Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages

Abstract

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-alphabeta receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

Figure 1. MNV-1-Specific Staining In Vivo Occurs in Cells of the MΦ Lineage

Immunohistochemistry was performed on liver (A and B) and spleen (C and D) sections from STAT1^−/− mice 2 d after oral infection. MNV-1-specific staining was seen in Kupffer cells of infected livers when probed with MNV-1 immune (A) but not preimmune (B) serum. A selected Kupffer cell lining the sinusoid is indicated by an arrowhead. MNV-1-specific staining consistent with MΦ was seen in red pulp (C) and marginal zone (D) in the spleen. The arrow indicates a cell with MΦ morphology. No staining was observed in tissues from mice infected for 1 d, in infected tissues incubated with preimmune serum, or in mock-infected tissues incubated with immune serum. RP, red pulp; WP, white pulp.

Figure 2. MNV-1 from Brain Homogenate Replicates in Cells of the DC and MΦ Lineage In Vitro

BMDCs and BMMΦ, as well as MEFs from wt or STAT1^−/− mice, and RAW 264.7 cells were infected with a MOI of 0.05. (A) MNV-1 causes CPE in permissive cells. MNV-1- or mock-infected cells were observed by light microscopy 2 d postinfection. The boxed area is magnified further to show the border of the plaque. (B) Infected cell lysates were analyzed in two to four independent experiments by plaque assay at various timepoints postinfection to calculate standard deviations. For wt BMMΦ, MNV-1 growth was detected in two out of four experiments.

Figure 3. Characterization of the Triple Plaque-Purified Strain MNV-1.CW1

(A–C) MNV-1.CW1 purified on CsCl density gradients was visualized by (A) negative staining electron microscopy, (C) Coomassie staining, and (B) Western blot analysis with a polyclonal anti-MNV-1-capsid antibody. Molecular weight markers are indicated in kiloDaltons. (D) Specific binding of mAb A6.2 to two different concentrations of CsCl-purified MNV-1 particles in an enzyme-linked immunosorbent assay. (E) Neutralization of MNV-1 from brain homogenate and MNV-1.CW1 by mAb A6.2 but not the isotype control (10H2) mAb in a plaque neutralization assay. The assay was repeated three times to calculate standard deviations. The limit of detection is indicated by the dashed line. (F) Timecourse of viral RNA synthesis in RAW 264.7 cells. Northern blot analysis of viral RNA from cells infected with MNV-1.CW1 (MOI of 2.0) or mock-infected cells. The size of RNA markers in kilobases is shown on the left. The positions of subgenomic- and genomic-length RNA are indicated on the right. This timecourse is a representative of two independent experiments. (G) Timecourse of viral protein synthesis in infected RAW 264.7 cells. MNV-1-specific proteins were precipitated from radiolabeled cell lysates of MNV-1.CW1-infected RAW 264.7 cells (MOI of 2.0) at indicated times after infection. The size of the proteins in kiloDaltons is indicated.

Figure 4. Ultrastructural Studies of MNV-1.CW1-Infected RAW 264.7 Cells

Cells were infected with MNV-1.CW1 (P3) (MOI of 2.0) (D–L) or mock-infected (A–C) and processed for electron microscopy 12 (D–F), 18 (G–I), or 24 (A–C; J–L) h.p.i. MNV-1 particles are indicated by arrows and confronting membranes by arrowheads. VA, vesiculated areas; Nuc, nucleus; rER, rough endoplasmic reticulum. Scale bars, 200 nm for (A), (D), (G), and (J); 500 nm for (B), (E), (H), and (K); 2 μm for (C), (F), (I), and (L).

Figure 5. Critical Role for STAT-1 in Limiting MNV-1 Growth In Vitro

(A) MNV-1.CW1 has no defect in viral growth in vitro. Growth curves (MOI of 0.05) were performed two or three times with MNV-1.CW1 (P3) on indicated cells to calculate standard deviations. (B) MNV-1 growth in MΦ is controlled by STAT-1 and Type I IFNs. BMMΦ of the indicated genotype were infected with MNV-1.CW1 (P3) at the indicated MOI. The experiment was performed twice to calculate standard deviations. The p-values for PKR versus wt infection at MOI 0.05 and 2.0, 0.8867 and 0.1616, respectively, are not significant. Statistical analysis was performed using the paired t-test (GraphPad Prism, version 3.03).

Figure 6. Changes in Virulence of Plaque-Purified MNV-1 over Multiple Passages Are Associated with Limited Amino Acid Changes

(A) Serial passage of MNV-1.CW1 in cell culture causes attenuation. STAT1^−/− mice were infected orally with the indicated virus dose. The number of mice analyzed is indicated in parentheses. (B) Summary of sequence analysis of MNV-1 over several passages. The nucleotide and amino acid differences between the indicated viruses are shown (for detail see Table 1).