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Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast
1Children's Memorial Institute of Education and Research, Northwestern University Medical School, Chicago, Illinois 60614, USA.
Abstract:
Cell cycle G2 arrest, nuclear localization, and cell death induced by human immunodeficiency virus type 1 Vpr were examined in fission yeast by using a panel of Vpr mutations that have been studied previously in human cells. The effects of the mutations on Vpr functions were highly similar between fission yeast and human cells. Consistent with mammalian cell studies, induction of cell cycle G2 arrest by Vpr was found to be independent of nuclear localization. In addition, G2 arrest was also shown to be independent of cell killing, which only occurred when the mutant Vpr localized to the nucleus. The C-terminal end of Vpr is crucial for G2 arrest, the N-terminal alpha-helix is important for nuclear localization, and a large part of the Vpr protein is responsible for cell killing. It is evident that the overall structure of Vpr is essential for these cellular effects, as N- and C-terminal deletions affected all three cellular functions. Furthermore, two single point mutations (H33R and H71R), both of which reside at the end of each alpha-helix, disrupted all three Vpr functions, indicating that these two mutations may have strong effects on the overall Vpr structure. The similarity of the mutant effects on Vpr function in fission yeast and human cells suggests that fission yeast can be used as a model system to evaluate these Vpr functions in naturally occurring viral isolates.
Insights
Fission yeast effectively models human immunodeficiency virus type 1 Vpr functions, including cell cycle G2 arrest and cell death. Specific Vpr regions and mutations impact these cellular processes, validating yeast as a research system.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Human immunodeficiency virus type 1 (HIV-1) Vpr protein induces cell cycle G2 arrest and cell death.
- Understanding Vpr's functions is crucial for developing antiviral strategies.
Purpose of the Study:
- To investigate the functions of HIV-1 Vpr, including cell cycle G2 arrest, nuclear localization, and cell death, in a fission yeast model system.
- To compare the effects of Vpr mutations in fission yeast with their known effects in human cells.
- To identify key structural regions of Vpr responsible for its cellular functions.
Main Methods:
- Utilized a panel of well-characterized Vpr mutations previously studied in human cells.
- Introduced these Vpr mutations into fission yeast (Schizosaccharomyces pombe).
- Assessed Vpr-induced cell cycle G2 arrest, nuclear localization, and cell death in fission yeast.
Main Results:
- Vpr mutation effects on G2 arrest, nuclear localization, and cell death were highly conserved between fission yeast and human cells.
- Vpr-induced G2 arrest was independent of nuclear localization but dependent on the C-terminal end of Vpr.
- Cell killing by Vpr required nuclear localization and involved a distinct region of the protein.
- N- and C-terminal deletions and specific point mutations (H33R, H71R) significantly disrupted Vpr functions, indicating the importance of overall Vpr structure.
Conclusions:
- Fission yeast serves as a robust and predictive model system for studying HIV-1 Vpr functions.
- The study elucidates the distinct structural requirements for Vpr-mediated G2 arrest, nuclear localization, and cell death.
- Findings support the use of fission yeast for evaluating Vpr functions in diverse viral isolates.