Specific Sites of N-Linked Glycosylation on the Hemagglutinin of H1N1 Subtype Influenza A Virus Determine Sensitivity to Inhibitors of the Innate Immune System and Virulence in Mice
Abstract:Oligosaccharides on the hemagglutinin (HA) and neuraminidase of influenza A virus (IAV) are a target for recognition by lectins of the innate immune system, including soluble surfactant protein-D and the macrophage mannose receptor on airway macrophages. Glycans attached to the head of H1 subtype of IAV differ markedly in number and location. A reverse genetic approach was used to define the importance of particular N-glycosylation sites on H1 in determining sensitivity to innate immune defenses and virulence … Show more
“…Alternatively, high-mannose glycans at Asn 136 might be orientated in a manner that is prohibitive to effective recognition by MBL and SP-D. For seasonal H1N1, loss of a high-mannose glycan from Asn 104 has been associated with resistance to conglutinin, a bovine-specific collectin (58). Addition of Asn 104 and Asn 144 together, but not either site alone, to PR8 HA was associated with enhanced sensitivity to MBL and SP-D (20), suggesting that for seasonal H1 IAV both sites express glycans recognized by collectins but that multivalent interactions increase binding avidity and therefore anti-IAV activity.…”
Section: Discussionmentioning
confidence: 99%
“…Site-directed mutagenesis was used to add or remove N-linked glycosylation sites from the Auck/09 HA (20 …”
Section: Generation Of Iav Glycosylation Mutants Using Rgmentioning
confidence: 99%
“…SP-D and MBL bind to HA and NA (15,18,19), with the majority of binding to HA. Sitedirected mutagenesis and reverse genetics (RG) approaches have demonstrated the importance of particular N-glycans on the head of seasonal H1 (20) and H3 IAV (21,22) as ligands for MBL and SP-D.…”
Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the viral hemagglutinin (HA). Glycans on the head of HA promote virus survival by shielding antigenic sites, but highly glycosylated seasonal IAV are inactivated by soluble lectins of the innate immune system. In 2009, human strains of pandemic H1N1 [A(H1N1)pdm] expressed a single glycosylation site (Asn104) on the head of HA. Since then, variants with additional glycosylation sites have been detected, and the location of these sites has been distinct to those of recent seasonal H1N1 strains. We have compared wild-type and reverse-engineered A(H1N1)pdm IAV with differing potential glycosylation sites on HA for sensitivity to collectins and to neutralizing Abs. Addition of a glycan (Asn136) to A(H1N1)pdm HA was associated with resistance to neutralizing Abs but did not increase sensitivity to collectins. Moreover, variants expressing Asn136 showed enhanced growth in A(H1N1)pdm-vaccinated mice, consistent with evasion of Ab-mediated immunity in vivo. Thus, a fine balance exists regarding the optimal pattern of HA glycosylation to facilitate evasion of Ab-mediated immunity while maintaining resistance to lectin-mediated defenses of the innate immune system.
“…Alternatively, high-mannose glycans at Asn 136 might be orientated in a manner that is prohibitive to effective recognition by MBL and SP-D. For seasonal H1N1, loss of a high-mannose glycan from Asn 104 has been associated with resistance to conglutinin, a bovine-specific collectin (58). Addition of Asn 104 and Asn 144 together, but not either site alone, to PR8 HA was associated with enhanced sensitivity to MBL and SP-D (20), suggesting that for seasonal H1 IAV both sites express glycans recognized by collectins but that multivalent interactions increase binding avidity and therefore anti-IAV activity.…”
Section: Discussionmentioning
confidence: 99%
“…Site-directed mutagenesis was used to add or remove N-linked glycosylation sites from the Auck/09 HA (20 …”
Section: Generation Of Iav Glycosylation Mutants Using Rgmentioning
confidence: 99%
“…SP-D and MBL bind to HA and NA (15,18,19), with the majority of binding to HA. Sitedirected mutagenesis and reverse genetics (RG) approaches have demonstrated the importance of particular N-glycans on the head of seasonal H1 (20) and H3 IAV (21,22) as ligands for MBL and SP-D.…”
Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the viral hemagglutinin (HA). Glycans on the head of HA promote virus survival by shielding antigenic sites, but highly glycosylated seasonal IAV are inactivated by soluble lectins of the innate immune system. In 2009, human strains of pandemic H1N1 [A(H1N1)pdm] expressed a single glycosylation site (Asn104) on the head of HA. Since then, variants with additional glycosylation sites have been detected, and the location of these sites has been distinct to those of recent seasonal H1N1 strains. We have compared wild-type and reverse-engineered A(H1N1)pdm IAV with differing potential glycosylation sites on HA for sensitivity to collectins and to neutralizing Abs. Addition of a glycan (Asn136) to A(H1N1)pdm HA was associated with resistance to neutralizing Abs but did not increase sensitivity to collectins. Moreover, variants expressing Asn136 showed enhanced growth in A(H1N1)pdm-vaccinated mice, consistent with evasion of Ab-mediated immunity in vivo. Thus, a fine balance exists regarding the optimal pattern of HA glycosylation to facilitate evasion of Ab-mediated immunity while maintaining resistance to lectin-mediated defenses of the innate immune system.
“…All research complied with the University of Melbourne's Animal Experimentation Ethics guidelines and policies. At various times postinfection, mice were culled, and samples including blood, bronchoalveolar lavage fluid (BALF), thymus, and lungs were collected as described (28,30). Plaque assay was used to determine titers of infectious virus in clarified homogenates prepared from the lungs of IAV-infected mice.…”
Section: Binding and Infection Of La-4 And Mdck Cells By Influenza VImentioning
confidence: 99%
“…Subconfluent monolayers of mouse LA-4 airway epithelial cells or MDCK cells in eight-well chamber slides (Lab-Tek; Nunc) were incubated with increasing dilutions of IAV for 1 h at 37˚C, washed three times, and incubated in serum-free media, as described (28). At 7-8 h postinfection, cells were fixed in 80% (v/v) acetone and stained with anti-NP mAb MP3.1092.IC7, followed by FITC-conjugated rabbit anti-mouse Ig (Dako).…”
Section: Binding and Infection Of La-4 And Mdck Cells By Influenza VImentioning
The long pentraxin, pentraxin 3 (PTX3), can play beneficial or detrimental roles during infection and disease by modulating various aspects of the immune system. There is growing evidence to suggest that PTX3 can mediate antiviral activity in vitro and in vivo. Previous studies demonstrated that PTX3 and the short pentraxin serum amyloid P express sialic acids that are recognized by the hemagglutinin (HA) glycoprotein of certain influenza A viruses (IAV), resulting in virus neutralization and anti-IAV activity. In this study, we demonstrate that specificity of both HA and the viral neuraminidase for particular sialic acid linkages determines the susceptibility of H1N1, H3N2, and H7N9 strains to the antiviral activities of PTX3 and serum amyloid P. Selection of H3N2 virus mutants resistant to PTX3 allowed for identification of amino acid residues in the vicinity of the receptor-binding pocket of HA that are critical determinants of sensitivity to PTX3; this was supported by sequence analysis of a range of H3N2 strains that were sensitive or resistant to PTX3. In a mouse model of infection, the enhanced virulence of PTX3-resistant mutants was associated with increased virus replication and elevated levels of proinflammatory cytokines in the airways, leading to pulmonary inflammation and lung injury. Together, these studies identify determinants in the viral HA that can be associated with sensitivity to the antiviral activities of PTX3 and highlight its importance in the control of IAV infection.
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