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Discovery of a proteinaceous cellular receptor for a norovirus

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Science  26 Aug 2016:
Vol. 353, Issue 6302, pp. 933-936
DOI: 10.1126/science.aaf1220

New insights into norovirus entry

There's no escaping norovirus when you have it—the symptoms from this gastroenteritis-causing virus, though brief, are often debilitating. Preventing infections will rely on improving our understanding of how norovirus enters host cells. Orchard et al. show that the entry of murine norovirus (MNoV) into host cells requires a protein called CD300lf. In cell culture, mouse cells needed to express CD300lf in order for MNoV binding, entry, and replication to occur. Deleting the gene encoding CD300lf in mice protected them against MNoV infection. Human cells expressing CD300lf allowed MNoV to break the species barrier, a finding that may lead to new insights into the infectivity of this virus.

Science, this issue p. 933

Abstract

Noroviruses (NoVs) are a leading cause of gastroenteritis globally, yet the host factors required for NoV infection are poorly understood. We identified host molecules that are essential for murine NoV (MNoV)–induced cell death, including CD300lf as a proteinaceous receptor. We found that CD300lf is essential for MNoV binding and replication in cell lines and primary cells. Additionally, Cd300lf−/− mice are resistant to MNoV infection. Expression of CD300lf in human cells breaks the species barrier that would otherwise restrict MNoV replication. The crystal structure of the CD300lf ectodomain reveals a potential ligand-binding cleft composed of residues that are critical for MNoV infection. Therefore, the presence of a proteinaceous receptor is the primary determinant of MNoV species tropism, whereas other components of cellular machinery required for NoV replication are conserved between humans and mice.

Noroviruses (NoVs) are nonenveloped positive-sense RNA viruses (1, 2). Because of the strict species tropism of viruses in the NoV genus and the lack of robust replication of human norovirus (HNoV) in animal models, murine norovirus (MNoV) has emerged as a model system to uncover basic mechanisms of NoV biology in vitro and in vivo (1, 38). MNoV can establish persistent enteric infection, enabling studies of the interplay between viral persistence, resident enteric microorganisms, and the host immune system (46). The capacity of HNoV and MNoV to bind cells and the susceptibility of humans to HNoV have been linked to expression of cell-surface and secreted carbohydrates (1, 913), whereas another member of the Caliciviridae family uses a proteinaceous receptor (14, 15). Host factors, including receptors required for NoV infection and pathogenesis, have largely defied molecular identification; their discovery would aid in understanding mechanisms of NoV replication, vaccination, species tropism, and enteric viral persistence. To identify host molecules required for MNoV infection, we undertook an unbiased forward genetic approach (fig. S1).

MNoV replicates and induces cell death in murine macrophage-like cells, including the microglial BV2 cell line, allowing the identification of genes essential for MNoV replication by means of CRISPR-Cas9 technology. We introduced four independent genome-wide subpools of single-guide RNAs (sgRNAs) from the murine Asiago library into BV2 cells stably expressing Cas9 and then infected the cells with MNoV strains that either cause acute systemic infection (MNoVCW3) or persistent enteric infection (MNoVCR6) (1618). sgRNA sequences from the surviving cells were sequenced and analyzed using the STARS gene-ranking algorithm (Fig. 1A) (17). sgRNAs targeting Cd300lf (CLM-1, CMRF35, MAIR-V, and LMIR3), a gene that encodes a cell-surface immunoglobulin (Ig) domain–containing molecule within a family of proteins involved in binding lipids, were most significantly enriched for both MNoV strains (Fig. 1A and tables S1 to S3) (19). We generated two independent BV2ΔCD300lf clones (where the delta signifies disruption of the Cd300lf gene); the growth of MNoVCW3 and MNoVCR6 was abolished in both clones, whereas the replication of other viruses was unaffected (Fig. 1B and fig. S2). We also validated the importance of several additional molecules that were predicted by our screen to play a role in MNoV-induced cell death (Fig. 1A and fig. S3). Taken together, these data provide a systematic overview of the molecules required for NoV replication in these cells.

Fig. 1 CRISPR screen identifies CD300lf as necessary for MNoV infection.

(A) A heat map showing enrichment of genes in the two indicated conditions. Genes are color-coded on the basis of their STARS score. (B) Wild-type BV2 cells and two independently derived CD300lf-deficient clones (clone 1 and clone 2) transduced with an empty vector or a vector expressing CD300lf were challenged with MNoVCW3 (top) or MNoVCR6 (bottom) at a multiplicity of infection (MOI) of 0.05. Viral production was assessed by plaque assay (PFU, plaque-forming unit). Shown are means ± SEM for data pooled from three independent experiments. L.O.D., limit of detection.

