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Comparative Study
. 2001 Apr;75(8):3791-801.
doi: 10.1128/JVI.75.8.3791-3801.2001.

Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes

Y Zhu  1 , H A Gelbard, M Roshal, S Pursell, B D Jamieson, V Planelles
Affiliations
Comparative Study

Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes

Y Zhu et al. J Virol. 2001 Apr.

Abstract

All primate lentiviruses known to date contain one or two open reading frames with homology to the human immunodeficiency virus type 1 (HIV-1) vpr gene. HIV-1 vpr encodes a 96-amino-acid protein with multiple functions in the viral life cycle. These functions include modulation of the viral replication kinetics, transactivation of the long terminal repeat, participation in the nuclear import of preintegration complexes, induction of G2 arrest, and induction of apoptosis. The simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagm) contains a vpr homologue, which encodes a 118-amino-acid protein. SIVagm vpr is structurally and functionally related to HIV-1 vpr. The present study focuses on how three specific functions (transactivation, induction of G2 arrest, and induction of apoptosis) are related to one another at a functional level, for HIV-1 and SIVagm vpr. While our study supports previous reports demonstrating a causal relationship between induction of G2 arrest and transactivation for HIV-1 vpr, we demonstrate that the same is not true for SIVagm vpr. Transactivation by SIVagm vpr is independent of cell cycle perturbation. In addition, we show that induction of G2 arrest is necessary for the induction of apoptosis by HIV-1 vpr but that the induction of apoptosis by SIVagm vpr is cell cycle independent. Finally, while SIVagm vpr retains its transactivation function in human cells, it is unable to induce G2 arrest or apoptosis in such cells, suggesting that the cytopathic effects of SIVagm vpr are species specific. Taken together, our results suggest that while the multiple functions of vpr are conserved between HIV-1 and SIVagm, the mechanisms leading to the execution of such functions are divergent.

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Figures

FIG. 1
FIG. 1
Expression vectors used for analysis of cell cycle distribution and transactivation. (A) Dual expression vectors for vpr variants and the reporter gene, thy-1. Arrows denote promoters. SV40 p(A), simian virus 40 polyadenylation signal; CMV IE, human cytomegalovirus immediate-early promoter; vprHIV-1-FS and vprAGM-FS, carboxy-terminal truncations of vprHIV-1 and vprAGM, generated by introducing frameshift mutations at codons 64 and 85, respectively. (B) Luciferase reporter vectors in which the viral LTR serve as a basal promoters.
FIG. 2
FIG. 2
Flow cytometric analysis of vpr-induced G2 arrest. Cells were transfected with control plasmid (pCMV-thy) or dual expression vectors encoding the murine thy-1 gene and the indicated vpr version (top of each histogram). At 48 h after transfection, the cells were analyzed for thy-1 expression and DNA content. Where indicated, the cells were treated by adding 2 mM caffeine to the medium 1 h posttransfection. Transfected cells were distinguished from untransfected ones by Thy-1 expression and were electronically gated. Histograms depict the cell cycle profile of gated Thy-1-positive cells only. The left peaks constitute cells in G1, and the right peaks constitute cells in G2/M; cells in S phase are between the G1 and G2 peaks. The frequencies of cells in different stages of the cell cycle were calculated using Multicycle AV software (Phoenix Flow Systems, San Diego, Calif.).
FIG. 3
FIG. 3
Transactivation of vprHIV-1 and vprAGM on the viral LTR. Cos-7 (A) and SKBR3 (B) cells were transiently transfected with a luciferase reporter construct (LTRAGM-Luc or LTRHIV-1-Luc) and a vpr expression construct. The structures of all constructs, except the control plasmid, are shown in Fig. 1. The control plasmid was pCMV-thy. At 48 h posttransfection, the cells were lysed in 100 μl of lysis buffer and a 20-μl volume was used to measure luciferase activity. Experiments were normalized to light units measured with control plasmid, to which an arbitrary value of 1 was assigned. All experiments were performed in triplicate, and standard deviations are shown as error bars.
FIG. 4
FIG. 4
Effect of caffeine on transactivation. SKBR3 (A and B) or Cos-7 (C) cells were transiently transfected with vprHIV-1 (A) or vprAGM (B and C) vectors and 2 mM caffeine was added to the medium 1 h later; at 72 h posttransfection, the cells were lysed and analyzed for luciferase activity as described in the legend to Fig. 3.
FIG. 5
FIG. 5
Induction of G2/M arrest by taxol leads to LTR transactivation. (A) SKBR3 cells were incubated in medium containing 90 nM taxol [(+) taxol] or medium alone [(−) taxol] and were assayed for cell cycle profile after 48 h. (B) Cells were transfected with the indicated reporter constructs; at 1 h posttransfection they were incubated in medium containing taxol or medium alone, and at 72 h they were lysed and analyzed for luciferase activity. (C) SKBR3 cells were transfected with the indicated plasmids and treated with 90 nM taxol or medium alone; at 72 h posttransfection they were lysed and analyzed for luciferase activity.
FIG. 6
FIG. 6
Viral vectors used for transduction of vprAGM, vprHIV-1, and mHSA. (A) Lentivirus transfer vectors were constructed by incorporating the indicated genes into the defective vector, D102; the vpr open reading frame was eliminated by mutating the translational start codon, which overlaps with vif; an XbaI restriction endonuclease site was introduced for convenient cloning of desired genes into this region of the genome; an EcoRI site was a present and was used as the downstream cloning site; discontinuous lines depict splicing donors (SD) and acceptors (SA). (B) The lentivirus packaging construct, pCMVΔR8.2-Δvpr, was described previously (4).
FIG. 7
FIG. 7
Induction of apoptosis by and subcellular localization of vprHIV-1 and vprAGM. (A) Apoptosis induction in Cos-7 cells. The cells were either mock infected or infected with one of the indicated lentivirus vectors and assayed for apoptosis at 72 h postinfection, using the TUNEL method; levels of infection were quantitated by measuring the frequency of GFP-positive cells and were between 80 and 95% for all experiments. (B) Apoptosis induction in HeLa cells. Experiments were performed in parallel with those in panel A. (C) Subcellular localization of GFP-vpr fusion proteins. Expression vectors bearing indicated fusion proteins were transiently transfected into HeLa or Cos-7 cells and visualized using fluorescence microscopy after 24 h.
FIG. 8
FIG. 8
Effect of caffeine on the induction of apoptosis by vprHIV-1 and vprAGM. Infections and apoptosis measurements were performed as described in the legend to Fig. 7; TUNEL-positive (apoptotic) cells were visually counted, and the levels of apoptosis were expressed as a percentage of the total cells counted; the percentage of apoptotic cells in mock infections was always lower than 5%. All experiments were done in triplicate, and standard deviations are shown. (A) SKBR3 cells. (B) Cos-7 cells. (C) Sup-T1 cells.
FIG. 9
FIG. 9
Proposed model to summarize the relationships among various functions of vprHIV-1 and vprAGM. The observed effects of drugs are depicted in grey. Caffeine inhibits vprHIV-1 G2 arrest and also its downstream effects, apoptosis and transactivation. For vprAGM, caffeine inhibits G2 arrest but not apoptosis or transactivation. Transactivation by vprAGM is due, in part, to a cell cycle-independent mechanism, and therefore, caffeine has little or no effect on transactivation. However, the SIVagm LTR is also responsive to cell cycle arrest since taxol can induce transactivation. Because treatment with caffeine does not significantly affect transactivation, the significance of G2 arrest for vprAGM is uncertain (question mark).
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