scholarly journals Effect of Hemagglutinin Glycosylation on Influenza Virus Susceptibility to Neuraminidase Inhibitors

2005 ◽  
Vol 79 (19) ◽  
pp. 12416-12424 ◽  
Author(s):  
Vasiliy P. Mishin ◽  
Dmitri Novikov ◽  
Frederick G. Hayden ◽  
Larisa V. Gubareva
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Dominant Role ◽  
Neuraminidase Inhibitors ◽  
Virus Binding ◽  
Receptor Binding Site ◽  
Virus Susceptibility ◽  
Infected Cells

ABSTRACT Inhibition of neuraminidase (NA) activity prevents release of progeny virions from influenza-infected cells and removal of neuraminic (sialic) acid moieties from glycans attached to hemagglutinin (HA). Neuraminic acid moieties situated near the HA receptor-binding site can reduce the efficiency of virus binding and decrease viral dependence on NA activity for replication. With the use of reverse genetics technique, we investigated the effect of glycans attached at Asn 94a, 129, and 163 on the virus susceptibility to NA inhibitors in MDCK cells and demonstrated that the glycan attached at Asn 163 plays a dominant role in compensation for the loss of NA activity.

scholarly journals Interdependence of Hemagglutinin Glycosylation and Neuraminidase as Regulators of Influenza Virus Growth: a Study by Reverse Genetics

2000 ◽  
Vol 74 (14) ◽  
pp. 6316-6323 ◽  
Author(s):  
Ralf Wagner ◽  
Thorsten Wolff ◽  
Astrid Herwig ◽  
Stephan Pleschka ◽  
Hans-Dieter Klenk
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Influenza Virus Infection ◽  
Simian Virus 40 ◽  
Host Cells ◽  
Receptor Binding Site ◽  
Virus Growth ◽  
Recombinant Viruses

ABSTRACT The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377–8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.

scholarly journals Neutrophils do not bind to or phagocytize human immune complexes formed with influenza virus

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1639-1646 ◽  
Author(s):  
DR Ratcliffe ◽  
J Michl ◽  
EB Cramer
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Epithelial Cells ◽  
Binding Site ◽  
Immune Complexes ◽  
Human Neutrophils ◽  
Sialic Acid Binding ◽  
Infected Cells ◽  
Neutrophil Adherence ◽  
Human Immune

Abstract Neutrophils appear to form the first line of defense against influenza virus, yet it is unclear how these leukocytes recognize influenza- infected cells. While demonstrating that neutrophils adhere specifically to the sialic acid-binding site on the hemagglutinin molecule (HA) on the surface of influenza-infected (WSN[H1N1]) epithelial cells and not to other viral or epithelial cell antigens, it was observed that human neutrophils do not recognize immune complexes formed with influenza virus. Intact antibodies (mouse monoclonal antibodies [MoAbs] IgG1 and IgG2b, human immune heat-inactivated serum [predominantly IgG1], and IgG purified from human immune serum) that block the sialic acid-binding site on HA significantly reduced (> 80%) neutrophil adherence to influenza-infected epithelial cells. Binding and phagocytosis of free influenza virions and neutrophil agglutination by influenza virus were completely prevented by these antibodies. Intact and F(ab')2 fragments of mouse MoAbs to other viral epitopes caused increased neutrophil adherence to infected cells. This binding was eliminated by F(ab'2) fragments of MoAbs against the sialic acid- binding site on HA, but not by saturating amounts of MoAbs, which block the neutrophil Fc receptors. Thus, it appears that human neutrophils show little ability to bind via their Fc receptors to the immune complexes formed with antibody and either influenza-infected epithelial cells or the free virion. These findings are in contrast to the general dogma, and are the first example of antibody opsonization reducing, rather than enhancing, neutrophil binding and phagocytosis of a pathogen.

scholarly journals Antiviral Activity and Underlying Action Mechanism of Euglena Extract against Influenza Virus

