Human Rhinovirus Type 2 Uncoating at the Plasma Membrane Is Not Affected by a pH Gradient but Is Affected by the Membrane Potential

Abstract

The minor receptor group human rhinovirus type 2 enters host cells by endocytosis via members of the low-density-lipoprotein receptor family. In late endosomes, it undergoes a conformational change solely induced by a pH of < or =5.6, resulting in RNA transfer across the endosomal membrane into the cytoplasm. To determine potential driving forces of this process, we investigated whether RNA penetration might depend on the pH gradient and/or the membrane potential between the acidic endosome lumen and the neutral cytoplasm. Since these parameters are difficult to assess in endosomes, we took advantage of the possibility of inducing structural changes, RNA release, and consequently infection from the plasma membrane. To manipulate the pH gradient, cell-bound virus was exposed to membrane-permeant or -impermeant acidic buffers at 4 degrees C, and this was followed by a shift to 34 degrees C in medium containing bafilomycin to prevent RNA release from endosomes. To manipulate the plasma membrane potential, similar experiments were carried out, but these included K(+) diffusion potentials in the presence of the K(+) ionophore valinomycin. We demonstrated that infection does not depend on a pH gradient but is enhanced by plasma membrane hyperpolarization compared to plasma membrane depolarization.