We selected CD300lf for further analysis because of its cell-surface expression and the importance of viral receptors in conferring permissiveness for viral replication and species tropism. Transfection of MNoVCW3 RNA into BV2ΔCD300lf cells was sufficient to restore MNoVCW3 production, demonstrating that CD300lf is essential for viral entry (Fig. 2A). Preincubation of cells with a polyclonal antibody targeting CD300lf (α-CD300lf) blocked MNoVCW3-induced cytopathic effects in BV2 cells (Fig. 2B). Similarly, incubating MNoVCW3 with recombinant CD300lf ectodomain (sCD300lf) neutralized MNoVCW3-induced cytopathic effects, whereas annexin V or phosphoserine treatment had no effect (Fig. 2C and fig. S4). Similar results were obtained for infection of the B cell line M12, bone marrow–derived dendritic cells, and bone marrow–derived macrophages, indicating the essential role of CD300lf in multiple cell types (Fig. 2D). These data together indicate that interactions between MNoV and CD300lf are essential for MNoV infection.

Fig. 2 CD300lf is a MNoV receptor.

(A) Indicated BV2 cells were transfected with MNoVCW3 RNA and harvested 12 hours posttransfection. Viral production was measured by plaque assay. (B) BV2 cells were incubated with either α-CD300lf or an isotype control before infection with MNoVCW3 at a MOI of 5.0. Cell viability was measured 24 hours postinfection. IC50, half maximal inhibitory concentration. (C) MNoVCW3 was incubated with either soluble sCD300lf or a control protein before infection of BV2 cells. Cellular viability was assayed 24 hours postinfection. (D) MNoVCW3 infection was inhibited by either cellular pretreatment with α-CD300lf or viral pretreatment with sCD300lf. Infection was measured by fluorescence-activated cell sorting for intracellular MNoV NS1/2 expression, and inhibition is relative to an isotype control (for α-CD300lf) or control protein (for sCD300lf). BMDM, bone marrow–derived macrophage; BMDC, bone marrow–derived cell. (E) A representative MNoVCW3 binding assay in complete media to indicated BV2 cell lines, as assayed by quantitative polymerase chain reaction. Binding assays were performed when cells or virus were preincubated with α-CD300lf or sCD300lf, respectively. Data were analyzed by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test; three independent experiments were performed in triplicate. (F) MNoVCW3 binding assay performed in phosphate-buffered saline (PBS) plus 10% fetal bovine serum (FBS) or derivatives as indicated. Data were analyzed by one-way ANOVA with Tukey's multiple comparison test; three independent experiments were performed in triplicate. Throughout, results are shown as means ± SEM for data pooled from three independent experiments [except in (E), where results are shown from one representative experiment]. ns, not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

To directly test whether CD300lf functions as a binding receptor for MNoV, we analyzed the attachment of MNoVCW3 to BV2 cells on ice. BV2ΔCD300lf cells were impaired in MNoVCW3 binding (Fig. 2E). Additionally, treating BV2 cells with α-CD300lf or preincubating virus with sCD300lf reduced MNoV binding (Fig. 2E). In BV2ΔCD300lf cells, binding of MNoVCW3 was not further inhibited by α-CD300lf or by premixing virus with sCD300lf (Fig. 2E). These results show that CD300lf mediates viral binding and is a functional receptor for MNoV.

Previous reports have suggested that carbohydrates facilitate the binding of MNoV and HNoV to host cells and control the susceptibility of humans to HNoV infection (1, 911). Therefore, we assessed the relative contribution of carbohydrates to MNoV attachment and infection. Unexpectedly, mice deficient in Fut2, which controls histo-blood group antigen (HBGA) secretor status, had similar MNoV loads to those of wild-type littermate controls (fig. S5A). Also, treating cells with the mannosidase I inhibitor kifunensine significantly reduced cell-surface carbohydrates but did not significantly alter MNoVCW3 binding to cells (fig. S5, B and C). These data suggest that, within the sensitivity of these assays, MNoV binding and infection are dependent on CD300lf but not on protein-associated glycans or secretor status.

In addition to CD300lf, we discovered that efficient MNoV binding to cells requires a cofactor found in serum (Fig. 2F). The cofactor is present in delipidated serum and is resistant to proteinase K and heat denaturation (95°C; Fig. 2F). Size fractionation of serum indicates that the cofactor is present in fractions with an average molecular weight of less than 5000 Da (Fig. 2F). Thus, the serum cofactor is most likely a small nonproteinaceous heat-stable molecule that interacts with MNoV and/or CD300lf to facilitate cellular binding.

