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CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

ABSTRACT: Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lf^F/F ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoV^CR6) in vivo In contrast, the nonpersistent MNoV strain CW3 (MNoV^CW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoV^CW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoV^CW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoV^CR6; that myelomonocytic cells are a major, but not exclusive, target cell of MNoV^CW3; and that STAT1 signaling restricts the cellular tropism of MNoV^CW3 This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis.IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoV^CR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoV^CW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoV^CW3 infection. Mortality associated with the MNoV^CW3 strain in Stat1^-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.

SUBMITTER: Graziano VR

PROVIDER: S-EPMC7925115 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Human norovirus is the leading cause of gastroenteritis worldwide, yet basic questions about its life cycle remain unanswered due to an historical lack of robust experimental systems. Recent studies on the closely related murine norovirus (MNV) have identified CD300LF as an indispensable entry factor for MNV. We compared the MNV susceptibilities of cells from different mouse strains and identified polymorphisms in murine CD300LF which are critical for its function as an MNV receptor. Bone marrow-derived macrophages (BMDMs) from I/LnJ mice were resistant to infection from multiple MNV strains which readily infect BMDMs from C57BL/6J mice. The resistance of I/LnJ BMDMs was specific to MNV, since the cells supported infection of other viruses comparably to C57BL/6J BMDMs. Transduction of I/LnJ BMDMs with C57BL/6J CD300LF made the cells permissible to MNV infection, suggesting that the cause of resistance lies in the entry step of MNV infection. In fact, we mapped this phenotype to a 4-amino-acid difference at the CC' loop of CD300LF; swapping of these amino acids between C57BL/6J and I/LnJ CD300LF proteins made the mutant C57BL/6J CD300LF functionally impaired and the corresponding mutant of I/LnJ CD300LF functional as an MNV entry factor. Surprisingly, expression of the I/LnJ CD300LF in other cell types made the cells infectible by MNV, even though the I/LnJ allele did not function as an MNV receptor in macrophage-like cells. Correspondingly, I/LnJ CD300LF bound MNV virions in permissive cells but not in nonpermissive cells. Collectively, our data suggest the existence of a cell type-specific modifier of MNV entry.IMPORTANCE MNV is a prevalent model system for studying human norovirus, which is the leading cause of gastroenteritis worldwide and thus a sizeable public health burden. Elucidating mechanisms underlying susceptibility of host cells to MNV infection can lead to insights on the roles that specific cell types play during norovirus pathogenesis. Here, we show that different alleles of the proteinaceous receptor for MNV, CD300LF, function in a cell type-dependent manner. In contrast to the C57BL/6J allele, which functions as an MNV entry factor in all tested cell types, including human cells, I/LnJ CD300LF does not function as an MNV entry factor in macrophage-like cells but does allow MNV entry in other cell types. Together, these observations indicate the existence of cell type-specific modifiers of CD300LF-dependent MNV entry.

Project description:Human noroviruses are the leading cause of acute gastroenteritis in humans. Noroviruses also infect animals, such as cows, mice, cats, and dogs. How noroviruses bind and enter host cells is still incompletely understood. Recently, the type I transmembrane protein CD300lf was identified as the murine norovirus receptor, yet it is unclear how the virus capsid and receptor interact at the molecular level. In this study, we determined the X-ray crystal structure of the soluble CD300lf (sCD300lf) and the murine norovirus capsid protruding domain complex at a 2.05-Å resolution. We found that the sCD300lf-binding site is located on the topside of the protruding domain and involves a network of hydrophilic and hydrophobic interactions. sCD300lf locked nicely into a complementary cavity on the protruding domain that is additionally coordinated with a positive surface charge on sCD300lf and a negative surface charge on the protruding domain. Five of six protruding domain residues interacting with sCD300lf were maintained between different murine norovirus strains, suggesting that sCD300lf was capable of binding to a highly conserved pocket. Moreover, a sequence alignment with other CD300 paralogs showed that the sCD300lf-interacting residues were partially conserved in CD300ld but variable in other CD300 family members, consistent with previously reported infection selectivity. Overall, these data provide insights into how a norovirus engages a protein receptor and will be important for a better understanding of selective recognition and norovirus attachment and entry mechanisms.IMPORTANCE Noroviruses exhibit exquisite host range specificity due to species-specific interactions between the norovirus capsid protein and host molecules. Given this strict host range restriction, it has been unclear how the viruses are maintained within a species between relatively sporadic epidemics. While much data demonstrate that noroviruses can interact with carbohydrates, recent work has shown that expression of the protein CD300lf is both necessary and sufficient for murine norovirus infection of mice and binding of the virus to permissive cells. Importantly, the expression of this murine protein by human cells renders them fully permissive for murine norovirus infection, indicating that at least in this case, host range restriction is determined by molecular events that control receptor binding and entry. Defining the atomic-resolution interactions between the norovirus capsid protein and its cognate receptor is essential for a molecular understanding of host-range restriction and norovirus tropism.

Project description:The intracellular fibroblast growth factors (iFGF/FHFs) bind directly to cardiac voltage gated Na+ channels, and modulate their function. Mutations that affect iFGF/FHF-Na+ channel interaction are associated with arrhythmia syndromes. Although suspected to modulate other ionic currents, such as Ca2+ channels based on acute knockdown experiments in isolated cardiomyocytes, the in vivo consequences of iFGF/FHF gene ablation on cardiac electrical activity are still unknown. We generated inducible, cardiomyocyte-restricted Fgf13 knockout mice to determine the resultant effects of Fgf13 gene ablation. Patch clamp recordings from ventricular myocytes isolated from Fgf13 knockout mice showed a ~25% reduction in peak Na+ channel current density and a hyperpolarizing shift in steady-state inactivation. Electrocardiograms on Fgf13 knockout mice showed a prolonged QRS duration. The Na+ channel blocker flecainide further prolonged QRS duration and triggered ventricular tachyarrhythmias only in Fgf13 knockout mice, suggesting that arrhythmia vulnerability resulted, at least in part, from a loss of functioning Na+ channels. Consistent with these effects on Na+ channels, action potentials in Fgf13 knockout mice, compared to Cre control mice, exhibited slower upstrokes and reduced amplitude, but unexpectedly had longer durations. We investigated candidate sources of the prolonged action potential durations in myocytes from Fgf13 knockout mice and found a reduction of the transient outward K+ current (Ito). Fgf13 knockout did not alter whole-cell protein levels of Kv4.2 and Kv4.3, the Ito pore-forming subunits, but did decrease Kv4.2 and Kv4.3 at the sarcolemma. No changes were seen in the sustained outward K+ current or voltage-gated Ca2+ current, other candidate contributors to the increased action potential duration. These results implicate that FGF13 is a critical cardiac Na+ channel modulator and Fgf13 knockout mice have increased arrhythmia susceptibility in the setting of Na+ channel blockade. The unanticipated effect on Ito revealed new FGF13 properties and the unexpected lack of an effect on voltage-gated Ca2+ channels highlight potential compensatory changes in vivo not readily revealed with acute Fgf13 knockdown in cultured cardiomyocytes.

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CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

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