why treatment of cells with neuraminidase would increase rbcs binding to hemagglutinin

Why are two subsets of neuraminidase differently acquired by viral immunology?

For experiments in which the effect of trypsin treatment was tested, after neuraminidase treatment, cells were washed and either mock treated or treated for 1 h ... with influenza virus HA as the binding protein (one to two RBCs per cell), we found that F protein-promoted fusion with SV5 HN as the binding protein was too rapid to measure with ...

What is the role of hemagglutinin antigen in the fight against viruses?

Jul 17, 2017 · The two surface glycoproteins of influenza A virus, hemagglutinin and neuraminidase, mediate a range of host interactions from receptor binding to viral release. As mentioned previously, the hemagglutinin binds to carbohydrates on the cell surface terminating in sialic acid. Neuraminidase, likewise, acts on sialic acid, cleaving the moiety from ...

What has been docked into the overlapped structures of hemagglutinin?

Partial receptor depletion of red blood cells was achieved by treatment of 2 ml of a 10% RBC solution in serum-free medium for 2 h at 37°C with 0 to 50 mU of Clostridium perfringens neuraminidase (type X from C. perfringens, catalog number N-2876; Sigma Scientific, St. Louis, MO) as described previously . Neuraminidase was then removed by ...

What is haemagglutination and how does it work?

Jan 17, 2007 · Partial removal of sialic acid receptors from RBCs. Partial receptor depletion of red blood cells was achieved by treatment of 2 ml of a 10% RBC solution in serum-free medium for 2 h at 37°C with 0 to 200 milliunits of Clostridium perfringens neuraminidase (type X from C. perfringens) (catalog no. N-2876; Sigma Scientific, St. Louis, MO) as described previously ().

What is the role of hemagglutinin and neuraminidase in influenza infection?

Does neuraminidase cleave hemagglutinin?

Why are hemagglutinin and neuraminidase particularly important to transmission for influenza A?

What does the neuraminidase do?

How does a neuraminidase inhibitor work?

Are hemagglutinin and neuraminidase receptors?

How does Tamiflu inhibit neuraminidase?

What increases the possibility of antigenic shift in influenza virus?

How does hemagglutinin bind to sialic acid?

What is the function of hemagglutinin HA and neuraminidase NA )?

Is neuraminidase a virulence factor?

What are the functions of hemagglutinin-neuraminidase?

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three different activities: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. These three discrete properties each affect the ability of HN to promote viral fusion and entry. For human parainfluenza type 3, one bifunctional site on HN can carry out both binding and neuraminidase, and the receptor mimic, zanamivir, impairs viral entry by blocking receptor binding. We report here that for Newcastle disease virus, the HN receptor avidity is increased by zanamivir, due to activation of a second site that has higher receptor avidity. Only certain receptor mimics effectively activate the second site (site II) via occupation of site I; yet without activation of this second site, binding is mediated entirely by site I. Computational modeling designed to complement the experimental approaches suggests that the potential for small molecule receptor mimics to activate site II, upon binding to site I, directly correlates with their predicted strengths of interaction with site I. Taken together, the experimental and computational data show that the molecules with the strongest interactions with site I—zanamivir and BCX 2798—lead to the activation of site II. The finding that site II, once activated, shows higher avidity for receptor than site I, suggests paradigms for further elucidating the regulation of HN′s multiple functions in the viral life cycle.

What is the second binding site of NDV?

The second binding site on NDV HN, revealed by cocrystallization with thiosialoside ( 28 ), was found to be located on the dimer interface of the molecule and made up of hydrophobic residues from both monomers. The involvement of the Arg516 side chain in the interaction with sialic acid suggested that Arg516, a residue that is strongly conserved among NDV isolates, is important for the function of site II ( 28 ). Mutations at this residue (R516A or R516S) result in HNs that, when coexpressed with F, are less efficient in fusion promotion ( 3 ), suggesting that site II may play a role in the HN fusion promotion function during viral entry ( 3, 28 ). However, these HNs mutated at site II (R516A or R516S) were found to bind RBCs similarly to WT HN in hemagglutination assays, a finding previously interpreted to mean that site II did not contribute to receptor-binding activity ( 3 ). The present finding that the NDV HN receptor-binding avidity is considerably increased with activation of site II by zanamivir occupation of site I suggests that site II may in fact contribute to receptor binding at specific times in the viral entry process.

