Two research groups have independently discovered that the HIV Vpr protein interacts with cellular proteins to form a type of protein complex called an E3 ubiquitin ligase. E3 ubiquitin ligases typically mark proteins for degradation and disposal by the cell’s proteasome (a sort of cellular house cleaning function). These new studies suggest that Vpr is hijacking this normal cellular process in a way that causes cellular proteins involved in regulation of the cell cycle to be degraded, potentially explaining Vpr’s well-described ability to cause cell cycle arrest (an inability of the cell to divide normally). These discoveries may pave the way toward identifying compounds that can block the interactions between Vpr and cellular proteins, preventing the formation of the E3 ubiquitin ligase described in these papers.
Abstracts:
PLoS Pathog 3(7): e85
HIV-1 Vpr-Mediated G2 Arrest Involves the DDB1-CUL4AVPRBP E3 Ubiquitin Ligase
Jean-Philippe Belzile, Ghislaine Duisit, Nicole Rougeau, Johanne Mercier, Andrés Finzi, Éric A. Cohen
Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)—components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4AVPRBP—were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest–defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4AVPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.
JVI Accepts, published online ahead of print on 11 July 2007
J. Virol. doi:10.1128/JVI.01380-07
DDB1 and Cul4A are required for HIV-1 Vpr-induced G2 arrest
Lindi Tan, Elana Ehrlich, and Xiao-Fang Yu*
Vpr-mediated induction of G2 cell cycle arrest has been postulated to be important for 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. Interaction of Vpr with DDB1 was inhibited when DCAF1 (VprBP) expression was reduced by 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.
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