, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8505, Japan. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). TMEV is classified into two subgroups, TO and GDVII, based on different biological activities. The DA strain and other members of the TO subgroup cause a biphasic disease with an acute, subclinical encephalomyelitis, followed by a chronic demyelination in the spinal cords of mice (Obuchi & Ohara, 1998; Roos, 2002; Oleszak et al., 2004). TMEV contains a large open reading frame (ORF), translated as a long precursor polyprotein that undergoes autoproteolytic processing to yield all of the viral proteins required to fulfil the viral life cycle. In the TO subgroup, an additional protein, designated L*, was found to be translated from an alternative ORF starting 13 nt downstream of the initiation codon AUG of the virus polyprotein ORF and ending in the VP2-encoding region (Kong & Roos, 1991). The L* ORF is conserved in all TO subgroup strains analysed (Michiels et al., 1995). In GDVII subgroup strains, the AUG initiation codon for the translation of the alternative ORF is substituted for ACG, therefore no L* synthesis occurs. The role of L* is still poorly understood and controversial. DA (DAFL3 clone; Roos et al., 1989) with a mutation at the L* initiation codon, designated DAL*-1, fails to synthesize L* and has attenuated demyelinating activity in the central nervous system (CNS), suggesting that L* plays a key role for viral persistence and demyelination (Chen et al., 1995b; Ghadge et al., 1998). A mutant virus from a different DA clone, DA1, which also fails to synthesize L*, has a weak influence on virus persistence (van Eyll & Michiels, 2000).
We have shown previously that L* is required for virus growth in cells of the macrophage lineage (Takata et al., 1998; Obuchi et al., 2000; Himeda et al., 2005), the target cell of DA persistence. We also generated polyclonal rabbit anti-L* antibody (
-L*) and demonstrated that L* is not incorporated into virions and is associated with microtubules in vitro (Obuchi et al., 2001). In vivo expression of L* in the CNS was confirmed by immunoprecipitation with -L* in the acute phase of infection. Additionally, expression of L* in the acute phase of infection was analysed immunohistochemically by using a mutant virus (DA/3xFLAGL*) expressing FLAG epitope-tagged L*. A double-immunolabelling study demonstrated that L* colocalizes with the viral antigen and is expressed exclusively in neurons (Asakura et al., 2002).
We extended our previous observation and found that the mutant virus DA/3xFLAGL* does not persist or cause demyelination in the CNS. Therefore, in this study, we generated a further two mutant viruses expressing FLAG epitope-tagged L* to analyse the expression of L* in vivo in the chronic phase of infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). As shown in Fig. 1, in a construct designated DA/3xFLAGL*, which was generated previously (Asakura et al., 2002), a 3xFLAG epitope sequence was tagged directly to the N terminus of L*, resulting in the disruption of the zinc-binding motif in L. A further two constructs, DA/3xFLAGL*4 and DA/3xFLAGL*5, were designed to express FLAG epitope-tagged L* and L with an intact zinc-binding motif. Namely, the spacer sequence, consisting of 9 nt (CTTGCAAAC), was inserted between the 3xFLAG epitope and L* sequences, in order not to disrupt the zinc-binding motif in L. The ORF coding for L* starts with two AUG codons in the same frame separated by 9 nt (van Eyll & Michiels, 2002). In DA/3xFLAGL*4, only the first AUG was changed to ACG, as shown in Fig. 1. Therefore, a truncated 15 kDa protein could be synthesized in addition to a full-length, 18 kDa L*. In DA/3xFLAGL*5, the second AUG codon was changed to ACG in order not to synthesize the truncated 15 kDa L*. Comparison of the two mutant viruses makes clear the influence of the truncated 15 kDa L*. For the above modification, an overlap-extension PCR was applied as described previously (Asakura et al., 2002). The clones with correctly sized insert were sequenced completely on both strands. Plasmid DNAs were linearized with XbaI (TOYOBO) and viral RNA was synthesized by using T7 RNA polymerase (Promega). BHK-21 cells were then transfected with the synthesized RNA by using Lipofectin (Gibco) according to the manufacturer's instructions. Virus was purified by a standard plaque technique, propagated on BHK-21 cells and used in the following experiments.