What is the second site of NDV HN?

The second receptor-binding site on NDV HN, located at the dimer interface, becomes activated when the globular head-binding site is occupied by zanamivir, a transition state analog of sialic acid, but does not become activated by the receptor analog 2,3-sialyllactose. Two possible explanations may be proposed for these findings. The lower binding avidity of 2,3-sialyllactose for site I, suggested by molecular modeling and calculated free energies of binding (Table#N#​ (Table1),#N#1 ), may be below the threshold required for activation of site II. A more interesting possibility is that generation of a transition state compound in active site I during receptor interaction leads to the activation of site II. Such a reaction intermediate, which would resemble zanamivir rather than 2,3-sialyllactose, may activate site II under physiological conditions. In this case, one could envision that after initial binding of NDV-HN via its bifunctional site I to a new cell and initiation of neuraminidase cleavage of receptor moieties, the second site, which binds more avidly but lacks neuraminidase, becomes activated. The timing would be propitious; at the time of viral entry, avid binding and fusion with the target cell are needed.

What is 293T in a cell?

293T (human kidney epithelial) cells were grown in Dulbecco's modification of Eagle's medium (Mediatech Cellgro) supplemented with 10% fetal bovine serum and antibiotics. For assays of cells in air at different pHs, the above medium was replaced with a CO 2 -independent medium (Invitrogen) which was adjusted following the manufacturer's instructions to the indicated pH ( 21 ).

Does NDV HN have avidity?

We have previously developed a quantitative receptor avidity assay ( 20, 25) and shown that NDV HN has low avidity for receptors on RBCs (either human or avian) compared to the HPIV3 HNs ( 24 ). This receptor avidity assay was then used to specifically measure the avidity of NDV HN site II and compare it to the avidity of site I. In this assay, receptor-depleted RBCs are bound to HN-expressing cells in a quantitative HAD (the higher the avidity, the greater receptor depletion is required to reduce binding). At 4°C, neither NDV nor HPIV3 neuraminidases are active and therefore cannot contribute to RBC release. Based on these results, in the presence of zanamivir, HAD should result from site II binding only, allowing its avidity to be assessed.

Who is the FDA for recombinant HA?

We would like to thank Dr. Maryna Eichelberger (FDA) and Dr. Manju Joshi (FDA) for providing the recombinant HA and members of the Division of Viral Products at the Center for Biologics Evaluation and Research for offering several helpful suggestions and insightful discussions.

How to address NA abundance in H1N1?

Our results demonstrate that the bottleneck of NA abundance in H1N1 virions can be addressed by changing the internal gene backbone or the polymerase gene segments in the CVVs. This approach preserves the NA and HA antigens and the increase in NA content induced higher NAI titres without significantly reducing the HAI titres, resulting in a more balanced NA and HA antibody responses. The improved balance against both antigens was more evident in the single immunizations with the lowest virion amounts, suggesting the initial NA antibody response in mice is limited by a quantity threshold rather than HA immunodominance. Future challenge experiments using immunizations with lower virion amounts will help to determine if the increase in NA content can decrease the antigen amount required for protection, and to define the NA antigen amounts that correlate with protection from strains with unmatched HAs.

What are the two reassortant backbones?

