HuNoV infection causes epithelial apoptosis and downregulates the level of tight junction proteins involved in sealing, leading to diarrhea29. Comparably, murine norovirus (MNV) infection also induces apoptosis in RAW264.7 cells30. In the absence of a robust cell culture system for HuNoV31, we used murine norovirus 1 (MNV-1), as a surrogate model for HuNoV, to test for the inhibition of virus-induced cell death by γ-PGA. We first determined the range of multiplicity of infection (MOI) of MNV in which viral infection-induced apoptosis is triggered during the course of viral infection. As shown in Fig. 1a, infection of RAW264.7 cells with MNV at an MOI of ≥0.005 produced ≥1 × 1011 viral RNA copies/ml of media within 30 h. With an MOI ≤0.0005, there was a >10-fold reduction in viral RNA levels, delaying the progress of cell death within 2 days. Accordingly, we used an MOI of 0.005 for infecting cells to assess the impact of γ-PGA. As shown in Fig. 1b, treatment with 2,000-kDa γ-PGA significantly inhibited MNV infection-induced cell death as visualized by live cell staining, with a 4.4-fold increase in IFN-β mRNA expression than in non-treated control cells. γ-PGA dose-dependently prevented virus-induced cell death resulting in increased cell viability (from approximately 40% to 80% at 200 nM; Fig. 1c), demonstrating the potent antiviral activity of this non-canonical TLR4 ligand against MNV.Figure 1Inhibition 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.