Demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus

Abstract

Demyelination is the pathologic hallmark of the human immune-mediated neurologic disease multiple sclerosis, which may be triggered or exacerbated by viral infections. Several experimental animal models have been developed to study the mechanism of virus-induced demyelination, including coronavirus mouse hepatitis virus (MHV) infection in mice. The envelope spike (S) glycoprotein of MHV contains determinants of properties essential for virus-host interactions. However, the molecular determinants of MHV-induced demyelination are still unknown. To investigate the mechanism of MHV-induced demyelination, we examined whether the S gene of MHV contains determinants of demyelination and whether demyelination is linked to viral persistence. Using targeted RNA recombination, we replaced the S gene of a demyelinating virus (MHV-A59) with the S gene of a closely related, nondemyelinating virus (MHV-2). Recombinant viruses containing an S gene derived from MHV-2 in an MHV-A59 background (Penn98-1 and Penn98-2) exhibited a persistence-positive, demyelination-negative phenotype. Thus, determinants of demyelination map to the S gene of MHV. Furthermore, viral persistence is insufficient to induce demyelination, although it may be a prerequisite for the development of demyelination.

FIG. 1

Schematic diagram of targeted recombination performed in this study. Recombination occurred between Alb4 and a synthetic RNA transcribed from vector pMHV2, in which the S gene of MHV-A59 was replaced with the S gene of MHV-2. Infection of Alb4 simultaneous with transfection with pMHV2 RNA resulted in the generation of two recombinant viruses, Penn98-1 and Penn98-2, which contained the MHV-2 S gene flanked by MHV-A59 sequences (as confirmed by DNA sequencing) and the inserted AvrII and SbfI restriction sites. Since both restriction sites derived from pMHV2 were present in the recombinant viruses, recombination must have occurred 5′ to the S gene. Black bars represent sequences derived from MHV-A59; white bars represent sequences derived from MHV-2; and gray bars represent plasmid sequences. ntd, nucleotide; M, membrane protein gene.

FIG. 2

RT-PCR analysis of putative recombinant viruses (RC2 to RC16). Following characterization and sequencing of the S gene region, the putative recombinant viruses RC2 and RC4 were later designated Penn98-1 and Penn98-2, respectively. (A) A 602-nucleotide region surrounding the Alb4 deletion was amplified with primers IZJ5 and IZJ6 from the genomic RNAs of putative recombinants as well as Alb4 and wild-type MHV-A59. The faster electrophoretic mobility of Alb4 corresponds to the deletion in the N gene, which does not exist in MHV-A59 and which has been repaired in all of the putative recombinant viruses tested. Lane M, size markers. (B) An 868-nucleotide fragment surrounding the AvrII restriction site was amplified from the genomes of putative recombinants, wild-type MHV-A59, and Alb4 using primers FIJ79 and MHV-2S1R and was digested with AvrII prior to electrophoresis. Undigested Alb4 and MHV-A59 are in contrast to the digested recombinant viruses, which contain the AvrII restriction site. (C) A 1,313-nucleotide fragment surrounding the SbfI restriction site was amplified from the genomes of putative recombinant viruses, wild-type MHV-A59, and Alb4 using Penn11F and RIJ84 and was digested with SbfI prior to electrophoresis. The digestion of the recombinant viruses with SbfI indicates the existence of this restriction site in all of the putative recombinant viruses, in contrast to Alb4 and MHV-A59, which do not contain this restriction site.

FIG. 3

Viral replication in the brains and livers of mice following i.c. injection with 5 PFU each of Penn98-1 and Penn98-2. Each time point represents the mean titer for two or three mice. Titers are expressed as log₁₀ PFU per gram of tissue.

FIG. 4

Immunohistochemical detection of viral antigen in brain sections 5 days postinfection using rabbit anti-MHV polyclonal antibodies. (Top) Section from a Penn98-1-infected brain showing antigen detection in numerous neurons and glial cells in an area of acute encephalitis similar to that caused by MHV-A59. (Bottom) Section from a Penn98-2-infected brain showing similar antigen detection. Alkaline phosphatase was the marker enzyme; fast red-naphthol phosphate was the substrate-chromogen reagent; magnification, ×148.

FIG. 5

Analysis of demyelination induced by recombinant viruses. Spinal cord sections were obtained from mice infected with different recombinant viruses and sacrificed 30 days after infection; the sections were stained with Luxol fast blue for myelin. Infection with wtR13 at 2,500 PFU resulted in extensive demyelination. Similar but smaller demyelinating lesions were seen in all mice infected with 5 PFU of wtR13. In contrast, all five mice infected with 5 PFU of Penn98-1 and all five mice infected with 5 PFU of Penn98-2 showed normal spinal cords at multiple levels tested.

FIG. 6

Analysis of viral persistence by RT-PCR amplification with MHV-specific primers, which amplify N gene sequences conserved in all sampled viruses. Mice were sacrificed during acute disease at 5 days postinfection (top panels) or during chronic demyelinating disease at 30 days postinfection (bottom panels). Infection and mock infection of L2 fibroblast cells were used as positive and negative controls, respectively (bottom left panel). Viruses were detected in livers, brains, and spinal cords of mice during acute infection. A spinal cord signal was detected in MHV-A59-, Penn98-1-, and Penn98-2-infected mice during chronic infection but not in MHV-2- and Penn98-1-infected mice. Lanes M, size markers.