title: The C-terminal domain of ERp29 mediates polyomavirus binding, unfolding, and infection.
abstract: Penetration of the endoplasmic reticulum (ER) membrane by polyomavirus (PyV) is a decisive step in virus entry. We showed previously that the ER-resident factor ERp29 induces the local unfolding of PyV to initiate the ER membrane penetration process. ERp29 contains an N-terminal thioredoxin domain (NTD) that mediates its dimerization and a novel C-terminal all-helical domain (CTD) whose function is unclear. The NTD-mediated dimerization of ERp29 is critical for its unfolding activity; whether the CTD plays any role in PyV unfolding is unknown. We now show that three hydrophobic residues within the last helix of the ERp29 CTD that were individually mutated to either lysine or alanine abolished ERp29's ability to stimulate PyV unfolding and infection. This effect was not due to global misfolding of the mutant proteins, as they dimerize and do not form aggregates or display increased protease sensitivity. Moreover, the mutant proteins stimulated secretion of the secretory protein thyroglobulin with an efficiency similar to that of wild-type ERp29. Using a cross-linking coimmunoprecipitation assay, we found that the physical interaction of the ERp29 CTD mutants with PyV is inefficient. Our data thus demonstrate that the ERp29 CTD plays a crucial role in PyV unfolding and infection, likely by serving as part of a substrate-binding domain.
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, 109 Zina Pitcher Place, Rm. 3043, Ann Arbor, MI 48109, USA.
authors.forename: Emily K
authors.initials: EK
authors.lastname: Rainey-Barger
authors.forename: Souren
authors.initials: S
authors.lastname: Mkrtchian
authors.forename: Billy
authors.initials: B
authors.lastname: Tsai
mesh_terms: D000818:Animals; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D047468:Immunoprecipitation; D008561:Membrane Fusion; D051379:Mice; D016296:Mutagenesis; D041681:NIH 3T3 Cells; D011120:Polyomavirus / Q000502:physiology*; D027601:Polyomavirus Infections / Q000821:virology*
chemical_list: C491737:Erp29 protein, mouse; D006360:Heat-Shock Proteins