DDB1 and Cul4A Are Required for Human Immunodeficiency Virus Type 1 Vpr-Induced G2 Arrest▿

Vpr-mediated induction of G2 cell cycle arrest has been postulated to be important for human immunodeficiency virus type 1 (HIV-1) replication, but the precise role of Vpr in this cell cycle arrest is unclear. In the present study, we have shown that HIV-1 Vpr interacts with damaged DNA binding protein 1 (DDB1) but not its partner DDB2. The interaction of Vpr with DDB1 was inhibited when DCAF1 (VprBP) expression was reduced by short interfering RNA (siRNA) treatment. The Vpr mutant (Q65R) that was defective for DCAF1 interaction also had a defect in DDB1 binding. However, Vpr binding to DDB1 was not sufficient to induce G2 arrest. A reduction in DDB1 or DDB2 expression in the absence of Vpr also did not induce G2 arrest. On the other hand, Vpr-induced G2 arrest was impaired when the intracellular level of DDB1 or Cullin 4A was reduced by siRNA treatment. Furthermore, Vpr-induced G2 arrest was largely abolished by a proteasome inhibitor. These data suggest that Vpr assembles with DDB1 through interaction with DCAF1 to form an E3 ubiquitin ligase that targets cellular substrates for proteasome-mediated degradation and G2 arrest

Differential Inhibition of Long Interspersed Element 1 by APOBEC3 Does Not Correlate with High-Molecular-Mass-Complex Formation or P-Body Association▿

The human cytidine deaminase APOBEC3G (A3G) and other APOBEC3 proteins exhibit differential inhibitory activities against diverse endogenous retroelements and retroviruses, including Vif-deficient human immunodeficiency virus type 1. The potential inhibitory activity of human APOBEC proteins against long interspersed element 1 (LINE-1) has not been fully evaluated. Here, we demonstrate inhibition of LINE-1 by multiple human APOBEC3 cytidine deaminases, including previously unreported activity for A3DE and A3G. More ancient members of APOBEC, cytidine deaminases AID and APOBEC2, had no detectable activity against LINE-1. A3A, which did not form high-molecular-mass (HMM) complexes and interacted poorly with P bodies, was the most potent inhibitor of LINE-1. A3A specifically recognizes LINE-1 RNA but not the other cellular RNAs tested. However, in the presence of LINE-1, A3A became associated with HMM complexes containing LINE-1 RNA. The ability of A3A to recognize LINE-1 RNA required its catalytic domain and was important for its LINE-1 suppression. Although the mechanism of LINE-1 restriction did not seem to involve DNA editing, A3A inhibited the accumulation of nascent LINE-1 DNA, suggesting interference with LINE-1 reverse transcription and/or integration or intracellular movement of LINE-1 ribonucleoprotein. Thus, association with P bodies or cellular HMM complexes could not predict the potency of APOBEC3 anti-LINE-1 activities. The catalytic domain of APOBEC3 proteins may be important for proper folding and target factors such as RNA or protein interaction in addition to cytidine deamination