Inhibition of norovirus infection-induced apoptosis by the TLR4 ligand γ-PGA. (a) Molecular structure of γ-PGA with γ-linkage (top). RT-qPCR quantification of viral genome titers in the culture media from RAW264.7 cells infected with increasing MOI of murine norovirus 1 (MNV-1). Approximately 60–70% cells were dead when the viral genome titer reached to a titer of 1 × 1011 copies/ml media denoted by the dotted line. (b) RT-qPCR quantification of the expression of IFN-β mRNA in RAW264.7 cells treated with 2,000-kDa γ-PGA (100 nM) for 18 h. Shown is the GAPDH-normalized IFN-β mRNA level relative to that in mock-treated cells. Presented below each bar are representative images of RAW264.7 cells stained with neutral red, 30 h post-infection with MNV-1 at an MOI of 0.005. (c) RAW264.7 cells infected with MNV-1 (MOI of 0.005) were treated with the indicated doses of 2,000-kDa γ-PGA. Cell viability was quantified by the MTS assay at 36 h post-infection. (d) MNV-1 (MOI of 0.005)-infected RAW264.7 cells were treated with the indicated concentrations of 2,000-kDa γ-PGA with or without 500-kDa γ-PGA. Viral genome and VP1 levels were analyzed 30 h later by RT-qPCR and immunoblotting (IB), respectively. In (c,d), data are mean ± s.d.