title: Functional genetic and biophysical analyses of membrane disruption by human adenovirus.
abstract: The identification of the adenovirus (AdV) protein that mediates endosome penetration during infection has remained elusive. Several lines of evidence from previous studies suggest that the membrane lytic factor of AdV is the internal capsid protein VI. While these earlier results imply a role for protein VI in endosome disruption, direct evidence during cell entry has not been demonstrated. To acquire more definitive proof, we engineered random mutations in a critical N-terminal amphipathic α-helix of VI in an attempt to generate AdV mutants that lack efficient membrane penetration and infection. Random mutagenesis within the context of the AdV genome was achieved via the development of a novel technique that incorporates both error-prone PCR and recombineering. Using this system, we identified a single mutation, L40Q, that significantly reduced infectivity and selectively impaired endosome penetration. Furthermore, we obtained biophysical data showing that the lack of efficient endosomalysis is associated with reduced insertion of the L40Q mutation in protein VI (VI-L40Q) into membranes. Our studies indicate that protein VI is the critical membrane lytic factor of AdV during cellular entry and reveal the biochemical basis for its membrane interactions.
authors.affiliation: Department of Immunology and Microbial Sciences, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Crystal L
authors.initials: CL
authors.lastname: Moyer
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Wiethoff
authors.forename: Oana
authors.initials: O
authors.lastname: Maier
authors.forename: Jason G
authors.initials: JG
authors.lastname: Smith
authors.forename: Glen R
authors.initials: GR
authors.lastname: Nemerow
mesh_terms: D000260:Adenoviruses, Human / Q000472:pathogenicity*; D019943:Amino Acid Substitution / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004252:DNA Mutational Analysis; D011992:Endosomes / Q000821:virology*; D005818:Genetic Engineering / Q000379:methods; D005827:Genetics, Microbial / Q000379:methods; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D053586:Virus Internalization
chemical_list: D036022:Capsid Proteins; D050505:Mutant Proteins