Overexpression of the α-2,6-Sialyltransferase in MDCK Cells Increases Influenza Virus Sensitivity to Neuraminidase Inhibitors

Abstract

No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration of sialyl-␣2,6-galactose (Sia[␣2,6]Gal)-containing virus receptors in conventional cell lines, replication of human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of Sia(␣2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased, whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neuraminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI.The neuraminidase (NA) of influenza A and B viruses cleaves the ␣-glycosidic linkages between sialic acid and the adjacent sugar and thus destroys virus receptors on the cell surface, extracellular inhibitors, and viral glycoproteins (reviewed in references 2 and 8). The NA activity is believed to be particularly important at the late stages of infection by preventing hemagglutinin (HA)-mediated self-aggregation and facilitating release of progeny virions from cells. Interaction of virions with cell-associated and soluble sialylglycoconjugates of the host is mediated by HA and NA in an antagonistic manner, which has to be carefully balanced to allow efficient virus replication (reviewed in reference 36).With increasing use of neuraminidase inhibitors (NAI) for influenza treatment, there is a need for a suitable methodology to monitor for emergence of virus resistance (32,34,38). In cell culture experiments, resistance to NAI results from mutation of either HA, NA, or both glycoproteins. Mutations in HA usually precede NA mutations and reduce virus affinity for sialic acid-containing receptors, thereby decreasing the dependency of the virus on NA function, whereas mutations in NA decrease the binding affinity of the inhibitor to the catalytic site (reviewed in references 19, 29, and 30). In a clinical setting, NA-mediated resistance seems to be the primary mechanism of resistance to NAI and can be easily and reliably monitored using an in vitro enzyme inhibition assay (32,34,38). Since the possibility cannot be excluded that the loss of sensitivit...

Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production

“…Recent publications have reported on the expression of the human siat1 gene (ST6Gal I) in MDCK cells that was associated with an increase in ␣2,6-linked sialic acid on glycoproteins (29)(30)(31). The researchers wanted to increase the number of 6-linked sialic acids on the cell surface to enhance influenza virus sensitivity to neuraminidase inhibitor, which is the key component of antiviral drugs for influenza.…”

Neuraminidase Inhibitor-Resistant Recombinant A/Vietnam/1203/04 (H5N1) Influenza Viruses Retain Their Replication Efficiency and Pathogenicity In Vitro and In Vivo

“…Madin-Darby canine kidney (MDCK) cells and human embryonic kidney cells transformed with large T antigen (293T cells) were obtained from the American Type Culture Collection (Manassas, VA). MDCK cells transfected with cDNA encoding human 2,6-sialyltransferase (MDCK-SIAT1 cells) were kindly provided by Mikhail N. Matrosovich and maintained as described previously (32,50).…”

“…To further evaluate the effect of the H274Y and N294S NA mutations on viral growth, we assayed the yields of the recombinant VN1203 (H5N1) and PR8 (H1N1) viruses after multiple replication cycles in MDCK cells (which exhibit more ␣-2,3-linked terminal sialic acid [SA] than ␣-2,6-linked terminal SA on the cell surface) and MDCK-SIAT1 cells (which exhibit more ␣-2,6-linked terminal SA than ␣-2,3-linked terminal SA on the cell surface) (32). At 12 hours …”

Decreased Neuraminidase Activity Is Important for the Adaptation of H5N1 Influenza Virus to Human Airway Epithelium

“…Previous studies have demonstrated that the functional balance between the receptor-binding HA activity and receptor-destroying NA activity of the surface influenza virus glycoproteins determines the pattern of emergence of NA inhibitor resistance (24,39,40). The disparate hemagglutinin (HA)-NA balance and the differences in sialic acid (SA) receptors between available continuous cell lines and human respiratory epithelial cells significantly limit the suitability of the commonly used cell cultures for phenotypic characterization of NA inhibitor resistance (21,24,39). Here, we used primary normal human bronchial epithelial (NHBE) cells, which possess human respiratory tract SA receptors; i.e., they express high concentrations of SA-␣2,6-galactose (Gal)-containing receptors and lesser amounts of SA-␣2,3-Gal receptors (22).…”