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.
Highlights
Human parainfluenza virus type 3 (HPIV3) is an important pathogen of viral upper and lower respiratory tract illnesses such as bronchiolitis, croup, and pneumonia that require hospitalization all over the world in children aged younger than 5 years [1,2,3,4]
The results showed that all mutant proteins in this domain which still acted as the DI-DII linker in the prefusion form detected at the cell surface were present at approximately wild-type levels, but they were deficient in fusion activity in all steps of the fusion process when coexpressed with their homologous HN proteins
To study the role of this domain in fusion activity of F protein, the prefusion HPIV3 F protein structural model was generated to identify the new location of this linker in it, and site-directed mutagenesis was utilized to replace each amino acid in this domain to form nine single mutations
Summary
Human parainfluenza virus type 3 (HPIV3) is an important pathogen of viral upper and lower respiratory tract illnesses such as bronchiolitis, croup, and pneumonia that require hospitalization all over the world in children aged younger than 5 years [1,2,3,4]. Fusion of the viral envelope with a host cell membrane is an essential step for its entry into the cytoplasm of the target cells to cause virus spread and the cell-cell fusion. It has evolved a membrane fusion mechanism that commonly involved two surface glycoproteins: the hemagglutinin-neuraminidase (HN) and the fusion (F) protein [6]. The HN protein is a type II tetramer glycoprotein that carries out three discrete but critical functions (receptor recognition, neuraminidase and activating the F protein to induce membrane fusion) at specific points in the process of viral entry [7,8,9]. The F protein is thought to directly execute membrane fusion after its homologous HN protein binding to a host receptor [10]
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