A dual reporter gene based system to quantitate the cell fusion of avian influenza virus H5N1

Su, Yan; Yang, Hongzhou; Zhang, Baojiang; Qi, Xiaoxuan; Tien, Po

doi:10.1007/s10529-007-9521-4

Cited by 5 publications

(8 citation statements)

References 20 publications

(12 reference statements)

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“…Our results indicated that the luciferase-based cell–cell fusion activity triggered by the DENV2 E protein was demonstrated to be feasible, but the luciferase expression levels were significantly lower compared to those reported for HCV E1E2 and Influenza virus HA proteins. 24 , 25 One possibility is that DENV prME may be expressed poorly on the cell surface. DENV contains an endoplasmic reticulum (ER) retention signal on the TM domain of the E protein that can cause prME proteins to remain mainly in the ER.…”

Section: Resultsmentioning

confidence: 99%

A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion

Lin

Huang

2020

Human Vaccines & Immunotherapeutics

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The class II membrane fusion induced by flavivirus E proteins is a unique pH-dependent membrane fusion process differently from the class I or III membrane fusion by other enveloped virus proteins. The fusion peptide on the DII of the flavivirus E proteins can insert into the cell membrane as a cell entry process besides the receptor bindings. A traditional assay using C6/36 mosquito cells infected by dengue viruses has been previously reported but did not provide efficient quantitation to measure the virus-triggered membrane fusion. Here we reported the development of a quantitative cell fusion assay for four serotypes of dengue viruses and the recently emerged Zika viruses. We used a pCI-neo vector encoding the prME genes of dengue and Zika viruses and investigated the cell fusion in transfected 293, Vero and CHO cells. Donor cells were co-transfection of the prME genes of dengue and Zika prME gene and T7 RNA polymerase to react with the indicator cells transfected with the luciferase gene under the control of the T7 promoter. Quantification of the virus-induced cell fusion was determined by the luciferase expression levels under a switch of pH from 7.4 to 5.4 in the co-cultured donor and indicator cells. The quantitative luciferase-based assay was applied to measure the anti-fusion activity by two monoclonal antibodies mAb 4G2 and mAb DB42 against dengue virus infections. This assay could quality as a quantitative bioassay for testing the potency of anti-fusion monoclonal antibodies.

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Section: Resultsmentioning

confidence: 99%

A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion

Lin

Huang

2020

Human Vaccines & Immunotherapeutics

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“…Fusion inhibition activity against pH1N1 and H5N1 viruses were determined by luciferase-based cell-cell fusion assays as previously described (32,33). In brief, effector cells (2 ϫ 10 4 293T cells in 96-well plates) and target cells (9 ϫ 10 5 293T cells in 6-well plates) were incubated overnight, followed by the transfection of pcDNA3.1-H5HA (KAN-1) or pcDNA3.1-pH1HA (RG-121) and pCAGT7pol plasmids into effector cells using TurboFect reagent (Thermo).…”

Section: Methodsmentioning

confidence: 99%

“…To examine fusion inhibition activity in antisera, we used luciferase reporter gene-based fusion assays to quantitate HA-induced pH-dependent cell-cell fusion levels as previously described (32,33). H5 WT-, H5 N484A-, H1 WT-, and H1 N503A-immunized sera were reacted with firefly and Renilla luciferase reporter gene-transfected 293T cells for 48 h at 37°C and examined for cell-cell fusion activity induced by H5HA [A/Thailand/1(KAN-1)/2004, KAN-1] or pH1HA (A/California/ 07/2009, RG-121).…”

Section: Stemmentioning

confidence: 99%

Unmasking Stem-Specific Neutralizing Epitopes by Abolishing N-Linked Glycosylation Sites of Influenza Virus Hemagglutinin Proteins for Vaccine Design

