Limits...
Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity.

Xie W, Wen H, Chu F, Yan S, Lin B, Xie W, Liu Y, Ren G, Zhao L, Song Y, Sun C, Wang Z - PLoS ONE (2015)

Bottom Line: Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F.Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface.Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, School of Public Health, Shandong University, Jinan, China.

ABSTRACT
Human parainfluenza virus type 3 (HPIV3) can cause severe respiratory tract diseases in infants and young children, but no licensed vaccines or antiviral agents are currently available for treatment. Fusing the viral and target cell membranes is a prerequisite for its entry into host cells and is directly mediated by the fusion (F) protein. Although several domains of F are known to have important effects on regulating the membrane fusion activity, the roles of the DI-DII linker (residues 369-374) of the HPIV3 F protein in the fusogenicity still remains ill-defined. To facilitate our understanding of the role of this domain might play in F-induced cell-cell fusion, nine single mutations were engineered into this domain by site-directed mutagenesis. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated F proteins. These mutants were analyzed for membrane fusion activity, cell surface expression, and interaction between F and HN protein. Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F. All of them resulted in a significant reduction in fusogenic activity in all steps of membrane fusion. Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface. Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F.

No MeSH data available.


Related in: MedlinePlus

Cell surface expression (CSE) of the mutant F proteins.Cell surface expression was measured by flow cytometry with an anti-F monoclonal antibody followed with an anti-mouse Alexa Fluor 488-conjugated antibody. (A) Representative fluorescent histograms for each mutant were shown. The x axis indicates the fluorescent intensity values shown in log scale and the y axis represents cell counts, respectively. The mean fluorescence intensity (MFI) of each mutant was calculated and listed below. (B) Quantitation of the cell surface expression level. The mean fluorescence intensity of cells expressing the mutated F proteins was represented as a percentage of that of the wt F subtracting background labeling of control cells transfected with the vector alone. The means and standard errors are from triplicate experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4549179&req=5

pone.0136474.g006: Cell surface expression (CSE) of the mutant F proteins.Cell surface expression was measured by flow cytometry with an anti-F monoclonal antibody followed with an anti-mouse Alexa Fluor 488-conjugated antibody. (A) Representative fluorescent histograms for each mutant were shown. The x axis indicates the fluorescent intensity values shown in log scale and the y axis represents cell counts, respectively. The mean fluorescence intensity (MFI) of each mutant was calculated and listed below. (B) Quantitation of the cell surface expression level. The mean fluorescence intensity of cells expressing the mutated F proteins was represented as a percentage of that of the wt F subtracting background labeling of control cells transfected with the vector alone. The means and standard errors are from triplicate experiments.

Mentions: To investigate whether the lack of fusion activity of these mutated F proteins was ascribed to their reduced levels of cell surface expression, fluorescence-activated cell sorting (FACS) analysis was executed with an anti-F monoclonal antibody as described in Materials and Methods to examine the relative cell surface expression levels of these mutated F proteins. Expression level was quantified by the mean fluorescence intensity per cell and expressed as a percentage of that of the wt F subtracting background labeling of control cells transfected with the vector alone. Fluorescent histograms of the wt or mutated F proteins were shown in Fig 6A. Quantitation of the cell surface expression, expressed as a percentage of the wt F protein level, are summarized in Fig 6B. Each of the mutated proteins gave a mean fluorescent intensity that was similar to that of the wt F protein, ranging from 93.1% to 105.9% of the wt F level. Therefore, the loss of fusion activity was not due to the diminished cell surface expression level.


Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity.

Xie W, Wen H, Chu F, Yan S, Lin B, Xie W, Liu Y, Ren G, Zhao L, Song Y, Sun C, Wang Z - PLoS ONE (2015)

Cell surface expression (CSE) of the mutant F proteins.Cell surface expression was measured by flow cytometry with an anti-F monoclonal antibody followed with an anti-mouse Alexa Fluor 488-conjugated antibody. (A) Representative fluorescent histograms for each mutant were shown. The x axis indicates the fluorescent intensity values shown in log scale and the y axis represents cell counts, respectively. The mean fluorescence intensity (MFI) of each mutant was calculated and listed below. (B) Quantitation of the cell surface expression level. The mean fluorescence intensity of cells expressing the mutated F proteins was represented as a percentage of that of the wt F subtracting background labeling of control cells transfected with the vector alone. The means and standard errors are from triplicate experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549179&req=5

pone.0136474.g006: Cell surface expression (CSE) of the mutant F proteins.Cell surface expression was measured by flow cytometry with an anti-F monoclonal antibody followed with an anti-mouse Alexa Fluor 488-conjugated antibody. (A) Representative fluorescent histograms for each mutant were shown. The x axis indicates the fluorescent intensity values shown in log scale and the y axis represents cell counts, respectively. The mean fluorescence intensity (MFI) of each mutant was calculated and listed below. (B) Quantitation of the cell surface expression level. The mean fluorescence intensity of cells expressing the mutated F proteins was represented as a percentage of that of the wt F subtracting background labeling of control cells transfected with the vector alone. The means and standard errors are from triplicate experiments.
Mentions: To investigate whether the lack of fusion activity of these mutated F proteins was ascribed to their reduced levels of cell surface expression, fluorescence-activated cell sorting (FACS) analysis was executed with an anti-F monoclonal antibody as described in Materials and Methods to examine the relative cell surface expression levels of these mutated F proteins. Expression level was quantified by the mean fluorescence intensity per cell and expressed as a percentage of that of the wt F subtracting background labeling of control cells transfected with the vector alone. Fluorescent histograms of the wt or mutated F proteins were shown in Fig 6A. Quantitation of the cell surface expression, expressed as a percentage of the wt F protein level, are summarized in Fig 6B. Each of the mutated proteins gave a mean fluorescent intensity that was similar to that of the wt F protein, ranging from 93.1% to 105.9% of the wt F level. Therefore, the loss of fusion activity was not due to the diminished cell surface expression level.

Bottom Line: Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F.Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface.Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, School of Public Health, Shandong University, Jinan, China.

ABSTRACT
Human parainfluenza virus type 3 (HPIV3) can cause severe respiratory tract diseases in infants and young children, but no licensed vaccines or antiviral agents are currently available for treatment. Fusing the viral and target cell membranes is a prerequisite for its entry into host cells and is directly mediated by the fusion (F) protein. Although several domains of F are known to have important effects on regulating the membrane fusion activity, the roles of the DI-DII linker (residues 369-374) of the HPIV3 F protein in the fusogenicity still remains ill-defined. To facilitate our understanding of the role of this domain might play in F-induced cell-cell fusion, nine single mutations were engineered into this domain by site-directed mutagenesis. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated F proteins. These mutants were analyzed for membrane fusion activity, cell surface expression, and interaction between F and HN protein. Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F. All of them resulted in a significant reduction in fusogenic activity in all steps of membrane fusion. Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface. Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F.

No MeSH data available.


Related in: MedlinePlus