Parvovirus H-1 NS1 Protein and ROS-Induced Apoptosis
The study shows that the nonstructural protein NS1 of Parvovirus H-1 triggers apoptosis (programmed cell death) in infected cells. This process is driven by an increase in reactive oxygen species (ROS) within the cells. The accumulation of ROS leads to cellular stress, ultimately resulting in apoptosis. The research highlights the role of NS1 in regulating cell death mechanisms through ROS production.
Culture & Maintenance
We use different cell lines to study the NS1 protein. These are the main cells we work with:
- HEK-293 T-REx Flp-In Cells:These cells are grown in DMEM with tetracycline-free fetal bovine serum, L-glutamine, penicillin, and streptomycin. We also add Blasticidin and Zeocin for selection.
- HeLa Cells : These are cultured in DMEM with similar additives, and we use Blasticidin and Zeocin to select for stable cell lines expressing the NS1 protein.
Plasmid Generation:
Plasmids such as pcDNA5-NS1-H1 and pcDNA4/TO-Flag-HA-NS1-H1 are used to express NS1. These plasmids are generated by PCR and cloning methods to ensure accurate expression.
Generation of Stable Cell Lines:
Cells are transfected with plasmids containing the NS1 gene. After transfection, cells are selected using antibiotics, and NS1 expression is induced by doxycycline.
Viral Infections:
Cells are infected with H-1 Parvovirus (H-1PV), grown in NB324K cells, to study the effects of the virus on the cells at different infection levels (MOI).
Protein Extraction and Analysis:
Proteins are extracted from induced and non-induced cells, followed by Western blotting to assess NS1 expression and analyze proteins like p53 and caspases, helping understand how NS1 affects cells.
Immunofluorescence and Cell Structural Analysis:
Immunofluorescence techniques are used to locate NS1 in cells using specific antibodies, providing insights into how NS1 affects cell structure and function.
Growth Curves and Cell Viability:
xCELLigence and MTT assays are used to track cell growth in response to NS1 expression, helping evaluate the effect of NS1 on cell survival and growth.
Flow Cytometry and Apoptosis Analysis:
Flow cytometry is employed to analyze changes in the cell cycle, apoptosis, and reactive oxygen species (ROS) levels, providing insights into how NS1 impacts cell stress and death.
Electron Microscopy:
Electron microscopy is used to examine the ultrastructure of cells, allowing detailed observation of changes caused by NS1 expression or H-1PV infection.