Kinetics of fusion and lipid transfer between virus receptor-containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay

Wunderli‐Allenspach, Heidi; Ott, Susanne

doi:10.1021/bi00460a005

Cited by 32 publications

(16 citation statements)

References 29 publications

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“…The lipid-mixing assay may indicate that the hairpin conformation, which is the point at which lipid mixing occurs, is achieved in the presence of these antibodies and that palivizumab and motavizumab block the transition from a hairpin conformation to a postfusion conformation. However, several reports suggest that R18 can passively transfer from virus to target cell by shear proximity of the membranes (5,36,46,54,55). Therefore, it is possible that R18 may transfer from virus to target cell upon the initial interaction of the F protein with the target cell membrane or the prehairpin conformation.…”

Section: Discussionmentioning

confidence: 99%

Respiratory Syncytial Virus-Neutralizing Monoclonal Antibodies Motavizumab and Palivizumab Inhibit Fusion

Huang¹,

Incognito²,

Cheng³

et al. 2010

J Virol

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Respiratory syncytial virus (RSV) is a major cause of virus-induced respiratory disease and hospitalization in infants. Palivizumab, an RSV-neutralizing monoclonal antibody, is used clinically to prevent serious RSV-related respiratory disease in high-risk infants. Motavizumab, an affinity-optimized version of palivizumab, was developed to improve protection against RSV. These antibodies bind RSV F protein, which plays a role in virus attachment and mediates fusion. Determining how these antibodies neutralize RSV is important to help guide development of new antibody drugs against RSV and, potentially, other viruses. This study aims to uncover the mechanism(s) by which palivizumab and motavizumab neutralize RSV. Assays were developed to test the effects of these antibodies at distinct steps during RSV replication. Pretreatment of virus with palivizumab or motavizumab did not inhibit virus attachment or the ability of F protein to interact with the target cell membrane. However, pretreatment of virus with either of these antibodies resulted in the absence of detectable viral transcription. These results show that palivizumab and motavizumab act at a point after F protein initiates interaction with the cell membrane and before virus transcription. Palivizumab and motavizumab also inhibited F protein-mediated cell-to-cell fusion. Therefore, these results strongly suggest that these antibodies block both cell-to-cell and virus-to-cell fusion, since these processes are likely similar. Finally, palivizumab and motavizumab did not reduce viral budding. Based on models developed from numerous studies of viral fusion proteins, our results indicate that these antibodies may prevent conformational changes in F protein required for the fusion process.

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Section: Discussionmentioning

confidence: 99%

Respiratory Syncytial Virus-Neutralizing Monoclonal Antibodies Motavizumab and Palivizumab Inhibit Fusion

Huang¹,

Incognito²,

Cheng³

et al. 2010

J Virol

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“…The dequenching signal of R18 fluorescence was measured 60 seconds after acidification with QuantaMaster QM-4SE spectrofluorometer (Photon Technology International, Lawrenceville, NJ, USA). The initial fluorescence of virus-liposome mixtures was set at 0% fusion, and the 100% fusion value was obtained by detergent lysis for each experiment using 0.1% of Triton X-100 [38]. …”

Section: Methodsmentioning

confidence: 99%

Targeting lentiviral vector to specific cell types through surface displayed single chain antibody and fusogenic molecule

Lei

Joo

Zarzar

et al. 2010

Virol J

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BackgroundViral delivery remains one of the most commonly used techniques today in the field of gene therapy. However, one of the remaining hurdles is the off-targeting effect of viral delivery. To overcome this obstacle, we recently developed a method to incorporate an antibody and a fusogenic molecule (FM) as two distinct molecules into the lentiviral surface. In this report, we expand this strategy to utilize a single chain antibody (SCAb) for targeted transduction.ResultsTwo versions of the SCAb were generated to pair with our various engineered FMs by linking the heavy chain and the light chain variable domains of the anti-CD20 antibody (αCD20) via a GS linker and fusing them to the hinge-CH2-CH3 region of human IgG. The resulting protein was fused to either a HLA-A2 transmembrane domain or a VSVG transmembrane domain for anchoring purpose. Lentiviral vectors generated with either version of the SCAb and a selected FM were then characterized for binding and fusion activities in CD20-expressing cells.ConclusionCertain combinations of the SCAb with various FMs could result in an increase in viral transduction. This two-molecule lentiviral vector system design allows for parallel optimization of the SCAb and FMs to improve targeted gene delivery.

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“…At pH 7.4, fluorescence of R18 began to increase gradually after 3 min of incubation. But this increase in the fluorescence was a result of nonspecific transfer of R18 as stated previouly by others [25]. Therefore the increase in the fluorescence during the first 3 min was considered to be a result of membrane fusion induced by HA.…”

Section: Fusion Activities Of Mutant Hasmentioning

confidence: 77%

The role of acidic residues in the ?fusion segment? of influenza A virus hemagglutinin in low-pH-dependent membrane fusion

Nobusawa

Hishida

Murata

et al. 1995

Archives of Virology

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To clarify the role of acidic amino acid residues in the "fusion segment" of hemagglutinin (HA) of influenza A virus (H1N1) in pH-dependent membrane fusion, we have constructed and expressed five mutant HA cDNAs in CV-1 cells by SV40-HA virus vectors (SVHA). Fusion activities of the five mutant HAs were examined by lipid mixing and polykaryon formation assays. In spite of the substitution of Gly and Lys for the acidic residues, all the mutants were found to retain their low-pH-dependent fusion activity by lipid mixing assay. Although SVHA-G19(HA(2)19D-->G), -K11 (HA(2)11E-->K) and -K19(HA(2)19D-->K) induced polykaryon formation at low pH as wild type HA did, SVHA-G11(HA(2)11E-->G) induced limited polykaryon formation and SVHA-G11,19 (HA(2)11E-->G, 19D-->G) did not. The substitution of Gly for Glu at position 11 inhibited widening of the initial fusion pore. However, Lys mutants induced the formation of an initial fusion pore and widened it at low pH where Lys residues might have positive charges. These results suggest that the neutralization of the charges on acidic residues in the "fusion segment" at low pH is not important for interaction of the "fusion segment" with the target lipid bilayer or for triggering the membrane fusion.

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Kinetics of fusion and lipid transfer between virus receptor-containing liposomes and influenza viruses as measured with the octadecylrhodamine B chloride assay

Cited by 32 publications

References 29 publications

Respiratory Syncytial Virus-Neutralizing Monoclonal Antibodies Motavizumab and Palivizumab Inhibit Fusion

Respiratory Syncytial Virus-Neutralizing Monoclonal Antibodies Motavizumab and Palivizumab Inhibit Fusion

Targeting lentiviral vector to specific cell types through surface displayed single chain antibody and fusogenic molecule

The role of acidic residues in the ?fusion segment? of influenza A virus hemagglutinin in low-pH-dependent membrane fusion

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