Next, we sought to assess the physiologic relevance of MNoV and CD300lf interactions. MNoV isolated from the spleens of MNoVCW3-infected mice remained sensitive to sCD300lf inhibition, indicating that sCD300lf neutralizes MNoV regardless of the source of the virus (Fig. 3A). To test the role of CD300lf and MNoV interactions in vivo, we incubated MNoVCW3 with either sCD300lf or a control protein before infection of Stat1−/− mice, which succumb to MNoVCW3 infection. In a dose-dependent manner, sCD300lf preincubation was able to protect from MNoV-induced lethality (Fig. 3, B and C). Last, we generated Cd300lf−/− mice to test the in vivo role of CD300lf in MNoV infection. Cd300lf−/− mice were resistant to MNoVCR6 infection compared with littermate controls (Fig. 3D). These data demonstrate that CD300lf is the primary physiological receptor for MNoV in vivo.

Fig. 3 CD300lf is a physiological MNoV receptor.

(A) MNoVCW3 harvested from the spleens of BL6/J mice or derived from BV2 cells was preincubated with sCD300lf before plaque assay. Inhibition is relative to a control protein. Shown are means ± SEM for data pooled from three independent experiments. (B and C) Survival of Stat1−/− mice after challenge with (B) 103 PFU or (C) 105 PFU of MNoVCW3 preincubated with either sCD300lf or control protein. Data were analyzed by a log-rank test (n = 11 mice per cohort, combined from three independent experiments). (D) Cd300lf−/− and littermate control mice were challenged with 106 PFU of MNoVCR6 perorally. Fecal shedding of MNoV genomes was monitored for 21 days postchallenge. Data were analyzed by repeated measures ANOVA; shown are means ± SEM (n = 9 or 10 mice per cohort, combined from two independent experiments). ***P < 0.001.

MNoV replicates in murine dendritic cells, macrophages, and B cells, but not in epithelial cells or human cells because of a restriction at viral entry (20, 21). Therefore, we tested whether expression of murine CD300lf was sufficient to confer susceptibility of HeLa cells to MNoV. As expected, HeLa cells transfected with a control plasmid were unable to support MNoV replication (Fig. 4A). In contrast, HeLa cells expressing murine but not human CD300lf were susceptible to MNoV (Fig. 4A). These results indicate that CD300lf expression is sufficient for MNoV growth in human cells and suggest that differences between human and murine CD300lf could contribute to MNoV species restriction.

Fig. 4 Structure-guided mapping identifies the CC′ loop and CDR3 of CD300lf as critical for MNoV infection.

(A) HeLa cells transiently transfected with indicated constructs were infected with either MNoVCW3 (left) or MNoVCR6 (right) at a MOI of 0.05. Viral production was measured by plaque assay at the indicated time points. GFP, green fluorescent protein. (B) HeLa cells transiently transfected with indicated CD300 constructs or TIM1 were infected with MNoVCW3 at a MOI of 5.0 and analyzed for expression of CD300 or TIM1 (FLAG) and MNoV NS1/2. (C) Recombinant ectodomains of indicated CD300 molecules (10 μg/ml) were preincubated with MNoVCW3 at a MOI of 5.0 before infection of BV2 cells. Cellular viability was assayed 24 hours postinfection. (D) HeLa cells transiently transfected with indicated CD300 constructs were infected with MNoVCW3 at a MOI of 5.0 and analyzed for expression of CD300 (FLAG) and MNoV NS1/2. CD300lf∆CT, CD300lf with truncation of cytoplasmic domain. In (A) to (D), results are shown as means ± SEM for data pooled from three independent experiments. (E) Ribbon diagram of murine CD300lf ectodomain with bound metal ion and Hepes. The β-sheets (cyan) are lettered as a canonical V-type Ig domain with the positions of the CDR-equivalent loops indicated. The disulfide bonds are shown in yellow. Numbers indicate amino acid positions. (F) Mapping of mutational results onto the CD300lf surface. Displayed in cyan are murine CD300lf residues replaced by human equivalent residues in CDR1, C′′, and DE loop that had no effect on infection. The CC′ loop and CDR3 mutations that diminished viral infection are shown in magenta.

In mice, there are eight CD300 family members (22). Therefore, we sought to determine whether other CD300 molecules are capable of functioning as MNoV receptors. After transfection into HeLa cells, only CD300lf and CD300ld conferred susceptibility to MNoV, whereas the expression of other CD300 family members or of the unrelated phosphatidylserine binding protein TIM1 was unable to support MNoV infection (Fig. 4B and fig. S6) (23). We further confirmed that expression of CD300lf or CD300ld, but not CD300lh, was sufficient for viral replication in human cells (Fig. 4A). However, in contrast to murine CD300lf, the recombinant ectodomains of murine CD300ld, murine CD300lh, or human CD300lf failed to neutralize viral infection (Fig. 4C). Additionally, BV2ΔCD300ld cells are susceptible to MNoV infection, suggesting that CD300ld can act as a MNoV receptor when ectopically expressed, but it is not a universal requirement (fig. S7). However, we cannot exclude the possibility that CD300ld may play a role in viral tropism in some circumstances in vivo. Experiments in Cd300lf−/− mice indicate that this putative role is not essential for intestinal infection or shedding.

