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. 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.
Human norovirus frequently causes outbreaks of acute gastroenteritis. Although discovered more than five decades ago, antiviral development has, until recently, been hampered by the lack of a reliable human norovirus cell culture system. Nevertheless, a lot of pathogenesis studies were accomplished using murine norovirus (MNV), which can be grown routinely in cell culture. In this study, we analyzed a sizeable library of nanobodies that were raised against the murine norovirus virion with the main purpose of developing nanobody-based inhibitors. We discovered two types of neutralizing nanobodies and analyzed the inhibition mechanisms using X-ray crystallography, cryo-electron microscopy (cryo-EM), and cell culture techniques. The first type bound on the top region of the protruding (P) domain. Interestingly, this nanobody binding region closely overlapped the MNV receptor-binding site and collectively shared numerous P domain-binding residues. In addition, we showed that these nanobodies competed with the soluble receptor, and this action blocked virion attachment to cultured cells. The second type bound at a dimeric interface on the lower side of the P dimer. We discovered that these nanobodies disrupted a structural change in the capsid associated with binding cofactors (i.e., metal cations/bile acid). Indeed, we found that capsids underwent major conformational changes following addition of Mg2+ or Ca2+. Ultimately, these nanobodies directly obstructed a structural modification reserved for a postreceptor attachment stage. Altogether, our new data show that nanobody-based inhibition could occur by blocking functional and structural capsid properties. IMPORTANCE This research discovered and analyzed two different types of MNV-neutralizing nanobodies. The top-binding nanobodies sterically inhibited the receptor-binding site, whereas the dimeric-binding nanobodies interfered with a structural modification associated with cofactor binding. Moreover, we found that the capsid contained a number of vulnerable regions that were essential for viral replication. In fact, the capsid appeared to be organized in a state of flux, which could be important for cofactor/receptor-binding functions. Blocking these capsid-binding events with nanobodies directly inhibited essential capsid functions. Moreover, a number of MNV-specific nanobody binding epitopes were comparable to human norovirus-specific nanobody inhibitors. Therefore, this additional structural and inhibition information could be further exploited in the development of human norovirus antivirals.
2324 Human norovirus frequently causes outbreaks of acute gastroenteritis. Although 25 discovered more than five decades ago, antiviral development has, until recently, been 26 hampered by the lack of a reliable human norovirus cell culture system. Nevertheless, a 27 lot of pathogenesis studies were accomplished using murine norovirus (MNV), which can 28 be grown routinely in cell culture. In this study, we analysed a sizeable library of 29 Nanobodies that were raised against the murine norovirus virion with the main purpose of 30 developing Nanobody-based inhibitors. We discovered two types of neutralizing 31 Nanobodies and analysed the inhibition mechanisms using X-ray crystallography, cryo-32 EM, and cell culture techniques. The first type bound on the top region of the protruding 33 (P) domain. Interestingly, the Nanobody binding region closely overlapped the MNV 34 receptor-binding site and collectively shared numerous P domain-binding residues. In 35 addition, we showed that these Nanobodies competed with the soluble receptor and this 36 action blocked virion attachment to cultured cells. The second type bound at a dimeric 37 interface on the lower side of the P dimer. We discovered that these Nanobodies 38 disrupted a structural change in the capsid associated with binding co-factors (i.e., metal 39 cations/ bile acid). Indeed, we found that capsids underwent major conformational 40 changes following addition of Mg 2+ or Ca 2+ . Ultimately, these Nanobodies directly 41 obstructed a structural modification reserved for a post-receptor attachment stage.42 Altogether, our new data show that Nanobody-based inhibition could occur by blocking 43 functional and structural capsid properties.3 44 AUTHOR SUMMARY 45 46 This research discovered and analysed two different types of MNV neutralizing 47 Nanobodies. The top-binding Nanobodies sterically inhibited the receptor-binding site, 48 whereas the dimeric-binding Nanobodies interfered with a structural modification 49 associated with co-factor binding. Moreover, we found that the capsid contained a 50 number of vulnerable regions that were essential for viral replication. In fact, the capsid 51 appeared to be organized in a state of flux, which could be important for co-factor/ 52 receptor binding functions. Blocking these capsid-binding events with Nanobodies 53 directly inhibited essential capsid functions. Moreover, a number of MNV-specific 54 Nanobody binding epitopes were comparable to human norovirus-specific Nanobody 55 inhibitors. Therefore, this additional structural and inhibition information could be further 56 exploited in the development of human norovirus antivirals. 4 57 INTRODUCTION 58 59 Norovirus belongs to the Caliciviridae family of non-enveloped, single-stranded positive-60 sensed RNA viruses [1]. The Norovirus genus comprises at least seven genogroups (GI-61 GVII), where GI, GII, and GIV infect humans [2]. Worldwide, human norovirus is one of 62 the leading causes of outbreaks of acute gastroenteritis [3-5]. There are still no antivirals 63 or ...
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