Mutational analysis of Vpr-induced G(2) arrest, nuclear localization, and cell death in fission yeast

Cell cycle G(2) arrest, nuclear localization, and cell death induced by hum an immunodeficiency virus type 1 Vpr were examined in fission yeast by usin g a panel of Vpr mutations that have been studied previously in human cells . The effects of the mutations an Vpr functions were highly similar between fission yeast and human cells. Consistent with mammalian cell studies, ind uction of cell cycle G(2) arrest by Vpr was found to be independent of nucl ear localization. In addition, G(2) 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 G(2) arrest, the N-termin al alpha-helix is important for nuclear localization, and a large part of t he Vpr protein is responsible for cell killing. It is evident that the over all structure of Vpr is essential for these cellular effects, as N- and C-t erminal deletions affected all three cellular functions. Furthermore, two s ingle point mutations (H33R and H71R), both of which reside at the end of e ach alpha-helix, disrupted all three Vpr functions, indicating that these t wo mutations may have strong effects on the overall Vpr structure. The simi larity of the mutant effects on Vpr function in fission yeast and human cel ls suggests that fission yeast can be used as a model system to evaluate th ese Vpr functions in naturally occurring viral isolates.