The mammalian APOBEC3 family of cytidine deaminases includes several members that possess potent antiretroviral activity. Human APOBEC3F and APOBEC3G are specifically incorporated into human immunodeficiency virus type 1 (HIV-1) progeny virions in the absence of virion infectivity factor (Vif), where they deaminate deoxycytidine to deoxyuridine on the minus strand of nascent reverse transcripts. Editing of the HIV-1 cDNA leads to its degradation or to G to A hypermutation of the integrated provirus. Here, we show that APOBEC3 proteins also restrict the activity of a distantly related long terminal repeat (LTR) retrotransposon. When expressed in the yeast Saccharomyces cerevisiae, human APOBEC3C, APOBEC3F, or APOBEC3G or mouse APOBEC3 potently inhibit replication of the Ty1 LTR retrotransposon. APOBEC3G interacts with Ty1 Gag and is packaged into Ty1 virus-like particles (VLPs) by a mechanism that closely resembles the one it uses to enter HIV-1 virions. Expression of APOBEC3G results in a reduced level of Ty1 cDNA integration and G to A editing of integrated Ty1 cDNA. Our findings indicate that APOBEC3G restricts Ty1 and HIV-1 by similar mechanisms and suggest that the APOBEC3 proteins target a substantially broader spectrum of retroelements than previously appreciated.
To determine why the duration of mitosis (DM) is less in Taxol than in nocodazole or Eg5 inhibitors we studied the relationship between Taxol concentration, the DM, and the mitotic checkpoint. We found that unlike for other spindle poisons, in Taxol the DM becomes progressively shorter as the concentration surpasses ∼0.5 µM. Studies on RPE1 and PtK2 expressing GFP/cyclin B or YFP/Mad2 revealed that cells ultimately satisfy the checkpoint in Taxol and do so faster at concentrations >0.5 µM. Inhibiting the aurora-B kinase in Taxol-treated RPE1 cells accelerates checkpoint satisfaction by stabilizing syntelic kinetochore attachments and reduces the DM to ∼1.5 h regardless of drug concentration. A similar stabilization of syntelic attachments by Taxol itself appears responsible for accelerated checkpoint satisfaction at concentrations >0.5 µM. Our results provide a novel conceptual framework for how Taxol prolongs mitosis and caution against using it in checkpoint studies. They also offer an explanation for why some cells are more sensitive to lower versus higher Taxol concentrations.
The genomic RNA of retroviruses and retrovirus-like transposons must be sequestered from the cellular translational machinery so that it can be packaged into viral particles. Eukaryotic mRNA processing bodies (P bodies) play a central role in segregating cellular mRNAs from the translational machinery for storage or decay. In this work, we provide evidence that the RNA of the Saccharomyces cerevisiae Ty1 retrotransposon is packaged into virus-like particles (VLPs) in P bodies. Ty1 RNA is translationally repressed, and Ty1 Gag, the capsid and RNA binding protein, accumulates in discrete cytoplasmic foci, a subset of which localize to P bodies. Human APOBEC3G, a potent Ty1 restriction factor that is packaged into Ty1 VLPs via an interaction with Gag, also localizes to P bodies. The association of APOBEC3G with P bodies does not require Ty1 element expression, suggesting that P-body localization of APOBEC3G and Ty1 Gag precedes VLP assembly. Additionally, we report that two P-body-associated 5 to 3 mRNA decay pathways, deadenylation-dependent mRNA decay (DDD) and nonsense-mediated decay (NMD), stimulate Ty1 retrotransposition. The additive contributions of DDD and NMD explain the strong requirement for general 5 to 3 mRNA degradation factors Dcp1, Dcp2, and Xrn1 in Ty1 retromobility. 5 to 3 decay factors act at a posttranslational step in retrotransposition, and Ty1 RNA packaging into VLPs is abolished in the absence of the 5 to 3 exonuclease Xrn1. Together, the results suggest that VLPs assemble in P bodies and that 5 to 3 mRNA decay is essential for the packaging of Ty1 RNA in VLPs.
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