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. 2005 Jun;79(11):7113-20.
doi: 10.1128/JVI.79.11.7113-7120.2005.

Viral expression of CCL2 is sufficient to induce demyelination in RAG1-/- mice infected with a neurotropic coronavirus

Taeg S Kim  1 , Stanley Perlman
Affiliations

Viral expression of CCL2 is sufficient to induce demyelination in RAG1-/- mice infected with a neurotropic coronavirus

Taeg S Kim et al. J Virol. 2005 Jun.

Abstract

Mouse hepatitis virus strain JHM causes a chronic demyelinating disease in susceptible strains of rodents. Demyelination does not develop in infected RAG1-/- (recombination activation gene-deficient) mice but can be induced by several experimental interventions, including adoptive transfer of virus-specific T cells or antibodies. A common feature of demyelination in these models is extensive infiltration of macrophages/microglia into the white matter. The data obtained thus far do not indicate whether macrophage/microglia infiltration, in the absence of T cells or antibody, is sufficient to mediate demyelination. To determine whether the expression of a single macrophage chemoattractant, in the context of virus infection, could initiate the demyelinating process, we engineered a recombinant coronavirus that expressed the chemokine CCL2/monocyte chemoattractant protein-1. CCL2 has been implicated in macrophage infiltration into the central nervous system and is involved in demyelination in many experimental models of demyelination. Extensive macrophage/microglia infiltration and demyelination has developed in RAG1-/- mice infected with this recombinant virus. Thus, these results suggest that the minimal requirement for demyelination is increased expression of a single macrophage-attracting chemokine in the context of an inflammatory milieu, such as that induced by a viral infection.

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Figures

FIG. 1.
FIG. 1.
Schematic diagram of recombinant J2.2 virus constructions. (A) Recombinant J2.2-v-1 (rJ2.2) was generated as described in Materials and Methods. (B) To engineer a recombinant virus that expressed CCL2, a CCL2-specific PCR product was cloned from RNA harvested from bone marrow cells and inserted into gene 4 of rJ2.2 using SbfI and MluI restriction sites. (C) As a control, a virus that encoded a truncated CCL2 protein was also generated (lys [AAG] to stop codon [TAG] at residue 52; rJ2.2.ΔCCL2).
FIG. 2.
FIG. 2.
Detection of CCL2 mRNA and protein in HeLa-MHVR cells infected with rJ2.2.CCL2. (A) CCL2 expression in cells infected with rJ2.2, rJ2.2.CCL2, or rJ2.2.ΔCCL2 was examined at 16 h p.i. by RT-PCR and agarose gel electrophoresis. Lanes: 1, 100-bp DNA ladder; 2, rJ2.2; 3, rJ2.2.CCL2; 4, rJ2.2.ΔCCL2. While the S gene was detected in all samples, the CCL2 gene was detected only in the cells infected with rJ2.2.CCL2 or rJ2.2.ΔCCL2. (B) Sequence analysis showed that the CCL2 gene amplified from rJ2.2.ΔCCL2-infected cells (lower panel) contained the introduced stop codon. (C) Expression of CCL2 protein was detected with anti-CCL2 antibody, as described in Materials and Methods. Whereas cells infected with rJ2.2, rJ2.2.CCL2, or rJ2.2.ΔCCL2 all expressed the viral N protein, CCL2 was detected only in cells infected with rJ2.2.CCL2.
FIG. 3.
FIG. 3.
Functional CCL2 was detected in the supernatants of cells infected with recombinant J2.2.CCL2. HeLa-MHVR cells were infected with rJ2.2, rJ2.2.CCL2, or rJ2.2.ΔCCL2 at an MOI of 0.5. Supernatants were harvested and analyzed as described in Materials and Methods. To confirm that CCL2 was responsible for the transmigration indicated in the figure, some samples were preincubated with anti-CCL2 antibody (100 μg/ml) for 30 min. THP-1 cells (5 × 105 cells/well) were placed on the top chamber of a transwell plate and incubated for 2 h. After the migration period, adherent and nonadherent cells were collected from the lower chambers of the transwell plate and the number of cells determined by counting each cell population for 1 min in a FACScan flow cytometer. Samples were analyzed in quadriplicate. A representative example of three independent experiments is shown.
FIG. 4.
FIG. 4.
Infection with rJ2.2.CCL2 resulted in delayed mortality compared to infection with rJ2.2.ΔCCL2. (A) Six-week-old RAG1−/− mice were infected i.c. with 500 PFU of either rJ2.2.CCL2 (n = 12) or rJ2.2.ΔCCL2 (n = 16). rJ2.2.CCL2-infected mice began to show clinical signs consistent with demyelination, including wobbly gait and hindlimb paresis, at 12 to 14 days p.i., and became moribund by 15 to 17 days p.i. In contrast, mice infected with rJ2.2.ΔCCL2 displayed signs consistent with encephalitis, including hunching, ruffled fur, and lethargy, at 9 to 11 days p.i. and became moribund by 14 days p.i. The fraction surviving at each day p.i. is shown. (B) To determine whether the CCL2 gene was deleted from rJ2.2.CCL2 or rJ2.2.ΔCCL2 after passage in mice, RNA was analyzed by RT-PCR using primers that flanked the inserted sequence. Only full-length products were detected.
FIG. 5.
FIG. 5.
Detection of demyelination in rJ2.2-infected RAG1−/− mice. RAG1−/− mice were infected i.c. with rJ2.2.CCL2 (A to C, G, and H) or rJ2.2.ΔCCL2 (D to F). Mice were harvested at 12 to 14 days p.i., and serial longitudinal sections (8 μm thick) of spinal cord were examined for demyelination (A and D), macrophage/microglia infiltration (B and E), and viral antigen (C and F) as described in Materials and Methods. Demyelination (A) and extensive macrophage/microglia infiltration into the white matter (B) were evident only in rJ2.2.CCL2-infected mice, but not in those infected with rJ2.2.ΔCCL2 (D and E). However, viral antigen was uniformly distributed throughout spinal cords in both rJ2.2.CCL2-infected (C) and rJ2.2.ΔCCL2-infected (F) mice, except in areas of demyelination (compare A and C). Axons were preserved (G) in areas of demyelination (A). No axonal staining was detected in the absence of anti-phosphoneurofilament antibody (H). Quantification of demyelination and the number of mice analyzed in these experiments are shown in Table 1. Scale bar, 250 μm.
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