Nutrients ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 3911
Author(s):  
Ayaka Nakashima ◽  
Yuka Horio ◽  
Kengo Suzuki ◽  
Yuji Isegawa
Keyword(s):  
Influenza Virus ◽  
Antiviral Activity ◽  
Defense Mechanisms ◽  
Mdck Cells ◽  
Influenza Virus Infection ◽  
Virus Titer ◽  
Prolonged Treatment ◽  
Action Mechanism ◽  
Cell Defense ◽  
Infected Cells

It is difficult to match annual vaccines against the exact influenza strain that is spreading in any given flu season. Owing to the emergence of drug-resistant viral strains, new approaches for treating influenza are needed. Euglena gracilis (hereinafter Euglena), microalga, used as functional foods and supplements, have been shown to alleviate symptoms of influenza virus infection in mice. However, the mechanism underlying the inhibitory action of microalgae against the influenza virus is unknown. Here, we aimed to study the antiviral activity of Euglena extract against the influenza virus and the underlying action mechanism using Madin–Darby canine kidney (MDCK) cells. Euglena extract strongly inhibited infection by all influenza virus strains examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. A time-of-addition assay revealed that Euglena extract did not affect the cycle of virus replication, and cell pretreatment or prolonged treatment of infected cells reduced the virus titer. Thus, Euglena extract may activate the host cell defense mechanisms, rather than directly acting on the influenza virus. Moreover, various minerals, mainly zinc, in Euglena extract were found to be involved in the antiviral activity of the extract. In conclusion, Euglena extract could be a potent agent for preventing and treating influenza.

scholarly journals Attenuation of Influenza A Virus mRNA Levels by Promoter Mutations

1998 ◽  
Vol 72 (8) ◽  
pp. 6283-6290 ◽  
Author(s):  
Ervin Fodor ◽  
Peter Palese ◽  
George G. Brownlee ◽  
Adolfo García-Sastre
Keyword(s):  
Influenza Virus ◽  
Transcription Initiation ◽  
Influenza A ◽  
Reverse Genetics ◽  
Mrna Levels ◽  
Double Mutation ◽  
Ribonucleoprotein Complexes ◽  
Virus Rna ◽  
Infected Cells

ABSTRACT We have engineered influenza A/WSN/33 viruses which have viral RNA (vRNA) segments with altered base pairs in the conserved double-stranded region of their vRNA promoters. The mutations were introduced into the segment coding for the neuraminidase (NA) by using a reverse genetics system. Two of the rescued viruses which share a C-G→A-U double mutation at positions 11 and 12′ at the 3′ and 5′ ends of the NA-specific vRNA, respectively, showed approximately a 10-fold reduction of NA levels. The mutations did not dramatically affect the NA-specific vRNA levels found in virions or the NA-specific vRNA and cRNA levels in infected cells. In contrast, there was a significant decrease in the steady-state levels of NA-specific mRNAs in infected cells. Transcription studies in vitro with ribonucleoprotein complexes isolated from the two transfectant viruses indicated that transcription initiation of the NA-specific segment was not affected. However, the majority of NA-specific transcripts lacked poly(A) tails, suggesting that mutations in the double-stranded region of the influenza virus vRNA promoter can attenuate polyadenylation of mRNA molecules. This is the first time that a promoter mutation in an engineered influenza virus has shown a differential effect on influenza virus RNA transcription and replication.

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

2003 ◽  
Vol 77 (15) ◽  
pp. 8418-8425 ◽  
Author(s):  
Mikhail Matrosovich ◽  
Tatyana Matrosovich ◽  
Jackie Carr ◽  
Noel A. Roberts ◽  
Hans-Dieter Klenk
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Sialic Acids ◽  
Neuraminidase Inhibitors ◽  
Human Influenza ◽  
Oseltamivir Carboxylate ◽  
Human Influenza Virus ◽  
Human Virus

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-α2,6-galactose (Sia[α2,6]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(α2,6)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.

scholarly journals Synergistic Antiviral Activity of S-033188/S-033447, a Novel Inhibitor of Influenza Virus Cap-Dependent Endonuclease, in Combination with Neuraminidase Inhibitors In Vitro

