A single amino acid substitution in the murine norovirus capsid protein is sufficient for attenuation in vivo

Abstract

Murine norovirus (MNV), a prevalent pathogen of laboratory mice, shares many characteristics with human noroviruses. Previous results indicated that passage of MNV1 in the macrophage cell line RAW 264.7 results in attenuation in STAT1-deficient mice (C. E. Wobus, S. M. Karst, L. B. Thackray, K. O. Chang, S. V. Sosnovtsev, G. Belliot, A. Krug, J. M. Mackenzie, K. Y. Green, and H. W. Virgin, PLoS. Biol. 2:e432, 2004). Sequence analysis revealed two amino acid differences between the virulent and attenuated viruses. Using an infectious cDNA clone of the attenuated virus, we demonstrated that a glutamate-to-lysine substitution at position 296 in the capsid protein (VP1) is sufficient to restore virulence in vivo, identifying, for the first time, a virus-encoded molecular determinant of norovirus virulence.

FIG. 1.

(A) Schematic representation of the MNV genome. The positions of the two amino acid substitutions identified in MNV.CW1 P1 and MNV.CW1 P3 which arise as a result of passage in tissue culture are highlighted. nt, nucleotide. (B and C) Selection pressure. The three recombinant viruses (P1-NS4, P1-VP1, and MNV.CW1.P1) generated in this study were passaged four times (P1 to P4 as indicated) at a high multiplicity of infection (MOI) (>2) in the murine macrophage RAW 264.7 cell line. At each passage, the sequences at genome positions 2151 and 5941 (indicated by arrows) were determined by reverse transcriptase PCR. Note that the sequence of CW1.P3 was not included, as this did not change at either position over multiple passages in tissue culture.

FIG. 2.

In vitro growth characteristics. (A) Single-step growth curve. RAW 264.7 cells were infected at an MOI of 2 and the total virus levels assayed by TCID₅₀ at 0, 6, 9, 12, 24, and 48 h postinfection (hpi). All assays were performed in triplicate; the average log titer is plotted together with standard deviation error bars. (B) Multistep growth curve. RAW 264.7 cells were infected at a reduced MOI (0.01) and the virus titers determined by TCID₅₀ (0, 6, 12, 24, 48, and 72 hpi). All assays were performed in triplicate; the average log titer is plotted together with standard deviation error bars. For both graphs, the time periods representing the viral growth phase analyzed by ANOVA are highlighted.

FIG. 3.

In vivo phenotype. STAT1^−/− mice were orally inoculated with 3 × 10⁴ PFU of sequence-verified passage 1 virus. The percent survival was recorded for 30 days following infection of 10 mice (5 males and 5 females) for each virus. Each differently shaded line represents a separate recombinant virus.