Norovirus Cell Tropism Is Determined by Combinatorial Action of a Viral Non-structural Protein and Host Cytokine

Abstract

Cellular tropism during persistent viral infection is commonly conferred by the interaction of a viral surface protein with a host receptor complex. Norovirus, the leading global cause of gastroenteritis, can be persistently shed during infection, but its in vivo cellular tropism and tropism determinants remain unidentified. Using murine norovirus (MNoV), we determine that a small number of intestinal epithelial cells (IECs) serve as the reservoir for fecal shedding and persistence. The viral non-structural protein NS1, rather than a viral surface protein, determines IEC tropism. Expression of NS1 from a persistent MNoV strain is sufficient for an acute MNoV strain to target IECs and persist. In addition, interferon-lambda (IFN-λ) is a key host determinant blocking MNoV infection in IECs. The inability of acute MNoV to shed and persist is rescued in Ifnlr1^-/- mice, suggesting that NS1 evades IFN-λ-mediated antiviral immunity. Thus, NS1 and IFN-λ interactions govern IEC tropism and persistence of MNoV.

Keywords: interferon-lambda; norovirus; persistence; reservoir; tropism.

Figure 1. Detection of intestinal epithelial cells infected by persistent MNoV in vivo

(A) Flow cytometry of NS1/2 and NS6/7 double staining. Colonic IECs from wild-type and Ifnlr1^−/− mice infected with CR6 were stained with control sera or anti-NS1/2 and anti-NS6/7 and analyzed. EpCam+/CD45− cells were pre-gated as IECs. (B) MNoV genome quantification by quantitative RT-PCR of RNA from sorted NS1/2 and NS6/7 double positive cells. N = 4 to 8 mice per group, combined from two independent experiments. (C–E) Quantification of MNoV+ IECs during CR6 infection in wild-type and Ifnlr1^−/− mice at (C) 7 dpi, (D) 14 dpi, and (E) 35 dpi. N = 5 to 13 mice per group, combined from two to four independent experiments. Dashed lines represent limit of detection. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (B) and Mann Whitney test (C–E). **P < 0.01, ***P < 0.001. See also Figure S1.

Figure 2. Immunofluorescence microscopy to visualize MNoV-infected IECs

(A–C) Proximal colons from wild-type (A, C) and Ifnlr1^−/−(B) mice infected with CR6 were analyzed at 14 dpi by immunofluorescence microscopy for detection of (A, B) NS1/2, NS6/7, DAPI and E-cadherin staining or (C) dsRNA (J2 antibody) and NS6/7, and Hoechst staining. Low frequency E-cadherin positive epithelial cells expressed the viral non-structural proteins NS1/2 and NS6/7 which were tightly co-localized in a punctated pattern throughout the cytoplasm. Below is a magnified inset represented by the white box. Dashed line represents the luminal interface. Scale bars represent 10 µm.

Figure 3. Regulation of IEC infection by adaptive immunity

(A) Time-course of MNoV genome copies shed into fecal pellets with time points at 7, 14, 21, 24, 28, and 35 days after CR6 or CW3 infection in Rag1^−/− mice. N = 9 to 31 mice per group, combined from three independent experiments. (B) MNoV genome copies in colon from wild-type or Rag1^−/− mice at 35 dpi. (C, D) Quantification of MNoV+ IECs from wild-type and Rag1^−/− mice at 14 dpi (C) and at 35 dpi (D). Dashed lines represent limit of detection. Statistical significance was determined by two-way ANOVA (A), one-way ANOVA followed by Tukey’s multiple-comparisons test (B) and Mann Whitney test (C, D). **P < 0.01, ***P < 0.001. See also Figure S2.

Figure 4. IEC tropism of persistent MNoV is NS1-dependent

(A) Schematic depicting the chimeric viruses used in this assay. (B–E) Wild-type mice were infected with the indicated viruses and analyzed at 14 dpi. (B, C) MNoV+ IECs from the proximal colon of infected mice were analyzed (B) and quantified (C) by flow cytometry. N = 5 mice per group, combined from two independent experiments. (D–E) MNoV genome copies in (D) stool and (E) colon. Dashed lines represent limit of detection. N = 5 to 13 mice per group, combined from two independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparisons test. **P < 0.01, ***P < 0.001, NS = not significant. See also Figure S3.

Figure 5. Differential IFN-λ sensitivity of chimeric MNoVs

(A–C) MNoV genome copies in tissues and fecal pellets. Wild-type mice were pre-treated with PBS or 3 µg of IFN-λ 1 day before infection, then infected with the indicated MNoV strains. MNoV genomes from spleen (A), stool (B), and colon (C) were quantified by qRT-PCR. N = 7 to 10 mice per group, combined from two independent experiments. Dashed lines represent limit of detection. Statistical significance was determined by Mann Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, NS = not significant. See also Figure S4.

Figure 6. IFN-λ does not control parenterally-introduced MNoV infection

(A–C) Wild-type mice were pre-treated with PBS or 3 µg of IFN-λ 1 day before infection, then infected with CR6 perorally (PO) or intraperitoneally (IP), and analyzed at 3 dpi. MNoV genomes from spleen (A), stool (B), and colon (C) were quantified by qRT-PCR. N = 9 to 10 mice per group, combined from two independent experiments. Dashed lines represent limit of detection. Statistical significance was determined by Mann Whitney test. *P < 0.05, ***P < 0.001, NS = not significant.

Figure 7. Complemented viral persistence of CR6^NS1-CW3 in Ifnlr1 knockout mice

(A–D) Wild type, Ifnlr1^−/−, Ifnar1^−/−, Rag1^−/−, Ifnlr1^f/f and Ifnlr1^f/f-Villincre mice were infected with CR6^NS1-CW3, and MNoV genomes from stool (A, C) and colon (B, D) were analyzed at 14 dpi by qRT-PCR. N = 6 to 13 mice per group, combined from two or three independent experiments. Dashed lines represent limit of detection. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. See also Figures S5 and S6.