The two single-gene reassortant virus backbones (WSN and PR8) encode for distinct HAs raising the question of whether the observed increase in the virion NA content is dependent on HA due to potential changes in the functional balance. Therefore, we generated two double-gene reassortant viruses where the HA genes were swapped while the NA (N1-BR18) remained identical ( Fig 3A ). In eggs, the virus with the WSN backbone and the HA from PR8 (WSN H1-PR8/N1-BR18) produced higher NA levels and slightly better HAU titres than the virus with the HA from WSN and the PR8 backbone ( Fig 3B ). Following purification, bands corresponding to oxidized and reduced NA were apparent for both viruses on Coomassie stained gels ( Fig 3C) and immunoblots ( Fig 3D ), which confirmed the NA levels were slightly higher in the WSN H1-PR8/N1-BR18 virions. The virion HA content was rather consistent, but somewhat lower M1 levels were observed in the WSN H1-PR8/N1-BR18 virions. In line with the Coomassie gel, WSN H1-PR8/N1-BR18 virions possessed ~15% more NA activity than PR8 H1-WSN/N1-BR18 virions, whereas the HAU titres were more than double ( Fig 3C ). These results implied that HA and one or more WSN backbone gene products support increased viral incorporation of recent N1s and that the HA from PR8 generates higher HAU titres with turkey red blood cells than the HA from WSN.

Does WSN cause higher NA?

To determine if a specific WSN backbone gene segment contributes to the higher NA virion incorporation, a series of PR8 N1-BR18 reassortant viruses carrying a different WSN internal gene segment were created and tested in eggs. Only the virus containing the PB1 gene segment from WSN produced higher NA levels and it also showed no observable loss in HAU titres ( Fig 3E ), indicating the viral polymerase from WSN likely contributes to the higher NA content in the virions. A similar analysis of a PR8 N1-BR18 reassortant virus carrying all the WSN polymerase subunits (PB1, PB2 and PA) together with NP, confirmed that the polymerase from WSN is largely responsible for the higher NA content in virions containing the WSN backbone ( Fig 3F ).

Is WSN a backbone?

Although WSN is a commonly used highly adapted lab strain like PR8, it has not received much consideration as a CVV backbone because of many reports showing it is neurovirulent in mice [ 48 – 50 ]. However, the neurovirulence trait has been linked to the NA in WSN [ 51, 52 ], requires intracerebral inoculation [ 48 ], and in CVVs both the HA and NA gene segments are replaced during generation. In addition, we have observed no clinical symptoms ( e. g. fever, weight loss, nasal symptoms or low energy) in ferrets that were inoculated with a series of identical HA and NA double-gene WSN and PR8 reassortants for another study and our results suggest polymerase components alone may be sufficient. We are not certain that using classical reassortment to create a CVV with a WSN backbone will improve viral growth and retain the higher NA virion content, but our results showing that the NA virion content is higher than an approved CVV and the viral protein yields are on par with the WT field isolate that is used to benchmark related CVVs, suggest further investigation is warranted.

What antibodies inhibit fusion?