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Immunoblotting.
Extracted proteins from BHK-21 cells infected with mutant viruses were separated by SDS-PAGE under reducing conditions on 15 % acrylamide gels and transferred to nitrocellulose membranes by electroblotting. The membranes were incubated with a mouse anti-FLAG M2 mAb (Sigma) and the mouse anti-VP1 capsid protein mAb DAmAb2 (kindly provided by Dr Raymond Roos, University of Chicago, IL, USA) overnight at 4 °C after blocking with Tris-buffered saline containing 5 % non-fat dry milk and 0.05 % Tween 20 for 1 h at room temperature. Bound antibody was then detected with biotinylated anti-mouse IgG and horseradish peroxidase-conjugated streptavidin (both from Jackson ImmunoResearch) using the Enhanced Chemiluminescence (ECL) system (Amersham Biosciences).
The expression of L* in each mutant virus was compared with an image analyser. L* expression was adjusted to the expression of capsid protein VP1. The relative ratios were compared with that of DA/3xFLAGL*.
Animal experiments.
SJL/J mice were purchased from Jackson Laboratories. The experiments were approved by the Kanazawa Medical University Institutional Animal Care and Use Committee. Four-week-old mice were injected intracerebrally with 2x105 p.f.u. virus in a 10 µl volume. At 21, 45, 90 and 180 days p.i., mice were sacrificed and perfused with physiological saline followed by 10 % formalin. Formalin-fixed brain tissues were dehydrated and embedded in paraffin. The entire spinal cord was also removed from the spinal canal, sectioned into lengths of a few millimetres and embedded in paraffin.
Histological study.
To detect inflammatory-cell infiltration and demyelination, haematoxylin–eosin (H–E) and Klüver–Barrera (K–B) stains were performed on 4 µm paraffin-embedded tissue sections. In addition, autoclave-pretreated tissue sections were subjected to the following immunohistochemical analysis. Briefly, the sections were incubated with DAmAb2 or anti-FLAG M2 mAb overnight at 4 °C, followed by incubation with peroxidase-conjugated secondary antibody (EnVision+; DAKO) for 30 min at room temperature and the bound antibodies were detected by 3,3'-diaminobenzidine tetrahydrochloride (DAB). To exclude non-specific bindings of secondary antibody with endogenous mouse antibodies, the serial sections were also stained without primary antibodies as a control. To identify L*-expressing cells, double labelling was also performed. The sections were subsequently incubated with biotinylated BS-1 lectin for macrophage/microglia (Vector Laboratories) or rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody for astrocytes (DAKO) overnight at 4 °C, followed by incubation with alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch) or secondary antibody (Histofine; Nichirei). The bound lectin or antibody was detected by using a New Fuchsin substrate kit (Nichirei). All sections were counterstained with haematoxylin.
Quantitative analysis of demyelinated lesions in spinal cords.
On each section with K–B staining, the areas of white matter or demyelinated lesion were quantified by using a digital image-analysis system (Mitani Corp.) attached to a Nikon photomicroscope. The ratio of the area of demyelinated lesion to the area of white matter was calculated.
RT-PCR.
One microgram of total RNA isolated from virus-infected SJL/J mouse spinal cords was denatured at 94 °C for 5 min, chilled on ice and reverse-transcribed into cDNA in a reaction mixture containing 50 U Moloney murine leukemia virus reverse transcriptase (Invitrogen), 10 µM dithiothreitol, 0.7 µM dNTPs and DA virus-specific primers (5' primer, DA940–960 nt; 3' primer, DA1311–1331 nt) at 42 °C for 60 min. The reaction was terminated by heating at 72 °C for 5 min. Two microlitres of the reaction mixture was subjected to the following PCR: 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 2 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The titres reached their peak at 12 h p.i. and decreased gradually thereafter. The peak titres of mutant viruses were similar to that of DA (Fig. 2a). In contrast, in L929 cells, DA/3xFLAGL* showed an approximately 2-log reduction of peak titre, as shown in Fig. 2(b). DA/3xFLAGL*4 and DA/3xFLAGL*5, carrying intact zinc-binding motifs, showed peak titres and growth patterns similar to those of DA.
