Inhibition of MNV by 2,000-kDa γ-PGA was further verified by measuring intracellular viral genome titers. γ-PGA reduced viral genome titer by up to 92% in cells infected with a low MOI (0.000005). Its inhibitory effect decreased slightly (86% inhibition) at an MOI of 0.05 (Fig. 2a). Additionally, it led to a 30% reduction in the level of Norwalk virus sub-genomic replicon (derived from a GI strain of HuNoV) at 100 nM. In HG23 cell line, 500-kDa γ-PGA had little impact on the replication of sub-genomic replicon (Fig. 2b). The HG23 replicon cell line derived from the human hepatocellular carcinoma cell line Huh7 normally fails to respond to TLR4 ligands because of the low-level expression of TLR4 expression together with the lack of the expression of its accessory proteins, CD14 and MD26. Thus, the insufficient, transient expression of TLR4 and the associated accessory proteins might have contributed to the relatively lower inhibitory effect. The critical role of these accessory proteins in γ-PGA sensing and TLR4-mediated IFN-β expression was further investigated in human embryonic kidney 293 T cells (HEK293T), which express no endogenous TLR4, MD2, and CD1433,34. By confocal microscopy, we found that γ-PGA sensing and intracellular entry occurred upon ectopic expression of both TLR4 and its two known accessory factors (Supplementary Fig. 2a–c). Substantial induction of IFN-β expression and IRF-3 activation were observed only when TLR4, MD2, and CD14 were expressed (Supplementary Fig. 2d). This TLR4-mediated IFN-β induction capability of γ-PGA was also verified in RAW264.7 cells in which RNAi-mediated depletion of TLR4 abrogated IFN-β expression and IRF-3 activation (Supplementary Fig. 2e).Figure 2Analysis of the antiviral activity of γ-PGA against mouse and human noroviruses. (a) RAW264.7 cells infected with the indicated MOI of MNV-1 were treated with 100 nM 2,000-kDa γ-PGA. Viral genome copy number and infectious virus titer in the culture media were determined by RT-qPCR at 40 h post-infection. (b) The HG23 cell line harboring a Norwalk virus sub-genomic replicon was transfected with plasmids expressing TLR4, MD2, and CD14 individually. After incubation for 6 h, transfected cells were treated with γ-PGA (100 nM) for 2 days, prior to RT-qPCR quantification of replicon RNA titer two days later. Neo, neomycin phosphotransferase-coding gene conferring resistance to G418. (c) RAW264.7 cells were pre-treated for 6 h before MNV-1 infection or treated after the viral infection with increasing concentrations of 2,000-kDa γ-PGA. Viral genome titer was determined at 30 h post-infection. (d,e) Inhibition of MNV-1 entry by γ-PGA. Q-dot (QD)-conjugated MNV-1, which was pre-incubated with 2,000-kDa γ-PGA (100 nM) for 30 min, was applied to RAW264.7 cells. Two hours after adsorption, viral entry was quantified by fluorescence microscopy (d), and the fluorescence intensity signals were quantified by image analysis using ImageJ software (e). BF, bright field image. Scale bar, 50 μm. Shown as an inset in (d) is the transmission electron micrograph of purified MNV particles negatively stained with uranyl acetate (Scale bar, 100 nM). (f) Expression of TLR1-9 in RAW264.7 cells infected with MNV-1 (MOI of 0.005). Shown are the GAPDH-normalized TLR expression levels relative to those in mock-treated cells, analyzed by RT-qPCR at 24 h post-infection. Results are from two independent experiments, each analyzed by triplicate assay. (g) Immunoblot assay representing the activation of IRF-3 by γ-PGA (100 nM) and MNV-1 infection (MOI of 0.005) without affecting TLR2, TLR3, and TLR4 expression levels. pIRF-3, phosphorylated IRF-3. In all panels, data are mean ± s.d. Statistical significance of differences between groups was determined via two-tailed unpaired Student’s t-test. *P < 0.05; **P < 0.01; n.s., not significant.