Sialic acid binding site of MCPyV VP1 is crucial for infectivity.
(A) Sialic acid binding site mutantions impair hemagglutination. Hemagglutination assays were performed by mixing a suspension of sheep red blood cells with varying doses of purified wild-type (WT) or VP1 mutant MCPyV capsids. (B) Pseudovirus transduction of sialic acid binding site mutants. Human A549 cells were inoculated with WT or mutant VP1 pseudovirions carrying an encapsidated GLuc reporter plasmid. Three days after inoculation, the culture supernatants were tested for GLuc activity (measured in relative light units, RLUs). (C) Cell attachment of sialic acid binding site mutants. A549 cell suspensions were mixed with 150 ng of WT or mutant VP1 capsids for 2 hours at 4°C. The cells were then washed and subjected to Western blotting using a polyclonal serum specific for MCPyV VP1. In the lanes labeled “Input,” 50 ng of VP1 was loaded into each lane. In the lanes labeled “Bound,” 2/3rds of the washed cell pellet was loaded into each lane. (D) Cell attachment of sialic acid binding site mutants depends on GAGs. A549 cell suspensions (5×104/well) were treated with a mixture of heparinase I (875 mU), heparinase III (87.5 mU), and chondroitinase ABC (35 mU) or mock treated in 50 µl of digestion buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 4 mM CaCl2 and 0.1% BSA) for one hour at 37°C. Then, 200 ng of WT or mutant VP1 capsids diluted in 200 µl of Opti-MEM were added and the mixture was incubated for 2 hours at 4°C. The cells were then washed and subjected to Western blotting using a polyclonal serum specific for MCPyV VP1.