Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway

PLoS Pathog. 2011 Mar;7(3):e1001329. doi: 10.1371/journal.ppat.1001329. Epub 2011 Mar 31.

Abstract

Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actomyosin / metabolism
  • Amiloride / analogs & derivatives
  • Amiloride / pharmacology
  • Cell Line, Tumor
  • Clathrin / metabolism
  • Dynamins / genetics
  • Dynamins / metabolism
  • Endocytosis* / drug effects
  • Fetal Blood
  • Gene Silencing
  • HeLa Cells
  • Host-Pathogen Interactions
  • Humans
  • Hydrazones / pharmacology
  • Influenza A virus / metabolism*
  • Pinocytosis* / drug effects
  • RNA, Small Interfering
  • Receptor Protein-Tyrosine Kinases / metabolism
  • Virus Internalization* / drug effects

Substances

  • Clathrin
  • Hydrazones
  • N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide
  • RNA, Small Interfering
  • Amiloride
  • Actomyosin
  • Receptor Protein-Tyrosine Kinases
  • Dynamins
  • ethylisopropylamiloride