INRA, Unité de Virologie et Immunologie Moléculaires, 78352 Jouy-en-Josas cedex, France1
INSERM U520, Institut Curie, section recherche, 12 rue Lhomond, 75005 Paris, France2
Author for correspondence: Abdenour Benmansour. Fax +33 1 34 65 25 91. e-mail abdenour{at}biotec.jouy.inra.fr
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Abstract |
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Introduction |
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Using differential display PCR, we have described vig1 as a gene induced during virus incubation of rainbow trout leukocytes with viral haemorrhagic septicaemia virus, a fish rhabdovirus (VHSV; Boudinot et al., 1999 ). Its human homologue, cig5, was similarly identified from primary skin cultures incubated with inactivated human cytomegalovirus (Zhu et al., 1997
). vig1 and cig5 sequences are very similar, suggesting a conserved function probably related to the host response to virus infection. The rat gene best5 identified in bone tissue from ovariectomized females (Grewal et al., 2000
) and mouse expressed sequence tags from different tissues display high levels of sequence similarity with vig1 and cig5. This conservation suggests that these genes, present in all vertebrates, may form a new family of genes implicated in the cell non-specific response. The vig1/cig5 gene family shares the MoaA sequence signature with several prokaryotic and eukaryotic genes. The MoaA motif (Prosite PDOC01009) is present in bacterial, plant and human proteins involved in the synthesis of enzymatic cofactors such as pyrroloquinoline quinone (PQQ3), haem D1 and molybdopterin (Hoff et al., 1995
; Reiss et al., 1998
). Such cofactors can play different roles. For example, a tetrahydrobiopterin cofactor modulates the activity of the inducible nitric oxide synthase. Accordingly, the vig1/cig5 gene family may be implicated in the synthesis of a cofactor participating in an enzymatic pathway induced in pathological conditions. However, the function of these genes remains to be elucidated.
The mouse appears as a more appropriate model to gain further insight into the function of such genes. To compare with the situation described in man and the rainbow trout, we used a rhabdovirus, vesicular stomatitis virus (VSV), and an alphaherpesvirus, pseudorabies virus (PrV), as virus inducers. We first confirmed that both viruses induced the mouse gene homologue to vig1 and cig5 (mvig). We then identified dendritic cells as the main cell population where the up-regulation of the virus-induced transcription took place. We also report that mvig induction is directly mediated by the VSV, but not by the PrV particle. Indeed, the strong up-regulation produced by PrV results only from type I interferon secretion. Finally we found that mvig induction could be also obtained with LPS. Thus, three pathways of induction are maintained in vertebrates for this gene family. Two pathways are related to the host response to viruses, and the third pathway depends on a strong activator from bacteria. The vig1/cig5 gene family may represent a new class of primitive mediators active during infection in vertebrates.
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Methods |
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Virus preparation.
The Bartha strain of PrV was propagated in porcine kidney cells (PK15) in serum-free MEM. Supernatants, collected after 2 days of infection, were clarified and the virus titre was determined on PK15 cells and expressed in TCID50/ml (1x108). The Indiana laboratory strain (Orsay) of VSV was grown for 24 h in BSR cells (a clone of BHK-21) in MEM supplemented with 2% FCS. The virus titre was determined by plaque titration and expressed in p.f.u./ml (typically 1x109). When necessary, VSV was inactivated with -propiolactone at 1/4000 for 1 h at room temperature and then overnight at 4 °C.
Dendritic cell preparation.
Positive selection of spleen dendritic cells was performed by magnetic cell sorting, with the VarioMACS (Miltenyi Biotech), using anti-CD11c (N418)-conjugated magnetic beads (#520-01) and a VS+ column. Positively selected cells were passed over a second selection column (MS+) to increase their purity. Purity of selected and depleted cells was verified by control staining of CD11c-positive cells with an FITC-conjugated antibody specific for hamster IgG(H+L) (Caltag). Rat anti-mouse class II antibody was TIB120 (ATCC no. 3480), and was used with an FITC-conjugated anti-rat kappa light chain (Pharmingen).
Bone marrow-derived dendritic cells were obtained as described by Winzler et al. (1997) . Briefly, bone marrow cells were cultured for 2 weeks in Iscoves modified Dulbeccos medium (Sigma) containing 10% heat-inactivated FBS (GIBCO), 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (Sigma), 50 µM 2-mercaptoethanol, in the presence of 30% conditioned medium from GM-CSF-producing NIH/3T3 cells (Regnault et al., 1999
; Winzler et al., 1997
). Antibodies specific for CD11c (N418), GR-1, CD86/B7-2(GL1) and CD40 (HM40-3) were from Pharmingen, and the anti-class II MHC was Y3P (ATCC no. 5168).