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, lower panel). L* was detectable by immunoblotting with anti-FLAG mAb in BHK-21 cells infected with each virus (Fig. 3a, upper panel). The expression of L* in DA/3xFLAGL*4- or DA/3xFLAGL*5-infected cells, adjusted to the expression of VP1 capsid protein, increased by approximately 30 % in comparison with that in DA/3xFLAGL*-infected cells (Fig. 3b).
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Transverse or longitudinal spinal-cord sections from infected mice were prepared and studied histologically. At 21 and 45 days p.i., less severe inflammatory-cell infiltration was observed in the spinal cords of mice infected with DA/3xFLAGL*4 or DA/3xFLAGL*5 than in those infected with wild-type DA by H–E staining. At 90 days p.i., however, similar inflammatory-cell infiltration was observed in all virus-infected groups (data not shown). At 21 and 45 days p.i., demyelinated lesions were not identified by K–B staining in the mice infected with mutant viruses. However, minor demyelinated lesions were occasionally observed at 90 days p.i. Extensive demyelinated lesions were identified in the entire spinal cords of mice infected with both DA/3xFLAGL*4 and DA/3xFLAGL*5 at 180 days p.i. As shown in Fig. 4, the lateral and anterior columns at thoracic-cord level were demyelinated in mice infected with both DA/3xFLAGL*4 (Fig. 4a) and DA/3xFLAGL*5 (Fig. 4b). These demyelinated areas were similar to those observed in mice infected with wild-type DA (data not shown). Serial-section staining with viral antigen revealed viral persistence in the spinal-cord white matter (Fig. 4a and b, insets). Double staining with anti-FLAG mAb and BS-1 lectin demonstrated that L* and lectin are colocalized, suggesting that L* is expressed in lectin-positive cells (macrophage/microglia) (Fig. 4c). Subsequent double staining demonstrated that L*-positive cells are GFAP-negative (Fig. 4d).
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, approximately 17–20 % of white matter was demyelinated in the spinal cords of mice infected with DA/3xFLAGL*4 or DA/3xFLAGL*5. Among these experimental groups, including the wild-type DA-infected group, there was no statistical significance in the percentage area of demyelinated lesion/area of white matter.
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, lanes 1 and 2). For wild-type DA, a band of the predicted size (392 bp) was detected (Fig. 5, lane 3). Further sequencing analysis of PCR products confirmed that these PCR products of 461 bp are derived from DA/3xFLAGL*4 and DA/3xFLAGL*5 (data not shown). These results indicated that there was no wild-type DA virus contamination in mice infected with DA/3xFLAGL*4 or DA/3xFLAGL*5.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ghadge et al., 1998; van Eyll & Michiels, 2002), although this hypothesis is controversial (van Eyll & Michiels, 2000). Our previous study (Asakura et al., 2002) demonstrated, by using a mutant virus, DA/3xFLAGL*, which expresses FLAG epitope-tagged L*, that L* is expressed in vivo in the acute phase of infection in the CNS. We extended our observation to detect the expression of L* in the chronic phase of infection. However, we did not find any inflammation or demyelination in the CNS of DA/3xFLAGL*-inoculated mice at 45 and 90 days p.i. In addition, RT-PCR detected no TMEV genome (data not shown).
L is synthesized by members of the genera Cardiovirus and Aphthovirus, but not by members of other genera of the family Picornaviridae. TMEV strains have an L protein composed of 76 aa located in the N terminus of the polyprotein. It is a highly acidic protein (Ohara et al., 1988). Kong et al. (1994) have observed that L of TMEV is required for viral spread in L929 cells, but not in non-interferon (IFN)-responsive BHK-21 cells. Based on the fact that DA (TMEV) L has a putative C-H-C-C zinc-binding motif, they demonstrated that DA virus with a mutation in the motif displayed a restricted viral infection in L929 cells. Chen et al. (1995a) extended this observation and demonstrated that L of TMEV is a metalloprotein and that zinc binds a C-H-C-C motif that is conserved among cardioviruses. In the DA subgroup, van Pesch et al. (2001) introduced point mutations in the zinc-binding motif without affecting the L* alternative ORF. They reported that L inhibits the production of IFN-/
by infected L929 cells and specifically inhibits transcription of the IFN-4 and IFN- genes, which are known be activated early in response to viral infection. They additionally demonstrated that mutation of the zinc finger was sufficient to block anti-IFN activity (van Pesch et al., 2001). A more recent study showed that L interferes with trafficking of the cytoplasmic IFN-regulatory factor 3, a factor critical for transcriptional activation of IFN-/ genes (Delhaye et al., 2004).