Stem-binding neutralizing antibodies have been postulated to inhibit the fusion process based on their interaction with the HA2 subunit and lack of activity in hemagglutination-inhibition (HAI) assays, which specifically detect antibodies that interfere with attachment of the virus to sialic acid receptors. Indirect evidence supporting this notion comes from biochemical studies showing that such antibodies can block the conformational changes of recombinant HA required for membrane fusion [6], [8], [10], or prevent the formation of syncytia in HA-expressing cells [7], [22]. Such a mechanism of action implies that these antibodies are internalized together with the virus and reach late endosomes, but this has so far not been shown. By using fluorescence single particle tracking methods we investigated the fate of viral particles and bound antibodies during infection of live cells ( Figure 1A) [23]. Movies of cells incubated with fluorescently labeled CR8020 mixed with H3N2 virus, and CR6261 mixed with H1N1 virus (CR8020 and CR6261 specifically bind Group 2 and Group 1 influenza A viruses, respectively. Table S1 ), reveal that stem-binding antibodies are indeed internalized in complex with the virus and transported along the microtubule cytoskeleton ( Figure 1B, 1C; Movies S1, S2 ). The joint and directed movement of internalized viruses and bound antibodies is evident from their high degree of co-localization over consecutive frames. This behavior was exclusively observed for viruses and bound stem-binding antibodies since head-binding antibodies prevent viral internalization to begin with and no evidence for the internalization of unbound antibody could be found ( Figure S1A, S1B; Movies S3, S5 ). Furthermore, pulse-labeling with a dye sensitive for low-pH vesicles, combined with single particle tracking in cells, demonstrated that virus-antibody complexes reach acidic late endosomes ( Figure 1D, 1E ). Prolonged tracking of cells that had internalized virus-antibody complexes allowed us to determine their individual fate. Following a pre-incubation with H3N2 virus, stem-binding bnAb CR8020 was observed co-localizing with viral particles inside cells at early time points ( Figure 1F, Table S2 ). Cells were imaged every 30 minutes for 15 hours after which they were fixed and probed for the expression of influenza nucleoprotein (NP), which was used as an indicator for infection. In cells that had internalized CR8020 in complex with the virus, no NP expression was detected ( Figure 1G ), indicating that the bound bnAb successfully prevented infection. In contrast, a comparable number of particles led to full infection in the control experiment in which H3N2 virus had been pre-incubated with a non-binding control antibody ( Figure 1H, 1I ). Similar results were obtained for the inhibition of infection by H1N1 virus following pre-incubation with CR6261 ( Figure S2 ).

How do monoclonal antibodies work?

Human monoclonal antibodies have been identified which neutralize broad spectra of influenza A or B viruses. Here, we dissect the mechanisms by which such antibodies interfere with infectivity. We distinguish four mechanisms that link the conserved hemagglutinin (HA) epitopes of broadly neutralizing antibodies to critical processes in the viral life cycle. HA-stem binding antibodies can act intracellularly by blocking fusion between the viral and endosomal membranes and extracellularly by preventing the proteolytic activation of HA. HA-head binding antibodies prevent viral attachment and release. These insights into newly identified ways by which the human immune system can interfere with influenza virus infection may aid the development of novel universal vaccines and antivirals.

Can calu3 cells be infected with trypsin?

Calu-3 cells support the propagation of influenza virus in the absence of trypsin, but cannot be infected by uncleaved virus. ( A) Calu-3 cells were infected with 10 TCID 50 cleaved A/Wisconsin/67/2005 (H3N2) influenza virus in the absence of trypsin. 24 hours after infection cells (nuclei blue) were fixed and stained for influenza NP (green) as indication for infection. ( B) 100 TCID 50 of uncleaved A/Wisconsin/67/2005 and A/Brisbane/59/2007 (harvested from MDCK cells in the absence of trypsin) were added to Calu-3 cells with or without trypsin. Uncleaved virus is not infectious but can be rendered infectious when treated with trypsin. Images (A and B) show an entire well.

Is R18 virus infectious?

R18 labeled influenza virus remain infectious. MDCK cells were infected with R18- or MOCK-labeled A/Puerto Rico/8/1934 (H1N1) or A/Aichi/2/1968-X31 (H3N2) and the number of infected cells (nucleus stained with DAPI, blue) for each virus was determined by staining for influenza NP expression (green).

What is the purpose of neuraminidase?

The main function of the Neuraminidase (NA) might be to remove receptors for influenza virus from newly formed virus particles so allowing these to be released and spread the infection. Another function of flu virus neuraminidase might be to destroy sialic acid containing inhibitors for the virus in the mucous secretions of the respiratory tract, so enabling the virus to more easily infect cells, and there may be other functions as yet undiscovered.

What is the stem N-glycan?

Studies have revealed that the stem N-glycans of HA is crucial for virus replication. In both culture systems, growth of virus lacking this glycans was completely blocked at 37 °C and inhibited at 33 °C. It is concluded that the stem N-glycans is indispensable for the formation of replication-competent influenza viruses.