Fig 2: Virus binding and CDHR3 glycosylation.Lysates from HeLa cells transfected (A) or transduced (B) with full-length cDNAs encoding Y529 or C529 CDHR3 proteins were reacted with C15 virus in the presence or absence of PNGaseF or neuraminidase as described in Methods. Immunoprecipitation and protein detection used an a-CDHR3 mAb reactive with the cytoplasmic domain. Captured virus was quantitated as in Fig 1. (C) As in B, transduced cell lysates were treated (or not) with biotinylated Sambucus nigra (S) or Maackia amurensis (M) lectins before extraction with streptavidin agarose beads and protein detection with a-CDHR3. (D) Plated stably transduced HeLa cells expressing FLAG-Y529 or C529 CDHR3 were biotinylated and labeled cell surface proteins were isolated with streptavidin agarose beads.

Fig 3: Dependence of CDHR3 and C15 binding on calcium.Lysates transfected with cDNAs encoding Y529 CDHR3 protein were reacted with C15 virus in buffer with 2mM calcium, 2mM EDTA or EGTA (A), or in calcium-free buffer (B) and immunoprecipitated with an a-CDHR3 mAb reactive with the cytoplasmic domain. (C) Purified rEC1-3 protein (100 pmol) was reacted with C15 virus in buffer with 2mM CaCl2 or 2 mM EDTA or EGTA. (D) rEC1-3, rEC1-2, and rEC1 (100 pmol) protein were first reacted with C15 virus in buffer with or without 2 mM CaCl2 for 1 h at 25 and then incubated another 2 hrs following the addition of 5 mM CaCl2 to designated samples. Immunoprecipitation was with an a-His mAb. Each panel (ABCD) is a separate experiment. Binding % is the observed C15 signal pixel count normalized to the CDHR3 protein (a-His) signal in the panel above (Total Lab 100) relative to positive control (100) in each unit.

Fig 4: Mapping RV-C binding domains in CDHR3.(A) Design of CDHR3 EC deletion constructs. (B) Cell lysates of HeLa cells transfected with these cDNAs were reacted with sucrose purified C15 virus (107 PFUe) as described in Methods. Western assays after immunoprecipitation with an a-His mAb detected CDHR3 (via a-FLAG) and captured virus (a-C15). Binding % is the observed C15 signal pixel count normalized to the CDHR3 protein (a-FLAG) signal in the panel above (Total Lab 100) relative to the WT CDHR3 lane (bold face).

Fig 5: CDHR3 glycosylation.HeLa cell lysates from cells transfected with cDNA for full-length CDHR3 (Y529) were gel fractionated. The CDHR3 band was treated (or not) with PNGaseF before trypsin digestion and analysis by mass spectrometry. Identified peptides are indicated: (+) deamide form of peptide; (+/-) amide and deamide forms. NetNGlyc strength is according to primary sequence data (* is low, *** is high). Protein numbering is from GenBank AIC58018, with EC delineation from the current structure model [23].