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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

Syncytium formation in monolayers coexpressing wt or mutated F and HN proteins.After 36 h posttransfection, BHK-21 monolayers transfected with vector alone, wt F alone, wt F and wt HN, or mutant F and wt HN were fixed with methanol and stained with Giemsa stain. (A) Photomicrographs from a representative expreriment. Red arrows indicate syncytia. (B) Quantification of syncytia produced by the mutated F proteins. Values were indicated as percentages of syncytium formation detected in cells transfected with wt F and wt HN proteins and are represented as the mean ± standard deviation (SD) from three separate experiments (*P<0.05, **P<0.01, ***P<0.001).
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pone.0136474.g002: Syncytium formation in monolayers coexpressing wt or mutated F and HN proteins.After 36 h posttransfection, BHK-21 monolayers transfected with vector alone, wt F alone, wt F and wt HN, or mutant F and wt HN were fixed with methanol and stained with Giemsa stain. (A) Photomicrographs from a representative expreriment. Red arrows indicate syncytia. (B) Quantification of syncytia produced by the mutated F proteins. Values were indicated as percentages of syncytium formation detected in cells transfected with wt F and wt HN proteins and are represented as the mean ± standard deviation (SD) from three separate experiments (*P<0.05, **P<0.01, ***P<0.001).

Mentions: We initially measured the extent of syncytium formation to evaluate an overall level of cell-cell fusion. HPIV3 wt or mutated F proteins were coexpressed in BHK-21 cells with their homologous HN proteins. After 36 h posttransfection, an inverted microscope was used to observe multinucleated giant cells and representative photomicrographs of syncytia were shown in Fig 2A. The cells expressing HPIV3 wt F protein or vector alone were used as negative controls. Under the microscope, the syncytia formed by these mutants in the presence of their homologous HN protein coexpression were not only smaller but also fewer in comparison to those produced by wt F and HN proteins. Quantification of syncytia, expressed as a percentage of those detected in cells coexpressing wt F and HN proteins, is shown in Fig 2B. When coexpressed with HN, all mutant F proteins displayed decreased levels of syncytium formation as compared with the HPIV3 wt F and HN proteins. The F proteins carrying individual K369E, S370A, V373A, V373T and P374T mutations almost abrogated their abilities to form syncytia, with extents less than 5.0% of the wt F and HN level. The remainder of the mutants in this domain (K369A, D371A, I372A and P374A) were capable of forming syncytia, though at lower levels than seen with the wt F protein.


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)

Syncytium formation in monolayers coexpressing wt or mutated F and HN proteins.After 36 h posttransfection, BHK-21 monolayers transfected with vector alone, wt F alone, wt F and wt HN, or mutant F and wt HN were fixed with methanol and stained with Giemsa stain. (A) Photomicrographs from a representative expreriment. Red arrows indicate syncytia. (B) Quantification of syncytia produced by the mutated F proteins. Values were indicated as percentages of syncytium formation detected in cells transfected with wt F and wt HN proteins and are represented as the mean ± standard deviation (SD) from three separate experiments (*P<0.05, **P<0.01, ***P<0.001).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4549179&req=5

pone.0136474.g002: Syncytium formation in monolayers coexpressing wt or mutated F and HN proteins.After 36 h posttransfection, BHK-21 monolayers transfected with vector alone, wt F alone, wt F and wt HN, or mutant F and wt HN were fixed with methanol and stained with Giemsa stain. (A) Photomicrographs from a representative expreriment. Red arrows indicate syncytia. (B) Quantification of syncytia produced by the mutated F proteins. Values were indicated as percentages of syncytium formation detected in cells transfected with wt F and wt HN proteins and are represented as the mean ± standard deviation (SD) from three separate experiments (*P<0.05, **P<0.01, ***P<0.001).
Mentions: We initially measured the extent of syncytium formation to evaluate an overall level of cell-cell fusion. HPIV3 wt or mutated F proteins were coexpressed in BHK-21 cells with their homologous HN proteins. After 36 h posttransfection, an inverted microscope was used to observe multinucleated giant cells and representative photomicrographs of syncytia were shown in Fig 2A. The cells expressing HPIV3 wt F protein or vector alone were used as negative controls. Under the microscope, the syncytia formed by these mutants in the presence of their homologous HN protein coexpression were not only smaller but also fewer in comparison to those produced by wt F and HN proteins. Quantification of syncytia, expressed as a percentage of those detected in cells coexpressing wt F and HN proteins, is shown in Fig 2B. When coexpressed with HN, all mutant F proteins displayed decreased levels of syncytium formation as compared with the HPIV3 wt F and HN proteins. The F proteins carrying individual K369E, S370A, V373A, V373T and P374T mutations almost abrogated their abilities to form syncytia, with extents less than 5.0% of the wt F and HN level. The remainder of the mutants in this domain (K369A, D371A, I372A and P374A) were capable of forming syncytia, though at lower levels than seen with the wt F protein.

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