We next sought to define the mechanism for MNoV entry via CD300lf and to determine how MNoV discriminates between mouse and human CD300lf proteins. Importantly, the intracellular domain of CD300lf was not required to make HeLa cells susceptible, indicating that species tropism is determined by the ectodomain (Fig. 4D). We determined the crystal structure of the CD300lf ectodomain at 1.6 Å resolution (Fig. 4E and table S4). Densities corresponding to a bound Hepes molecule, which was present in the purification buffers, and a coordinated metal were visible in a surface cleft formed between the CDR3 loop and the β-hairpin turn that connects the CC′ β-strands. CD300lf has been reported to mediate the phagocytosis of apoptotic cells through the calcium-dependent binding of lipids (2426). Hepes has chemical resemblance to a phospholipid headgroup (fig. S8). Our structure also reveals a metal coordinated primarily by CD300lf Asp98 and two CDR3 loop carbonyl oxygens. Although mutation of murine CD300lf Asp98 has been shown to disrupt apoptotic cell-surface binding, this mutant still allows MNoVCW3 infection of HeLa cells, suggesting that viral entry can occur in the absence of bound metal (Fig. 4D and fig. S8) (25, 27). The Ig domains of murine and human CD300lf share 59% sequence identity, and structurally the largest variation occurs in CDR3 and the CC′ loop (figs. S9 and S10) (27). Individual substitutions of human CC′ loop and CDR3 into murine CD300lf diminished and abolished MNoVCW3 infection of HeLa cells, respectively (Fig. 4, D and F). Additionally, purified recombinant proteins harboring CC′ loop and CDR3 human sequences failed to neutralize MNoV infection (Fig. 4C and fig. S11). However, the reciprocal CDR3 mutation (murine CDR3 into human CD300lf) was not sufficient for MNoV infection (Fig. 4D). Independently comparing murine CD300lf and CD300lh also indicated that the CD300lf CC′ loop and CDR3 sequences are necessary but not sufficient for receptor utilization by MNoV (fig. S12). These data provide a framework for understanding how MNoV discriminates between CD300 family members.

Our work establishes that CD300lf is a functional MNoV receptor that mediates binding to the cell surface and is both necessary and sufficient for viral entry and replication in vitro and in vivo. Because MNoV serves as a model system for understanding how viruses persist and shape the immune system, the modulation of receptor availability, either genetically or chemically, may foster understanding of immunomodulation, persistence, and tropism of MNoV. This work also enables the future study of MNoV replication in human cells, which may uncover novel mechanisms of viral replication and pathogenesis and allow a direct identification and mechanistic dissection of the cellular factors required for NoV replication across species. Additionally, our work has implications for understanding HNoV infections. HNoV binds to HBGA, and susceptibility to HNoV is correlated with host HBGA status; the studies that show this (12, 13) are the foundation for the hypothesis that glycans are HNoV receptors (9). However, HBGA alone cannot explain species tropism or the entry barrier for HNoV. In contrast, our data indicate that murine CD300lf is sufficient to explain tropism for MNoV and more broadly suggest the possibility that other NoVs use proteinaceous receptors in addition to small molecule cofactors that are present in serum or other biological fluids. It is intriguing that CD300 molecules can bind a range of host lipids and bacterial products (22, 28). It may therefore be that NoV cell and tissue tropism is determined in a combinatorial fashion by proteinaceous receptors interacting with permissiveness cofactors that are present at different sites.

Supplementary Materials

www.sciencemag.org/content/353/6302/933/suppl/DC1

Materials and Methods

Figs. S1 to S12

Tables S1 to S4

References (2939)

REFERENCES AND NOTES

Acknowledgments: We thank S. Karst and M. Diamond for providing valuable reagents and S. Handley and C. Desai for their helpful discussion and figure generation. We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, Missouri, for the use of the Genome Engineering and iPSC Center. CD300 constructs, cell lines, and mice are available from H.W.V. under a material transfer agreement with Washington University. The data from this study are tabulated in the main paper and in the supplementary materials. H.W.V., D.H.F., R.C.O., C.B.W., and C.A.N. are inventors on a patent application submitted by Washington University entitled “Receptor for norovirus and uses thereof” (U.S. Provisional Application 62/301,965). The atomic coordinates are deposited in the Protein Data Bank under accession code 5FFL. The Siteman Cancer Center is supported in part by National Cancer Institute Cancer Center Support Grant P30 CA091842. This work was supported by NIH grants U19 AI109725 (H.W.V.), 1F31CA177194 (B.T.M.), and 5T32CA009547 (M.T.B.).
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