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. 1998 Nov;72(11):8636-43.
doi: 10.1128/JVI.72.11.8636-8643.1998.

Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes

P Baudoux  1 , C Carrat, L Besnardeau, B Charley, H Laude
Affiliations

Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes

P Baudoux et al. J Virol. 1998 Nov.

Abstract

Transmissible gastroenteritis virus (TGEV), an enteric coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vivo and in vitro. Incubation of peripheral blood mononuclear cells with noninfectious viral material such as inactivated virions or fixed, infected cells leads to early and strong IFN-alpha synthesis. Previous studies have shown that antibodies against the virus membrane glycoprotein M blocked the IFN induction and that two viruses with a mutated protein exhibited a decreased interferogenic activity, thus arguing for a direct involvement of M protein in this phenomenon. In this study, the IFN-alpha-inducing activity of recombinant M protein expressed in the absence or presence of other TGEV structural proteins was examined. Fixed cells coexpressing M together with at least the minor structural protein E were found to induce IFN-alpha almost as efficiently as TGEV-infected cells. Pseudoparticles resembling authentic virions were released in the culture medium of cells coexpressing M and E proteins. The interferogenic activity of purified pseudoparticles was shown to be comparable to that of TGEV virions, thus establishing that neither ribonucleoprotein nor spikes are required for IFN induction. The replacement of the externally exposed, N-terminal domain of M with that of bovine coronavirus (BCV) led to the production of chimeric particles with no major change in interferogenicity, although the structures of the TGEV and BCV ectodomains markedly differ. Moreover, BCV pseudoparticles also exhibited interferogenic activity. Together these observations suggest that the ability of coronavirus particles to induce IFN-alpha is more likely to involve a specific, multimeric structure than a definite sequence motif.

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Figures

FIG. 1
FIG. 1
Induction of IFN-α by cell cultures expressing TGEV structural proteins. Vaccinia virus vT7-3-infected RK13 cells were transfected with Lipofectamine containing one or a combination of plasmids, each containing one of the TGEV genes, as indicated. The monolayers were fixed with 0.25% glutaraldehyde at 10 h postinfection and then incubated overnight with PBM cells (107/ml) for IFN induction. TGEV- or mock-infected ST cell monolayers were processed in the same way for comparison. The interferon titer in the maintenance medium was determined by an ELISA test specific for IFN-α.
FIG. 2
FIG. 2
Velocity centrifugation analysis of M protein material released from cells coexpressing TGEV M and E genes. The culture medium from vT7-3-infected, transfected, and labeled RK13 cells was centrifuged on a 20 to 45% linear sucrose gradient. The material present in each fraction was pelleted and analyzed directly by SDS–12% PAGE and autoradiography. The position (top of the peak) of labeled TGEV virions sedimented in a parallel gradient is shown by an open triangle. The sucrose concentrations in the fractions corresponding to the sedimentation peaks of pseudoparticles and virions, respectively, are shown (arrow).
FIG. 3
FIG. 3
Electron microscopy of TGEV pseudoparticles. Particles secreted from RK13 cells coexpressing the M and E genes were purified by a procedure similar to that used for TGEV virions (see Materials and Methods). Particles were viewed after immunogold labeling with an anti-M ectodomain antibody and negative staining. The insert shows two pseudoparticles at a twofold-higher magnification.
FIG. 4
FIG. 4
IFN-α-inducing activity of recombinant pseudoparticles and TGEV virions. Purified recombinant particles and virions were obtained as described in Materials and Methods. Based on the results of an ELISA, the suspensions were adjusted to have approximately the same M antigen content and then incubated with PBM cells at the indicated dilutions. The blocking effect of anti-M MAb 25.22 on IFN induction is shown on the right. The ratio of virions to PBM cells was estimated to be 100 ng per 2 × 105 cells at the 1/100 dilution.
FIG. 5
FIG. 5
Production of pseudoparticles by coexpression of heterologous and/or chimeric M and E genes of TGEV and BCV viruses. (a) Alignments of the sequences of mature TGEV and BCV M proteins (T and B, respectively) and of TGEV and BCV E genes (t and b, respectively). The predicted transmembrane segments are underlined in the TGEV sequences. Identical amino acids are in uppercase letters. In boldface are shown two short homologous regions selected to fuse TGEV and BCV sequences in the chimeric M or E gene constructs used in these experiments. Sequence data are from references (TGEV M), (BCV M), (TGEV E), and (BCV E). (b and c) Various pairs of homologous and chimeric constructs were expressed through the vT7-3 expression system. Particle assembly and secretion were assessed by monitoring the release of soluble, radiolabeled M material in the culture medium of transfected cells. Sedimentable material was collected by centrifugation through a glycerol cushion and analyzed by SDS–12% PAGE and autoradiography. The E chimera designated bt corresponds to the BCV amino-half sequence fused with the TGEV carboxy-half sequence, and tb is the reciprocal construct. The M chimera designated TBB corresponds to the TGEV N-terminal ectodomain fused with the remaining part of BCV M sequence, and vice versa for the BTT chimera. TBB and BTT chimeric proteins were expressed at equivalent levels as judged by immunoprecipitation assays of lysates of transfected cells with MAbs 25.22 and 3.60, respectively. Numbers on the left of panel b are molecular weights in thousands.
FIG. 6
FIG. 6
Induction of IFN-α by cell cultures producing homologous or chimeric pseudoparticles of TGEV and BCV. The experiments were performed as described in the legend to Fig. 1. The coexpressed genes are indicated as uppercase letters on the x axis for M constructs and by lowercase letters for E constructs. For panel b, an antibody specific for the ectodomain of TGEV M protein was added in the culture medium during the incubation with PBM cells.
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