Background: Viral protein R (Vpr) of HIV is known to cause G2/M phase cell cycle arrest, coinciding with increased transcription from the HIV-LTR. The mechanism of arrest is unknown, though it is thought to be mediated by Vpr interactions with cellular proteins. hVIP/mov34, a member of the eIF3 family of transcription regulators, was previously found to interact with Vpr. In addition, hVIP antisense causes cell cycle arrest similar to that seen with Vpr administration, indicating a potential role for hVIP in Vpr-mediated cell cycle arrest. Literature data cites phosphorylation as one of the main mechanisms employed in cell cycle control. Therefore, we hypothesize that Vpr may effect the phosphorylation status of hVIP to induce cell cycle arrest. In this study, we evaluated the effects of Vpr on hVIP phosphorylation and cell cycle regulation.

Methods: Cloning: A panel of hVIP mutants was created by PCR-based cloning into pcDNA3;1 containing a C-terminal His tag. Expression was tested via in vitro transcription/translation and all mutants expressed appropriate size protein. Phosphorylation studies: Hela cells were subjected to T7 mediated transfection with hVIP wild-type or mutant plasmid alone or with Vpr plasmid. Immunoprecipitation was performed on cell lysates using His (hVIP) specific antibody. Phosphorylated products were visualized by autoradiography.

Results: Immunoprecipitation experiments indicated that hVIP is phosphorylated in the cell. However, in the presence of Vpr, phosphorylation of wild-type hVIP is significantly decreased to background levels. Using a panel of hVIP mutants we mapped the phosphorylation domain of hVIP to the C terminal amino acids 244–341. Furthermore, the presence of Vpr decreased phophorylation of hvip244–341, suggesting that hVIP-Vpr interaction resides in this region.

Conclusions: Previous data indicates that Vpr-mediated cell cycle control may be exerted through the cellular protein hVIP/mov34. Here, we show that a potential mechanism of such control is altered phosphorylation of hVIP induced by the presence of Vpr. Specifically, altered phosphorylation is localized to the C terminal amino acids 244–341 of the protein, containing one predicted serine/threonine phosphorylation site. As the loss of Vpr-induced cell cycle arrest would be expected to lead to greater than 4-fold decrease in virus production, such specific data on the Vpr/hVIP interaction provides a potential site for rational HIV therapeutic design.