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S371-S371 ◽  
Author(s):  
Mitsutaka Kitano ◽  
Atsuko Yamamoto ◽  
Takeshi Noshi ◽  
Makoto Kawai ◽  
Ryu Yoshida ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Cultured Cells ◽  
H1n1 Virus ◽  
Mdck Cells ◽  
Active Form ◽  
Neuraminidase Inhibitors ◽  
Dependent Endonuclease ◽  
Influenza A H1n1

Abstract Background S-033447, an active form of orally available prodrug S-033188, is a novel small molecule inhibitor of cap-dependent endonuclease that is essential for influenza virus transcription and replication. In this study, we evaluated the inhibitory effect of S-033188 in combination with neuraminidase inhibitors on the replication of influenza A/H1N1 virus in cultured cells. Methods The inhibitory effects of S-033447 in combination with NA inhibitors on the cytopathic effect of A/PR/8/34 strain in Madin–Darby canine kidney cells cultured for 2 days were tested and EC50 were determined. The combination index (CI), which were obtained when S-033188 and NA inhibitor were added at the closest ratio of each EC50 value, were used for the evaluation of these combinational effects (Table 1). CI values were calculated by the Chou and Talalay method, in which combinational effect were determined according to the criteria as follows: synergistic if CI ≤ 0.8, additive if 0.8 < CI < 1.2, and antagonistic if CI ≥ 1.2. CI = (DA/A + B)/DA + (DB/A + B)/DB + (DA/A + B × DB/A + B)/(DA × DB) DA: the EC50 of S-033447 DB: the EC50 of NA inhibitor DA/A + B: the concentration of S-033447 giving 50% inhibition in combination with NA inhibitor at the closest ratio of each EC50 value DB/A + B: the concentration of NA inhibitor giving 50% inhibition in combination with S-033447 at the closest ratio of each EC50 value Results All CI values were lower than 0.8, under the condition that both S-033447 and NA inhibitor (oseltamivir acid, zanamivir hydrate, laninamivir, or peramivir trihydrate) were added at the closest ratio of each EC50 value (Table 1). Conclusion S-033447 in combination with oseltamivir acid, zanamivir hydrate, laninamivir, or peramivir trihydrate synergistically inhibited the replication of influenza A/H1N1 virus in MDCK cells. Disclosures All authors: No reported disclosures.

scholarly journals Entry of simian virus 40 is restricted to apical surfaces of polarized epithelial cells.

1988 ◽  
Vol 8 (8) ◽  
pp. 3391-3396 ◽  
Author(s):  
E T Clayson ◽  
R W Compans
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Apical Surface ◽  
Surface Binding ◽  
Virus Binding ◽  
Single Class ◽  
Infected Cells ◽  
Polarized Epithelial Cells

The uptake of simian virus 40 (SV40) by polarized epithelial cells was investigated by growth of cells on permeable supports and inoculation on either the apical or the basolateral surface. Binding of radiolabeled SV40 occurred on the apical but not the basolateral surfaces of permissive polarized Vero C1008 cells and nonpermissive polarized MDCK cells. When similar experiments were performed on nonpolarized Vero or CV-1 cells, virus binding occurred regardless of the direction of virus input. Electron micrographs of Vero C1008 cells infected at high multiplicities revealed virions lining the surfaces of apically infected cells, while the surfaces of basolaterally infected cells were devoid of virus particles. Analysis of the binding data revealed a single class of virus receptors (9 x 10(4) per cell) with a high affinity for SV40 (Kd = 3.76 pM) on the apical surfaces of Vero C 1008 cells. Indirect immunofluorescence studies revealed that synthesis of viral capsid proteins in Vero C1008 cells occurred only when input virions had access to the apical surface. Virus yields from apically infected Vero C1008 cells were 10(5) PFU per cell, while yields obtained from basolaterally infected cells were less than one PFU per cell. These results indicate that a specific receptor for SV40 is expressed exclusively on the apical surfaces of polarized Vero C1008 cells.

scholarly journals Sialic acid-dependent interaction of group B streptococci with influenza virus-infected cells reveals a novel adherence and invasion mechanism