Cell stimulation conditions.
Cells were adjusted to 25x106/ml, and infected with VSV or with PrV diluted 1/100 in culture medium. For each cell type tested, a control with no virus was included. Cells were incubated for 35 h at 37 °C. In addition, mock infections (i.e. incubation with supernatants from non-infected PK15 or BSR cells) were performed when relevant. Cycloheximide (CHX; Sigma) was used at 100 µg/ml. Anti-mouse /
interferon immunoglobulin (neutralizing titre of 5x105/ml against L-cell-derived
/
interferon) was provided by B. Dalton (SmithKline Beecham Pharmaceuticals). Normal sheep immunoglobulin was purchased from the National Veterinary Institute, Uppsala, Sweden. LPS from E. coli 026:B6 (Sigma) was used at 1 µg/ml.
mvig cloning and sequencing.
Total RNA isolated from lymph node cells according to the method of Chomczynski was treated with 10 U of DNase I (Boehringer Mannheim) in 0·1 M sodium acetate, 5 mM MgSO4 pH 5, with 60 U of ribonuclease inhibitor (RNaseOUT, GibcoBRL) for 1 h at 37 °C. DNase I was eliminated by phenol and chloroform extraction. RNA from each sample (5 µg) was reverse transcribed using 400 U of M-MLV reverse transcriptase with 7 mM of random hexanucleotide primers [pd(N)6, Pharmacia Biotech], 10 mM of each dNTP and 40 U of RNaseOUT.
A primer pair derived from the rat sequence (GenBank accession no. Y07704), MV1 forward 5' GCTCGGCTGCTGACCCTGTTCC 3' and MV1 reverse 5' AACAACAGTCTGTAGACAGC 3' (amplified product 1157 bp), was used to amplify a fragment encompassing most of the mvig ORF. PCR product was cloned using the TOPO TA cloning kit (Invitrogene), and several plasmid clones were used to derive a consolidated non-ambiguous sequence. To complete the ORF sequence, 5'RACE was performed on the RNA sample using the SMART RACE cDNA amplification kit (Clontech), according to the manufacturers instructions. The PCR product obtained with the universal primer (from the kit) and an MVIG-specific reverse-sense primer 5' TTAGGGTGGCTAGATCCCGGGAAGGAACAG 3' was cloned as described above. Sequences were assembled and aligned using the GCG package.
Normalized RTPCR assay.
An RTPCR assay was performed as described by Colle et al. (1997) . cDNA template and an internal
-actin DNA standard (1x106 copies) were diluted in 10 mM Tris, 50 mM KCl (pH 9 at 25 °C) with 100 pmol of each primer, 300 mM of each dNTP and 2·5 U of Taq polymerase (Promega). Amplification required 35 to 40 cycles as follows: 30 s at 94 °C, 30 s at 61 °C and 1 min at 72 °C. Aliquots of each PCR reaction were subjected to digestion with restriction enzymes specific for the wild-type template (BglII) or the standard template (ClaI) to allow the quantification of
-actin cDNA in the samples. mvig-Specific amplifications were then performed on normalized cDNA samples containing 1x106
-actin copies. Two pairs of primers specific for mvig were used: MV1forward 5' GCTCGGCTGCTGACCCTGTTCC 3' and MV1reverse 5' AACAACAGTCTGTAGACAGC 3' (amplified product 1157 bp), MV2forward 5' TTGTGCTGCCCCTTGAGGAAG 3' and MV2reverse 5' GTCCTCTTCCACGTTGAAACG 3' (amplified product 403 bp). PCR conditions were 94 °C for 5 min, and then 94 °C for 1 min, 56 °C (MV1 primers) or 45 °C (MV2 primers) for 1 min, and 72 °C for 2 min, for 25 to 30 cycles. PCR products were sequenced to verify the specificity of the amplification.