In this study, as shown in Fig. 2
, only the growth of DA/3xFLAGL* was restricted in L929 cells. In DA/3xFLAGL* virus, the epitope tag was inserted immediately after the initiation codon of L*, which is located 13 nt downstream of the initiation codon of L, i.e. in the middle of the zinc-binding motif, leading to complete disruption of zinc binding (Fig. 1). Although the data are not shown, RT-PCR demonstrated that transcription of the immediate-early IFN genes IFN-4 and INF- is preserved in DA/3xFLAGL*-infected L929 cells, suggesting strongly that disruption of the zinc-binding motif by insertion of the FLAG epitope induced no virus persistence or demyelination. The present data are supporting evidence that the zinc-binding motif within L plays an important role for TMEV persistence (van Pesch et al., 2001).
In order to analyse L* expression in vivo in the chronic phase of infection, we generated a further two mutant viruses expressing epitope-tagged L*. In these mutants, the zinc-binding motif within L is conserved, as shown in Fig. 1. As expected, these mutants persisted in the spinal cords of SJL/J mice and caused inflammation and demyelination.
As shown in Table 1, the mutant viruses caused demyelination at 180 days p.i., similar to that caused by wild-type DA. In addition, demyelination caused by the two mutant viruses was not significantly different. The data suggest that tagging of the FLAG epitope does not alter the biological activities of wild-type DA (virus persistence and demyelination), leading to the physiological expression of L*. The expression and localization of L* can be studied in mice inoculated with these mutant viruses.
When injected intracerebrally into SJL/J mice, mutant virus-specific RNA containing the epitope tag was identified in the spinal cords by RT-PCR (without wild-type DA virus contamination). Although the mice infected with DA/3xFLAGL*4 or -5 showed less severe clinical signs and pathological findings at 21, 45 and 90 days p.i. in comparison with wild-type DA-infected mice, after several months of infection, mice showed severe clinical signs, including spastic paraparesis and severe inflammatory demyelination in the spinal cords by histological study. This observation was further confirmed by quantitative analysis of demyelinated lesions at 180 days p.i. (Table 1).
The present study demonstrated that L* is expressed in lectin-positive and GFAP-negative cells (macrophage/microglia). Although the cell types where TMEV antigen resides in the chronic phase of infection are still to be confirmed, several reports suggest strongly that these are macrophages: the recovery of infectious virus from infiltrating mononuclear cells (Clatch et al., 1990), the predominant viral load in macrophages (Lipton et al., 1995) and clearance of virus by the depletion of infiltrating macrophages (Rossi et al., 1997). Therefore, it is suggested that, in the chronic phase of infection, L* is colocalized with TMEV capsid antigen in macrophage/microglia cells. As L* is reported to remain without being incorporated into virions (Obuchi et al., 2001), L* in the cytoplasm of macrophage/microglia cells may have some effect(s) on the biological activities of DA (persistence and demyelination) through interaction with some host factor(s) of macrophages.
The precise mechanisms of TMEV-induced persistent infection and demyelination are yet to be elucidated. The functions of L and L* are also not yet fully understood. The mutant viruses generated in the present study showed similar behaviour to that of wild-type DA both in vitro and in vivo, and L* was visualized in the chronic phase of infection. These viruses may be useful to pursue further elucidation of the role(s) of L and L*.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 July 2006;
accepted 2 April 2007.
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) and mutant (
, DA/3xFLAGL*; 
, DA/3xFLAGL*4; 
, DA/3xFLAGL*5) viruses on BHK-21 (a) and L929 (b) cells. Cells were infected with each virus (10 p.f.u. per cell). A mixture of culture supernatants and cell lysates of infected cells at various time points (0, 3, 6, 12, 24 and 48 h p.i.) was subjected to titre determination by plaque assay. Values represent the mean±SD of triplicate samples.
