HIV-1 Vpr Induces Cell Cycle G2 Arrest in Fission Yeast (Schizosaccharomyces pombe) through a Pathway Involving Regulatory and Catalytic Subunits of PP2A and Acting on Both Wee1 and Cdc25

Abstract

Viral protein R (Vpr) of human immunodeficiency virus type 1 induces G2 arrest in cells from distantly related eukaryotes including human and fission yeast through inhibitory phosphorylation of tyrosine 15 (Tyr15) on Cdc2. Since the DNA damage and DNA replication checkpoints also induce G2 arrest through phosphorylation of Tyr15, it seemed possible that Vpr induces G2 arrest through the checkpoint pathways. However, Vpr does not use either the early or the late checkpoint genes that are required for G2 arrest in response to DNA damage or inhibition of DNA synthesis indicating that Vpr induces G2 arrest by an alternative pathway. It was found that protein phosphatase 2A (PP2A) plays an important role in the induction of G2 arrest by Vpr since mutations in genes coding for a regulatory or catalytic subunit of PP2A reduce Vpr-induced G2 arrest. Vpr was also found to upregulate PP2A, supporting a model in which Vpr activates the PP2A holoenzyme to induce G2 arrest. PP2A is known to interact genetically in fission yeast with the Wee1 kinase and Cdc25 phosphatase that act on Tyr15 of Cdc2. Both Wee1 and Cdc25 play a role in Vpr-induced G2 arrest since a wee1 deletion reduces Vpr-induced G2 arrest and a direct in vivo assay shows that Vpr inhibits Cdc25. Additional support for both Wee1 and Cdc25 playing a role in Vpr-induced G2 arrest comes from a genetic screen, which identified genes whose overexpression affects Vpr-induced G2 arrest. For this genetic screen, a strain was constructed in which cell killing by Vpr was nearly eliminated while the effect of Vpr on the cell cycle was clearly indicated by an increase in cell length. Overexpression of the wos2 gene, an inhibitor of Wee1, suppresses Vpr-induced G2 arrest while overexpression of rad25, an inhibitor of Cdc25, enhances Vpr-induced G2 arrest. These two genes may be part of the uncharacterized pathway for Vpr-induced G2 arrest in which Vpr upregulates PP2A to activate Wee1 and inhibit Cdc25.

Viral infections and cell cycle G2/M regulation

“…None of the early checkpoint-specific mutants (rad1, rad3, rad9 and rad17) showed a significant effect on the induction of G2 arrest by Vpr [38,39,46]. Furthermore, mutations in both chk1 and cds1, which are thought to be the last steps specific for the checkpoint [1,18,24], also do not block Vprinduced G2 arrest [39,46]. Therefore, Vpr does not appear to use the DNA-damage or DNA-replication checkpoint pathways to induce G2 arrest in fission yeast.…”

“…The HIV-1 Vpr protein induces cell cycle G2 arrest [42], is a Vpr suppressor when overproduced [39]. The Cdc25 inhibitor rad25 [19], which is a homologue of human 14-3-3 proteins, enhances Vpr-induced G2 arrest when overproduced in fission yeast [39].…”

“…The Cdc25 inhibitor rad25 [19], which is a homologue of human 14-3-3 proteins, enhances Vpr-induced G2 arrest when overproduced in fission yeast [39]. Recent studies further show that Vpr binds to Cdc25C and to 14-3-3 in human cells [43][44][45].…”

“…1), it is unclear at the moment why human ATR and CHK1 are activated by Vpr but rad3 (the fission yeast homologue of ATR/ATM) or chk1/cds1 (CHK1/CHK2) double deletion in fission yeast does not block Vpr-induced G2 arrest [38,39]. One factor possibly contributing to the observed activation of ATR in mammalian cells is that retroviral integration appears to activate ATR [59], and the experiments showing activation of ATR by Vpr were done with lentiviral vectors which might therefore activate ATR to some extent independently of Vpr.…”

“…Caffeine is part of the methylxanthine family, and similar to the caffeine effect, another methylxanthine pentoxifylline (PTX) also inhibits Vpr-induced G2 arrest in mammalian cells [61]. Similarly, both PTX and caffeine suppress Vpr-induced G2 arrest in fission yeast [39,58] [our unpublished data]. Since PTX or caffeine inhibits Vpr-induced G2 arrest in fission yeast where the classic DNA checkpoints apparently play no role, these observations suggest molecular mechanisms other than the classic DNA checkpoints may be involved in activation of ATR and regulation of Cdc25 and Wee1.…”