Inhibition of clathrin-dependent endocytosis has multiple effects on humanrhinovirus serotype 2 cell entry

Minor group human rhinoviruses (exemplified by human rhinovirus serotype 2 (HRV2)) use members of the low density lipoprotein receptor family for cell entry; all these receptors possess clathrin-coated pit localization signal s. Viral infection should thus be inhibited under conditions of impaired cl athrin-mediated endocytosis. However, Madshus et at reported an increase in the cytopathic effect of HRV2 infection in HEp-2 cells upon suppression of clathrin-dependent endocytosis by hypotonic shock and potassium depletion (Madshus, I. H., Sandvig, K., Olsnes, S., and van Deurs, B. (1987) J. Cell, Physiol. 131, 14-22.) To resolve this apparent contradiction, we investiga ted the binding, internalization, conformational changes, and productive un coating of HRV2 in HeLa cells subjected to hypotonic shock and potassium de pletion. This treatment led to an increase in HRV2 binding, with internaliz ation being barely affected. The generation of C-antigenic particles requir ing pH less than or equal to5.6 was strongly reduced due to an elevation of the pH in endosomal compartments. However, K+ depletion only slightly affe cted de novo viral protein synthesis, suggesting that productivity of viral RNA in the cytoplasm is enhanced and thus compensates for the reduction in C-antigenic particles. The distinct steps in the entry pathway of HRV2 are thus differently influenced by potassium depletion. Viral internalization under conditions of inhibited clathrin-dependent endocytosis without the ne ed to disturb the ionic milieu was confirmed in HeLa cells overexpressing t he nonfunctional dynamin-1 mutant K44A. Unexpectedly, overexpression of dyn amin-1 K44A resulted in elevated endosomal pH compared with overexpression of wild-type dynamin.