Sequence analysis of the spike protein gene of murine coronavirus variants: Study of genetic sites affecting neuropathogenicity

Abstract

Mouse hepatitis virus (MHV), a coronavirus, causes encephalitis and demyelination in susceptible rodents. Previous investigations have shown that the MHV spike (S) protein is a critical determinant of viral tropism and pathogenicity in mice and rats. To understand the molecular basis of MHV neuropathogenesis, we studied the spike protein gene sequences of several neutralization-resistant variants of the JHM strain of MHV, which were selected with monoclonal antibodies (MAbs) specific for the S protein. We found that variant 2.2-V-1, which was selected with MAb J.2.2 and primarily caused demyelination, had a single point mutation at nucleotide (NT) 3340, as compared to the parental JHM virus, which predominantly caused encephalitis. This site was in the S2 subunit of the S protein. In contrast, variant 7.2-V-1, which was selected with MAb J.7.2 and primarily caused encephalitis, had two point mutations at NT 1766 and 1950, which were in the S1 subunit. Finally, the double mutant 2.2/7.2-V-2, which was selected with both MAbs J.2.2 and J.7.2, and was attenuated with respect to both virulence and the ability to cause demyelination, had a deletion spanning from NT 1523 to 1624 in the S1 and a point mutation at NT 3340 in the S2. We conclude that at least two regions of the S protein contribute to neuropathogenicity of MHV. We have also isolated a partial revertant of 2.2-V-1, which was partially resistant to MAb J.2.2 but retained the same neuropathogenicity as the variant 2.2-V-1. This revertant retained the mutation at NT 3340, but had a second-site mutation at NT 1994, further confirming that NT 3340 contributed to the pathogenic phenotype of MHV. By comparing these results with MHV variants isolated in other laboratories, which had mutations in other sites on the S gene and yet retained the demyelinating ability, we suggest that the ability of JHM viruses to induce demyelination is determined by the interaction of multiple sites on the S gene, rather than the characteristics of a single, unique site. Our study also revealed the possible presence of microheterogeneity of S gene sequence, particularly in the S1 region, in these viruses. The sequence microheterogeneity may also contribute to the differences in their biological properties.

Antibody Targeting of the CC Chemokine Ligand 5 Results in Diminished Leukocyte Infiltration into the Central Nervous System and Reduced Neurologic Disease in a Viral Model of Multiple Sclerosis

“…infections (34). Age-matched (5-7 wk old) C57BL/6 mice (H-2 b background) were used for all experiments (National Cancer Institute, Bethesda, MD).…”

Bovine Ephemeral Fever in Taiwan

“…Each virus was purified by 3 rounds of limited dilution, expanded, and cleared by centrifugation at 2,000 ϫ g for 25 min at 4 C. The supernatant containing viral particles was used for RNA extraction by mixing 0.25 ml of virus suspension with 0.75 ml of Trizol . b Half of the pelleted RNA was used for one reverse transcription reaction, 26 23 where GF1 were 5Ј-ATGTTCA-AGGTCCTCATAATTACC-3Ј (1-24) and GR1 were 5Ј-TAATGATCAAAGAACCTATCATCAC-3Ј (1,847-1,871). The reaction mixture was denatured at 94 C for 4 minutes, followed by 30 cycles of amplification at 94 C for 45 seconds, 52 C for 60 seconds, and 72 C for 90 seconds, with a final extension at 72 C for 10 minutes.…”

No evidence for quasispecies populations during persistence of the coronavirus mouse hepatitis virus JHM: sequence conservation within the surface glycoprotein gene S in Lewis rats.

“…Results from previous studies have indicated that the S gene of coronaviruses plays an important role in virulence and pathogenicity (Taguchi et al, 1985 ;Dalziel et al, 1986 ;Fleming et al, 1986 ;Wege et al, 1988 ;Morris et al, 1989 ;Parker et al, 1989 ;LaMonica et al, 1991 ;Wang et al, 1992). Therefore, we investigated whether coronavirus replication in brain tissue of persistently infected Lewis rats results in an accumulation or selection of S gene variants.…”

“…Several studies have suggested that the S and HE proteins are important determinants for the neuropathogenicity of the virus and for its ability to infect a variety of cell types (Taguchi et al, 1985 ;Morris et al, 1989 ;Parker et al, 1989 ;Wang et al, 1992 ;LaMonica et al, 1991 ;Fazakerley et al, 1992 ;Yokomori et al, 1993). The S protein in particular elicits neutralizing antibodies, binds to cell receptors and causes cell fusion.…”