title: Mutagenesis studies of the H5 influenza hemagglutinin stem loop region.
abstract: Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry.
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Antanasijevic
authors.affiliation: Microbiotix Inc., Worcester, Massachusetts 01605.
authors.forename: Arnab
authors.initials: A
authors.lastname: Basu
authors.affiliation: Microbiotix Inc., Worcester, Massachusetts 01605.
authors.forename: Terry L
authors.initials: TL
authors.lastname: Bowlin
authors.affiliation: the Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, Illinois 60208, and.
authors.forename: Rama K
authors.initials: RK
authors.lastname: Mishra
authors.affiliation: the Department of Microbiology and Immunology, University of Illinois, Chicago, Illinois 60612.
authors.forename: Lijun
authors.initials: L
authors.lastname: Rong
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607, caffrey@uic.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Caffrey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D018360:Crystallography, X-Ray; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D053586:Virus Internalization / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; C109963:hemagglutinin, avian influenza A virus
keywords: Antiviral Agent; Glycoprotein Structure; Influenza; Site-directed mutagenesis; Virus Entry