Fig 1: The VP3-C220F and VP1-S66N mutations reduce capsid stability. Graphs show the fractions of surviving virus as determined by endpoint titration. Mutants were more sensitive to all stress conditions, where the VP3-C220F (G2274U) mutant was the most sensitive one in all assays. (A) Resistance to low pH. Virus was incubated on dialysis filter membranes on PBS or acetate buffers under the indicated pH conditions. (B) Resistance to elevated temperatures. Virus was incubated at the indicated temperatures for 5 min. (C) Sensitivity to soluble ICAM-1 receptor domain. Indicated concentrations of soluble ICAM-1 were added to virus and incubated at 37°C for 30 min. (D) Virus sensitivity to endosomal-like ionic conditions. Virus was diluted in Dulbecco’s PBS (DPBS) or into a neutral pH endosomal-like buffer and incubated at 42°C for 5 min.
Fig 2: Differential susceptibility of the wt and the interprotomeric RV-A16 mutants VP3-C220F and VP1-S66N to cumulative destabilization by receptor binding, endosomal-like ionic conditions, and low pH. The data support a model in which the incoming virus particles are exposed to a series of uncoating cues in a stepwise manner, as originally established with human adenovirus (107). The initial cue occurs by ICAM-1 binding to the virion and destabilizes the particle by releasing the pocket factors. Upon endocytic uptake, the virion is exposed to a particular endosomal ionic environment with concentrations of Na+, K+, Cl−, Ca2+, and Mg2+ ions roughly intermediate between the extracellular medium and the cytosol, as well as progressively increasing proton concentration (53). While wt RV-A16 has a relatively high threshold for RNA uncoating, the RV-A16 mutants adapted to pH-neutral endosomes have a lower threshold. Mutant particles are readily inactivated by just one type of cue, such as ICAM-1 binding or endosomal-like ionic conditions, unlike wt RV-A16, which remains stable under these conditions. This indicates that the VP3-C220F and VP1-S66N mutants adapted to cells lacking low pH endosomes by reducing their capsid stability.
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