2018 ◽  
Vol 20 (4) ◽  
pp. e12818 ◽  
Author(s):  
Jie Tong ◽  
Yuguang Fu ◽  
Nai-Huei Wu ◽  
Manfred Rohde ◽  
Fandan Meng ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Group B Streptococci ◽  
Invasion Mechanism ◽  
Infected Cells ◽  
Group B ◽  
Dependent Interaction

scholarly journals Inhibition of release of lentivirus particles with incorporated human influenza virus haemagglutinin by binding to sialic acid-containing cellular receptors

2001 ◽  
Vol 82 (10) ◽  
pp. 2485-2494 ◽  
Author(s):  
Valerie Bosch ◽  
Beatrice Kramer ◽  
Tanya Pfeiffer ◽  
Lilian Stärck ◽  
David A. Steinhauer
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Binding Site ◽  
Lipid Membrane ◽  
Surface Proteins ◽  
Human Influenza ◽  
Receptor Binding Site ◽  
Human Influenza Virus ◽  
Cellular Receptors ◽  
Influenza Virus A

Mutants of the haemagglutinin (HA) gene of human influenza virus A/Aichi/2/68 (H3N2) encoding HA proteins that are proteolytically cleaved intracellularly, defective in binding to cellular receptors or defective for acylation within the cytoplasmic C terminus have been generated. Here, the properties of these mutated HA molecules are described and their incorporation into the lipid membrane of released human immunodeficiency virus (HIV)-like particles is analysed. It is demonstrated that, when produced from cells coexpressing any of the binding-competent Aichi-HA molecules, release of HIV-like particles into the extracellular medium is reduced and the particles that are released fail to incorporate Aichi-HA. These blocks in release and incorporation, respectively, can both be overcome. The release of normal amounts of particles with incorporated HA can be achieved either by mutation of the receptor-binding site on the Aichi-HA molecule or by removal of sialic acid from surface proteins with neuraminidase. In contrast, as a result of blockage of the sialic acid-binding site by sialidated oligosaccharides on the HA itself, the HA of influenza virus A/FPV/Rostock/34 (H7N1) is efficiently incorporated into HIV-like particles. These results, namely that particle release can be inhibited by interactions between the incorporated glycoprotein and the cell surface and/or that interactions with other cellular components can be inhibitory to incorporation into retrovirus envelopes, probably reflect general principles that may hold for many viral and cellular glycoproteins.

scholarly journals Neutrophils do not bind to or phagocytize human immune complexes formed with influenza virus

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1639-1646
Author(s):  
DR Ratcliffe ◽  
J Michl ◽  
EB Cramer
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Epithelial Cells ◽  
Binding Site ◽  
Immune Complexes ◽  
Human Neutrophils ◽  
Sialic Acid Binding ◽  
Infected Cells ◽  
Neutrophil Adherence ◽  
Human Immune

Neutrophils appear to form the first line of defense against influenza virus, yet it is unclear how these leukocytes recognize influenza- infected cells. While demonstrating that neutrophils adhere specifically to the sialic acid-binding site on the hemagglutinin molecule (HA) on the surface of influenza-infected (WSN[H1N1]) epithelial cells and not to other viral or epithelial cell antigens, it was observed that human neutrophils do not recognize immune complexes formed with influenza virus. Intact antibodies (mouse monoclonal antibodies [MoAbs] IgG1 and IgG2b, human immune heat-inactivated serum [predominantly IgG1], and IgG purified from human immune serum) that block the sialic acid-binding site on HA significantly reduced (> 80%) neutrophil adherence to influenza-infected epithelial cells. Binding and phagocytosis of free influenza virions and neutrophil agglutination by influenza virus were completely prevented by these antibodies. Intact and F(ab')2 fragments of mouse MoAbs to other viral epitopes caused increased neutrophil adherence to infected cells. This binding was eliminated by F(ab'2) fragments of MoAbs against the sialic acid- binding site on HA, but not by saturating amounts of MoAbs, which block the neutrophil Fc receptors. Thus, it appears that human neutrophils show little ability to bind via their Fc receptors to the immune complexes formed with antibody and either influenza-infected epithelial cells or the free virion. These findings are in contrast to the general dogma, and are the first example of antibody opsonization reducing, rather than enhancing, neutrophil binding and phagocytosis of a pathogen.

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