Influenza A virus entry into cells lacking sialylated N-glycans.

Influenza A virus (IAV) enters host cells after attachment of its hemagglutinin (HA) to surface-exposed sialic acid. Sialylated N-linked glycans have been reported to be essential for IAV entry [Chu VC, Whittaker GR (2004) Proc Natl Acad Sci USA 102:18153-18158], thereby implicating the requirement for proteinaceous receptors in IAV entry. Here we show, using different N-acetylglucosaminyl transferase 1 (GnT1)-deficient cells, that N-linked sialosides can mediate, but are not required for, entry of IAV. Entry into GnT1-deficient cells was fully dependent on sialic acid. Although macropinocytic entry appeared to be affected by the absence of sialylated N-glycans, dynamin-dependent entry was not affected at all. However, binding of HA to GnT1-deficient cells and subsequent entry of IAV were reduced by the presence of serum, which could be reversed by back-transfection of a GnT1-encoding plasmid. The inhibitory effect of serum was significantly increased by inhibition of the viral receptor-destroying enzyme neuraminidase (NA). Our results indicate that decoy receptors on soluble serum factors compete with cell surface receptors for binding to HA in the absence of sialylated N-glycans at the cell surface. This competition is particularly disturbed by the additional presence of NA inhibitors, resulting in strongly reduced IAV entry. Our results indicate that the balance between HA and NA is important not only for virion release, but also for entry into cells.

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

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.