Liu

Jan

Huang

et al. 2016

J Virol

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Influenza virus hemagglutinin (HA) protein consists of two components, i.e., a globular head region and a stem region that are folded within six disulfide bonds, plus several N-linked glycans that produce a homotrimeric complex structure. While N-linked glycosylation sites on the globular head are variable among different strains and different subtypes, N-linked glycosylation sites in the stem region are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Since the HA stem region is constituted by an HA1 N-terminal part and a full HA2 part, we expressed a series of recombinant HA mutant proteins with deleted N-linked glycosylation sites in the HA1 stem and HA2 stem regions of H5N1 and pH1N1 viruses. Unmasking N-glycans in the HA2 stem region (H5 N484A and H1 N503A) was found to elicit more potent neutralizing antibody titers against homologous, heterologous, and heterosubtypic viruses. Unmasking the HA2 stem N-glycans of H5HA but not H1HA resulted in more CR6261-like and FI6v3-like antibodies and also correlated with the increase of cell fusion inhibition activity in antisera. Only H5 N484A HA2 stem mutant protein immunization increased the numbers of antibody-secreting cells, germinal center B cells, and memory B cells targeting the stem helix A epitopes in splenocytes. Unmasking the HA2 stem N-glycans of H5HA mutant proteins showed a significantly improvement in the protection against homologous virus challenges but did so to a less degree for the protection against heterosubtypic pH1N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.

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“…Reporter fusion assay. The assay was performed as described previously (Su et al, 2008). 293T cells were transfected using Nanofectin (PAA Laboratories) with pDZ expression plasmids encoding HA (2 mg), pTM1-FFLuc encoding firefly luciferase (FF-Luc) under the control of the T7 promoter (1 mg) and pRL-SV40 encoding renilla-luciferase (Ren-Luc) under the control of the simian virus 40 (SV40) promoter (50 ng).…”

Section: Methodsmentioning

confidence: 99%

“…To further analyse the fusogenic activity of HA, a fusion assay was established using transfected HA-expressing cells (Su et al, 2008). To this end, 293T cells were co-transfected with expression constructs for HA and a T7-driven Human erythrocytes were incubated with lvPR8 and hvPR8 viruses, respectively, or were mock treated.…”

Section: Fusion Activity Of Hvpr8mentioning

confidence: 99%

Altered receptor specificity and fusion activity of the haemagglutinin contribute to high virulence of a mouse-adapted influenza A virus

Koerner

Matrosovich

Haller

et al. 2012

Journal of General Virology

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The viral haemagglutinin (HA) and the viral polymerase complex determine the replication fitness of a highly virulent variant of influenza A virus strain A/PR/8/34 (designated hvPR8) and its high pathogenicity in mice. We report here that the HA of the hvPR8 differs from the HA of a low virulent strain (lvPR8) by the efficiency of receptor binding and membrane fusion. hvPR8 bound to 2,6-linked as well as 2,3-linked sialic acid-containing receptors, whereas lvPR8 bound exclusively to 2,3-linked sialic acids with high avidity. Remarkably, hvPR8 infected its target cells faster than lvPR8 and tolerated an elevated pH for efficient membrane fusion. In spite of these differences, both viruses targeted type II but not type I pneumocytes in the lung of infected mice. The HA of hvPR8 differs from that of lvPR8 by 16 aa substitutions and one insertion. Mutational analyses revealed that amino acid at HA position 190 (H3 numbering) primarily determined the specificity of receptor binding, while the insertion at position 133 influenced the avidity of receptor binding. Both amino acid positions also strongly influenced viral virulence. Furthermore, leucine at position 78 and glutamine at position 354 were critical determinants of increased fusion activity and virulence of hvPR8. Our data suggest that the HA of hvPR8 enhances virulence by mediating optimal receptor binding and membrane fusion thereby promoting rapid and efficient viral entry into host cells.

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A dual reporter gene based system to quantitate the cell fusion of avian influenza virus H5N1

Cited by 5 publications

References 20 publications

A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion

A quantitative luciferase-based cell–cell fusion assay to measure four-serotype dengue virus E protein-triggered membrane fusion

Unmasking Stem-Specific Neutralizing Epitopes by Abolishing N-Linked Glycosylation Sites of Influenza Virus Hemagglutinin Proteins for Vaccine Design

Altered receptor specificity and fusion activity of the haemagglutinin contribute to high virulence of a mouse-adapted influenza A virus

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