Influenza A virus entry into cells lacking sialylated N-glycans

Abstract

Influenza A virus (IAV) enters host cells after attachment of its hemagglutinin (HA) to surface-exposed sialic acid. Sialylated Nlinked 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. (IAV) is an enveloped, negative-strand RNA virus that causes respiratory and/or intestinal infections in a variety of animal hosts, including birds and mammals. The IAV envelope contains two glycoproteins: the hemagglutinin (HA) protein, which is responsible for virus cell attachment and fusion, and the neuraminidase (NA) protein, which is the receptordestroying enzyme essential for release of the virus from the host cell after budding. IAV attaches to host cells by binding of HA to sialic acids (SIA) present on the host cell surface. Attachment is followed by entry via endocytic routes delivering the virus to the acidic environment of the late endosome that triggers HA-mediated fusion. The interactions of HA with SIA likely are important determinants not only of attachment, but also of virus uptake, intracellular trafficking, and fusion.Cell surface glycan composition is complex and varies between host and cell type. Sialylated glycans are attached to membrane phospolipids or to membrane proteins via asparagine (N-linked glycans) or serine/threonine (O-linked glycans) residues. How and to what extent binding of IAV to specific sialoglycans determines subsequent endocytosis remains unclear. N-linked glycans have been shown to be required for IAV entry, even though O-linked glycans and/or glycolipids permit efficient sialic acid-dependent binding of IAV to cells devoid of sialylated N-linked glycans (2). Thus, it appears that beyond the well-established requirement for binding to either α2-6 SIAs of human IAVs or α2-3 SIAs of avian IAVs, a specific subset of glyc...

Enterovirus D68 receptor requirements unveiled by haploid genetics

“…Analysis of the number of infected cells showed that SLC35A1 KO and CMAS KO cells were highly resistant to influenza A virus (IAV), whereas ST3GAL4/ST6GAL1 DKO cells were partially resistant ( Fig. 2A), consistent with the broad Sia specificity of IAV (19). In contrast, coxsackievirus-B3, which does not require Sia, could infect all cell lines ( Fig.…”

“…Furthermore, the identification of MGAT5, which forms GlcNAc-β1,6-Man linkages, suggested that EV-D68 specifically recognizes N-linked glycans containing a β1,6-linked antenna. It should be noted that Sia-galactose-GlcNAc, although mainly expressed on N-linked glycans, also occurs on O-linked glycans and glycolipids and that we observed that HEK293S cells, which lack complex N-linked glycans (19), are susceptible to EV-D68. Further proof that EV-D68 does not merely bind any sialylated glycan but has a preference for specific glycans stems from our observation that erythrocytes of several donors could be agglutinated by IAV but not by EV-D68.…”

Envelope Glycoprotein Binding to the Integrin α 4 β 7 Is Not a General Property of Most HIV-1 Strains

“…It is possible that the modified N-glycan moieties on AN-1gp120 result in a change in protein conformation that exposes the V2 loop LDV/LDI in a conformation optimal for ␣ 4 ␤ 7 reactivity while reducing CD4 binding. A possible explanation for why the gp120s produced in 293S/GnT1 Ϫ/Ϫ cells did not bind ␣ 4 ␤ 7 could be that CHO lec1.1 Ϫ/Ϫ cells carry an additional glycosylation defect (43). Our attempts to produce gp120s in CHO lec1.1 Ϫ/Ϫ cells were unsuccessful.…”

The Macrophage Galactose-Type Lectin Can Function as an Attachment and Entry Receptor for Influenza Virus

“…In these studies, a mutant CHO line lacking a functional N-acetylglucosaminyltransferase I (GnT1) gene (Lec1 cells) was shown to be resistant to IAV infection despite efficient SIA-dependent binding of virus to the cell surface (13). Recent studies confirmed Lec1 cells to be resistant to IAV infection but reported efficient entry of IAV into other GnT1-deficient cell lines (5), leading the authors to propose that Lec1 cells harbor additional defects that affect IAV entry in the absence of sialylated N-linked glycans.…”

“…In addition, recent studies identified macropinocytosis as an alternative entry route for IAV (4)(5)(6). Following internalization, virus is trafficked through the endosomal maturation pathway until endosomal acidification triggers a conformational change in the viral HA, exposing its fusion peptide and facilitating fusion of viral and endosomal membranes (7).…”