A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a “raised” conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection.Instituto de VirologíaFil: Salmen, Wilhelm. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Hu, Liya. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Bok, Marina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnologicas; ArgentinaFil: Bok, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Chaimongkol, Natthawan. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Caliciviruses Section; Estados UnidosFil: Ettayebi, Khalil. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Sosnovtsev, Stanislav V. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Caliciviruses Section; Estados UnidosFil: Soni, Kaundal. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Ayyar, B. Vijayalakshmi. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Shanker, Sreejesh. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Neill, Frederick H. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Sankaran, Banumathi. Berkeley Center for Structural Biology. Molecular Biophysics and Integrated Bioimaging. Lawrence Berkeley Laboratory; Estados UnidosFil: Atmar, Robert L. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Atmar, Robert L. Baylor College of Medicine. Department of Medicine; Estados UnidosFil: Estes, Mary K. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Estes, Mary K. Baylor College of Medicine. Department of Medicine; Estados UnidosFil: Green, Kim Y. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Caliciviruses Section; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virologia e Innovaciones Tecnologicas (IVIT); ArgentinaFil: Parreño, Gladys Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Prasad, B. V. Venkataram. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Prasad, B. V. Venkataram. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados Unido

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection

Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however, previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains.Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme, while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals, while HIEs from secretors are permissive to infection. However, whether FUT2 expression alone is critical for infection remains unproven, since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication, we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies, these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status