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Results |
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mvig is induced in the dendritic cell line D2SC1
The availability of several established cell lines representative of the different lineages of cells present in the mouse spleen allowed us to investigate the nature of the cells expressing mvig. For this purpose, we performed a series of infection experiments using VSV and PrV and a collection of representative cell lines. The lymphoma EL-4 (Shevach et al., 1972 ) and the T cell hybridoma B3Z (Shastri & Gonzalez, 1993
) represent the T cell lineage. The immature B cell line WEHI-231 (Gutman et al., 1981
) and the B lymphoma A20 (Kim et al., 1979
) represent the B lineage. A macrophage cell line (P388D1) and dendritic cell line D2SC1 (Paglia et al., 1993
; Lutz et al., 1994
) were representative of other antigen-presenting cells. The fibroblast line L-929 (ATCC, CCL-1, RF33956) was used as a non-lymphoid/non-myeloid control. The L-929 cell line is known to be permissive to VSV infection. Each cell line was incubated for 5 h with VSV, PrV or virus-free medium and mvig mRNA accumulation was assessed by an RTPCR assay normalized on the basis of quantitative
-actin expression. As shown in Fig. 2
, mvig induction was observed only in D2SC1. mvig was not induced in mock-infected D2SC1 (data not shown). These results suggest that the induction could be restricted to dendritic cells.
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mvig is induced by VSV and PrV in fresh bone marrow-derived dendritic cells
To further ascertain that mvig is induced by viruses in cells of the dendritic lineage, we used bone marrow-derived dendritic cells (Winzler et al., 1997 ; Regnault et al., 1999
). Briefly, dendritic cells were prepared by culturing bone marrow cells for 10 days in medium supplemented with supernatant of GM-CSF-producing fibroblasts. Following this treatment dendritic-specific marker CD11c (N418) was expressed by the bone marrow-derived cells while granulocytic-specific marker GR-1 was not expressed (data not shown), indicating that these cells belong effectively to the dendritic lineage (Fig. 4A
). In addition, the majority of cells expressed low levels of MHC class II and B7-2 molecules, and were CD40-negative, indicating an immature state.
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The bone marrow-derived dendritic cells were subjected to VSV or PrV for 5 h. A strong accumulation of mvig transcript was observed in virus-treated cells, while mvig transcript was barely detectable in the non-treated cells (Fig. 4B). We also verified that mvig is not induced in mock-infected bone marrow-derived dendritic cells (data not shown). These results confirmed that viruses can induce mvig in the dendritic lineage.
We have observed mvig expression in non-infected CD11c-positive cells obtained by mechanical separation. This result suggested that mvig expression may be also linked to a non-specific activation of dendritic cells. To further investigate this point, we searched for mvig expression in activated dendritic cells. LPS is a known powerful bacterial activator of dendritic cells. We used LPS to activate bone marrow-derived dendritic cells and then tested for mvig expression. Fig. 4(C) shows that mvig was strongly induced in these cells as early as 2 h after activation.
mvig induction is directly mediated by the VSV particle while the induction by PrV is mediated through the /
interferon pathway
We have previously shown that VHSV, a fish rhabdovirus, could induce vig1 via an interferon-independent pathway, and that a fish interferon-like compound was highly efficient in vig1 induction (Boudinot et al., 1999 ). In the murine model, VSV, a mammalian rhabdovirus, seems to share similar properties. Indeed, the up-regulation of mvig transcript level in D2SC1 cells was not abolished by addition of CHX, and CHX alone had no effect on the mvig mRNA level (Fig. 5A
, lanes 2 and 3), indicating that mvig induction did not require the de novo synthesis of any protein intermediate. Moreover, both inactivated and live VSV strongly induced mvig transcript accumulation in the dendritic cell line D2SC1, showing that this modulation did not require virus replication either (data not shown). To completely exclude the effect of interferon, we infected D2SC1 cells in the presence of anti-mouse
/
interferon antibodies (Fig. 5A
, lane 4). While anti-interferon antibodies completely abolished mvig induction by PrV (see below), they had no effect on the mvig induction obtained in the presence of VSV. We also checked that the anti-interferon antibody used alone cannot induce mvig (Fig. 5A
, lane 6), and that non-infected cells did not express mvig (Fig. 5A
, lane 7).
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Taken together, these results clearly indicate that viruses can use either of two distinct pathways to induce mvig. One is independent of the interferon pathway and seems to be mediated directly by the virus particle, the other proceeds through the well-known virus-induced interferon pathway.
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Discussion |
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MVIG protein displays a high sequence similarity with VIG1 and CIG5, and shares the conserved motifs identified for VIG1 and related proteins (Boudinot et al., 1999 ). The strong conservation of the mvig family in vertebrates suggests that the function of these genes may be conserved, and therefore should be of importance. This idea is supported by the fact that vig1 (trout), mvig (mouse) and cig5 (human) are induced by viruses.
