CD300lf is the primary physiologic receptor of murine norovirus but not human norovirus

Abstract

Murine norovirus (MNoV) is an important model of human norovirus (HNoV) and mucosal virus infection more broadly. Viral receptor utilization is a major determinant of cell tropism, host range, and pathogenesis. The bona fide receptor for HNoV is unknown. Recently, we identified CD300lf as a proteinaceous receptor for MNoV. Interestingly, its paralogue CD300ld was also sufficient for MNoV infection in vitro. Here we explored whether CD300lf is the sole physiologic receptor in vivo and whether HNoV can use a CD300 ortholog as an entry receptor. We report that both CD300ld and CD300lf are sufficient for infection by diverse MNoV strains in vitro. We further demonstrate that CD300lf is essential for both oral and parenteral MNoV infection and to elicit anti-MNoV humoral responses in vivo. In mice deficient in STAT1 signaling, CD300lf is required for MNoV-induced lethality. Finally, we demonstrate that human CD300lf (huCD300lf) is not essential for HNoV infection, nor does huCD300lf inhibit binding of HNoV virus-like particles to glycans. Thus, we report huCD300lf is not a receptor for HNoV.

Fig 1. CD300lf is necessary for MNoV^CW3 infection ex vivo and in vivo.

(A-C) BMDMs were generated from Cd300lf^-/- and Cd300lf^+/- littermate controls. (A) BMDMs were challenged with MNoV^CW3 (MOI = 0.05) and viral replication was measured by plaque assay 24 hpi. (B) Cd300lf^+/- and Cd300lf^-/- BMDMs were challenged with MNoV^CW3 (MOI = 5) and expression of the MNoV non-structural protein NS1/2 was measured by flow cytometry. (C) Quantification of NS1/2 expression. (D-E) Cd300lf^+/- and Cd300lf^-/- littermates were challenged with 10⁶ PFU PO MNoV^CW3. 24 hpi, virus was measured by both (D) plaque assay and (E) qPCR in the MLN, spleen, distal ileum, and proximal colon. Data was analyzed by Mann-Whitney test. Shown are means ± SEM. NS, not significant; *P<0.05; **P<0.01; ***P<0.001; L.O.D., limit of detection. Experiments in (A-C) where performed at least two independent times each in triplicate. Data in (D-E) are pooled from two independent experiments with at least three mice per group.

Fig 2. CD300lf is necessary and sufficient for infection by diverse MNoV strains.

(A) CD300lf WT and KO BV2 cells were infected with MNoV strains CW3, WU23, CR3, CR7, MNV-3, and S99 at a MOI of 5. CD300lf KO BV2 cells were protected from virus-induced cell death for all MNoV strains. (B) Mouse CD300ld (muCD300ld) and CD300lf (muCD300lf) were overexpressed in human HeLa cells by transient transfection. Cells were challenged with MNoV strains at a MOI of 5 for 24 hours and then infection was quantified by MNoV NS6/7 expression by flow cytometry. (C-H) Cd300lf^+/- or Cd300lf^-/- mice were challenged with 10⁶ PFU PO CW3, WU23, CR6, CR3, or CR7 for seven days. MNoV was detectable in the (C) feces, (D) MLN, (E) spleen, (F) ileum, and (G) colon of Cd300lf^+/- but not Cd300lf^-/- mice. (H) To test whether CR6 was shed in feces below the limit of detection by qPCR, we gavaged Stat1^-/- mice with feces from Cd300lf^+/- or Cd300lf^-/- mice challenged with MNoV^CR6 from (C). Fecal pellets from Cd300lf^-/- mice, did not establish detectable infection in Stat1^-/- mice. Data is pooled from two to four independent experiments. Data was analyzed by Mann-Whitney test. Shown are means ± SEM. NS, not significant; *P<0.05; **P<0.01; ***P<0.001; L.O.D., limit of detection.

Fig 3. CD300lf is required to generate humoral response after oral MNoV^CW3 challenge.

Sera was collected from Cd300lf^+/- and Cd300lf^-/- mice 14 days after challenge with 10⁶ PFU PO MNoV^CW3. (A) The maximal protection (1:10 sera dilution) and (B) sera IC₅₀ was measured by in vitro MNoV^CW3 neutralization assay in BV2 cells. (C) Cd300lf^+/- generated significantly increased anti-MNoV IgG (C) and IgM (D). Data is pooled from two independent experiments. Data was analyzed by Mann-Whitney test. Shown are means ± SEM. NS, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. L.O.D., limit of detection.

Fig 4. CD300lf is essential for oral MNoV transmission in Stat1^-/- mice.

(A) Cd300lf^+/+Stat1^-/- (N = 8) and Cd300lf^+/-Stat1^-/- (N = 4) mice challenged with 10⁶ PFU PO MNoV^CW3 succumbed to infection by 5 and 6 dpi, respectively. In contrast, all Cd300lf^-/-Stat1^-/- (N = 10) mice survived for at least 21 dpi. (B-F) Cd300lf^+/+Stat1^-/-, Cd300lf^+/-stat1^-/-, and Cd300lf^-/-Stat1^-/- mice were challenged with 10⁶ PFU PO MNoV^CR6. All mice survived infection. Viral genomes were quantified in the (B) feces, (C) MLN, (D) spleen, (E) ileum, and (F) colon at seven dpi. Data is pooled from at least three independent experiments. Data was analyzed by Mann-Whitney test and Kaplan-Meier curves were generated for survival experiments. Shown are means ± SEM. NS, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. L.O.D., limit of detection.

Fig 5. CD300lf is essential for pathogenesis of parenterally transmitted MNoV in STAT1 deficient mice.

(A) Mice were challenged with 10⁷ PFU IP MNoV^CW3. Cd300lf^-/-Stat1^-/- mice (N = 9 mice) survived infection in contrast to Cd300lf^+/+Stat1^-/- (N = 15 mice) and Cd300lf^+/-Stat1^-/- (N = 9 mice) littermates. (B) Mice were challenged with 10⁷ PFU IP MNoV^CR6. Cd300lf^-/-Stat1^-/- mice (N = 6 mice) survived infection in contrast to Cd300lf^+/+Stat1^-/- (N = 9 mice) and Cd300lf^+/-Stat1^-/- (N = 3 mice) littermates. (C) MNoV genomes were quantified from the MLN, spleen, ileum, and colon of Cd300lf^-/-Stat1^-/- mice seven days post-challenge with 10⁷ PFU IP MNoV^CR6. Data was analyzed by Kaplan-Meier curve for survival experiments. Data is pooled from at least three independent experiments with 1–5 mice per group. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. L.O.D., limit of detection.

Fig 6. Human CD300lf is not a HNoV entry factor.

(A) Human CD300lf and human CD300ld Fc-fusion proteins do not prevent binding of HNoV virus-like particles (VLPs) to pig gastric mucin for GI.1 Norwalk, GI.3 Desert Shield Virus, GII.4.1997, and GII.4.2012. (B) Polyclonal antibody against human CD300lf does not prevent HNoV GII.4 replication in HIEs relative to an IgG1 control. (C) Pre-incubating HNoV GII.4 with a human CD300lf Fc-fusion protein does not prevent HNoV replication in HIEs relative to a control protein (RV NSP4). Data in (A) is representative of at least two independent replicates each performed in duplicate. Data in (B and C) is pooled from three independent experiments with each condition and time point performed in triplicate wells of HIE cultures. Shown are means ± standard deviation.