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. 1999 Apr;73(4):3236-45.
doi: 10.1128/JVI.73.4.3236-3245.1999.

Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast

M Chen  1 , R T Elder, M Yu, M G O'Gorman, L Selig, R Benarous, A Yamamoto, Y Zhao
Affiliations

Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast

M Chen et al. J Virol. 1999 Apr.

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.

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Figures

FIG. 1
FIG. 1
Effects of VprLAI on G2 arrest, cell survival, and nuclear localization in fission yeast. (A) Flow cytometric analyses 40 h after transfer to medium with (vpr repressed) or without (vpr expressed) thiamine, showing the shift of G1 cells in the medium with thiamine (+T) to the G2 stage of the cell cycle in medium without thiamine (−T). (B) Qualitative assay for cell killing by Vpr. The plate with thiamine at the top represses the nmt1 promoter, and cells form normal-size colonies after incubation for 3 to 4 days. Cells with the VprNL4-3 plasmid (a) are on the left side, and cells with the VprLAI plasmid (b) are on the right. On the plate without thiamine shown at the bottom, the vpr gene is induced, and both VprNL4-3 and VprLAI prevent the formation of normal-size colonies. (C) The morphological changes induced by VprLAI differ somewhat from those induced by VprNL4-3. The cultures with thiamine for both VprNL4-3 (a) and VprLAI (b) show the normal Calcofluor staining pattern, with weak overall staining of the cell wall and intense staining of the septum forming at the site of eventual cell division. One cell in each panel shows this strong staining of the septum. The cultures without thiamine at the bottom (vpr expressed) both show large increases in chitin staining compared to normal cells. However, for VprNL4-3 (a), this increased chitin deposition occurs at the protruding ends of the cells while cells with VprLAI have very thick chitin deposits near the center of the cell, where the septum normally forms. (D) The GFP-VprNL4-3 fusion protein localizes around the rim of the nucleus and shows little overlap with nuclear DNA. A cell 17 h after induction of Vpr was stained for DNA with the vital stain Hoechst 33342 (Chikashige, 1994 no. 8). The third panel, in color, shows GFP-Vpr in the false color of red and DNA staining in the false color of green with little overlap between the two colors.
FIG. 2
FIG. 2
Effects of Vpr mutations on nuclear localization and G2 arrest. (A) Expression of mutant Vpr proteins. Extracts from induced (without thiamine) point mutant Vpr proteins (a) and mRNA from deletion mutant proteins (b) were isolated and subjected to Western and Northern blotting analyses, respectively. The arrow indicates the 15-kb band expected for Vpr. +T, with thiamine; −T, without thiamine; WT, wild type. (B) Mutations which affect localization of the GFP-Vpr fusion protein. (a) Localization of GFP not fused to Vpr showing uniform distribution throughout the cell. (b) Localization of GFP-VprLAI showing a ring or a ring of dots around the nucleus with little detectable labelling of the cytoplasm. Panels c to j show the results of those Vpr mutations which change the localization pattern: c, E25K; d, H33R; e, F34I; f, H71R; g, N15 deletion; h, N27 deletion; i, C63 deletion; j, C77 deletion. (C) Effects of Vpr mutations on G2 arrest. Representative flow cytometry analyses of the cell cycle are shown for four mutations. The upper panels are for cultures with thiamine, where vpr is repressed, and the bottom panels are for cultures without thiamine, where vpr is expressed.
FIG. 3
FIG. 3
Positions of amino acid substitutions which affect a Vpr function. The tertiary structure of Vpr is from reference . The amino acid is shown in blue when the change reduces an activity, the amino acid is shown in yellow when no activity is observed, and the amino acid is shown in red when wild-type or increased activity is observed. Structural domains implicated as being important for a function are shown in green. (a) Substitutions affecting G2 arrest. (b) Substitutions affecting localization to the nuclear rim. (c) Substitutions affecting cell killing.
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