In the rainbow trout, vig1 is induced by a fish rhabdovirus, VHSV, either directly or via soluble factors from a conditioned medium containing an interferon-like activity (Boudinot et al., 1999 ). In humans, cig5 was described as a cytomegalovirus-induced gene in human primary skin cultures (Zhu et al., 1997
). As in the fish, the accumulation of this transcript can be obtained through two pathways, either directly mediated by the virus, or via the type I interferons. We show here that mvig is also induced through two different pathways. VSV still induces mvig in the presence of anti-mouse
/
interferon antibodies or CHX, showing that mvig induction does not require any de novo protein synthesis and, specifically, no interferon. Furthermore, mock-infected cells do not express mvig. Although we cannot completely rule out direct mvig induction through cross-reactive factors released by VSV-producing cells, we consider that direct induction is the most probable hypothesis. Indeed, direct virus induction of the human mvig homologue was clearly demonstrated (Zhu et al., 1997
). While VSV-mediated induction does not require any protein intermediate synthesis, PrV-mediated induction is strictly dependent on interferon. Since anti-mouse
/
antibodies completely abolish the accumulation of mvig mRNA, it could be inferred there is no alternative to the interferon pathway for PrV. In contrast, Zhu et al. (1997)
observed both direct and interferon-mediated induction with another herpesvirus, the cytomegalovirus.
mvig can be induced in the dendritic cell line D2SC1, in dendritic cells purified from the spleen, and in bone marrow-derived dendritic cells. On the contrary, fibroblast, macrophage or lymphoid cell lines failed to show any accumulation of mvig transcript. Moreover, mvig induction was not observed in dendritic cell-depleted spleen cells. These results therefore strongly suggest that mvig induction by viruses is restricted to cells of the dendritic lineage. Tissue distribution of mvig homologues is consistent with this hypothesis: cig5 was induced in human skin primary cultures, which probably contain Langerhans cells (Zhu et al., 1997 ), and trout vig1 was expressed in lymphoid organs (Boudinot et al., 1999
).
Dendritic cells are professional antigen-presenting cells that are distributed in almost all tissues (Banchereau & Steinman, 1998 ; Steinman, 1999
). They rapidly pervade inflamed tissues, take up antigen, and differentiate upon inflammatory signals to reach a mature activated stage. Mature dendritic cells migrate to lymphatic organs (Sallusto et al., 1998
; Sozzani et al., 1998
), where they have the unique capacity to prime naive T cells for an antigen-specific immune response (Inaba & Steinman, 1985
; Flamand et al., 1994
; Porgador & Gilboa, 1995
; Zitvogel et al., 1996
). Thus, they are likely to play an essential role in the initiation of the specific T helper function and T cytotoxic antiviral responses (Klagge & Schneider-Schaulies, 1999
). Although mvig function remains enigmatic, its dendritic-targeted induction by VSV and PrV suggests a link with antiviral responses. This idea is reinforced by the conservation of two induction pathways, and by the diversity of the virus families already known to induce the genes of the mvig family. This redundancy may reflect the general importance of the vig1 family in the host response to viruses. Interestingly, the direct induction of the mvig gene family by different viruses (Zhu et al., 1997
; Boudinot et al., 1999
; this report) is reminiscent of the concept of pattern recognition receptors invoked for the initiation of the immune response against evolutionarily distant pathogens (Medzhitov & Janeway, 1997
).
In fact, LPS also induces mvig, indicating that the expression of this gene may be a more general feature of pathogen-activated dendritic cells. It is interesting to note that particular interferon-stimulated genes are also involved in the response to bacterial infection. LPS induces a p38-dependent activation of IRF3, leading to the expression of different interferon-stimulated genes (Navarro & David, 1999 ). mvig could be considered a member of these genes induced by both interferon and LPS. Finally, the characteristics of the mvig/cig5/vig1 gene family may illustrate the connectivity between several transduction pathways leading to the inflammatory responses.
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Acknowledgments |
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We thank Dr Y. Gaudin for VSV, Dr J.-H. Colle for the internal -actin DNA standard and Dr P. Ricciardi-Castagnoli for the D2SC1 cell line. We gratefully acknowledge Drs A. Regnault and J. Kanellopoulos for helpful discussions, and Dr S. Matthews for proofreading the manuscript.
P.B., S.R. and S.S. contributed equally to this work.
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Footnotes |
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b Present address: Unité de Biochimie Cellulaire, FRE 2219 CNRS, Université Pierre et Marie Curie, 9 quai Saint Bernard, 75005 Paris, France.
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Received 7 June 2000;
accepted 26 July 2000.