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. 2008 Jul 18;4(7):e1000108.
doi: 10.1371/journal.ppat.1000108.

MDA-5 recognition of a murine norovirus

Stephen A McCartney  1 , Larissa B Thackray, Leonid Gitlin, Susan Gilfillan, Herbert W Virgin, Marco Colonna
Affiliations

MDA-5 recognition of a murine norovirus

Stephen A McCartney et al. PLoS Pathog. .

Erratum in

  • PLoS Pathog. 2008 Oct;4(10). doi:10.1371/annotation/3ce83911-9ccf-4452-a690-2816d0e94c10.. Virgin Iv, Herbert W [corrected to Virgin, Herbert W]

Abstract

Noroviruses are important human pathogens responsible for most cases of viral epidemic gastroenteritis worldwide. Murine norovirus-1 (MNV-1) is one of several murine noroviruses isolated from research mouse facilities and has been used as a model of human norovirus infection. MNV-1 infection has been shown to require components of innate and adaptive immunity for clearance; however, the initial host protein that recognizes MNV-1 infection is unknown. Because noroviruses are RNA viruses, we investigated whether MDA5 and TLR3, cellular sensors that recognize dsRNA, are important for the host response to MNV-1. We demonstrate that MDA5-/- dendritic cells(DC) have a defect in cytokine response to MNV-1. In addition, MNV-1 replicates to higher levels in MDA5-/- DCs as well as in MDA5-/- mice in vivo. Interestingly, TLR3-/- DCs do not have a defect in vitro, but TLR3-/- mice have a slight increase in viral titers. This is the first demonstration of an innate immune sensor for norovirus and shows that MDA5 is required for the control of MNV-1 infection. Knowledge of the host response to MNV-1 may provide keys for prevention and treatment of the human disease.

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Conflict of interest statement

Washington University holds US patent 7,041,444 B2 (murine calicivirus; 9 May 2006) and has pending patent applications related to the field. Washington University and HWV receive income based on licenses for MNV technology.

Figures

Figure 1
Figure 1. MDA5 is required for cytokine response to MNV in vitro.
Bone marrow-derived dendritic cells from wild type (WT), TLR3 deficient (TLR3 KO), or MDA5 deficient (MDA5 KO) mice were infected in vitro with MNV at the indicated MOI. Cell culture supernatants were harvested 24 hours after inoculation, and examined for IFNα by ELISA (A) or for IL-6 (B), MCP-1 (C), or TNFa (D) by cytokine bead array. Data shown is the average of three independent experiments. Statistical analysis was done using student's t test where * = p<0.05, ** = p<0.01, and *** = p<0.001.
Figure 2
Figure 2. MNV replicates more efficiently in MDA5 and TLR3 KOs in vivo.
Wild type (129 or B6), MDA5−/− (MDA5), or TLR3−/− (TLR3) mice were inoculated perorally with 3×107 PFU MNV-1.CW3 or mock infected with media only. Organs were harvested 3 days after infection and viral titers of Spleen (A), Mesenteric Lymph Node (MLN) (B), Proximal Intestine (C), and Distal Intestine (D) were determined by plaque assay. Statistical significance was calculated using the Mann Whitney test, where *** = p<0.001, * = p<0.05. Mock-infected animals showed no detectable MNV-1 at all time-points tested. Data shown is from 6–9 animals. Dashed line indicates limit of detection of the assay.
Figure 3
Figure 3. MDA5 deficiency leads to increased MNV titers in vitro.
Bone marrow-derived dendritic cells from wild type (WT), MDA5−/−, or TLR3−/− mice were inoculated with MNV at an MOI of 5 (A) or 0.05 (B) or pre-treated with 20 U IFNα and then inoculated with an MOI of 0.05 (C). Viral titers were done at 6 hour time-points for each sample and statistical significance was determined using student's t test. There was no significant difference between WT and TLR3−/− titers, statistical significance is marked between WT and MDA5−/− titers where * = p<0.05. Data shown is the average of four independent experiments (A and B) or three independent experiments (C). In (D) supernatants from WT BMDC infected at MOI 0.05 were harvested at various time-points and tested for IFNβ by ELISA. Data shown is the average of three independent experiments.
Figure 4
Figure 4. Proteinase K and RNase abrogates MDA5 recognition of viral RNA.
WT, MDA5−/−, or TLR3−/− BMDCs were mock transfected or transfected with RNA purified from MNV virions that was treated with RNase A (RNase), Proteinase K (PK) or untreated. After 20 hours, supernatants were harvested and cytokines analyzed by ELISA for IFNβ (B) or IFNα (C). Data shown is from three independent experiments and statistics were calculated by student's t test, where * = p<0.05, ** = p<0.01. Treated and untreated RNA were electrophoresed on an agarose gel to visualize degradation (A).
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