Functional and Structural Characterization of Synthetic HIV-1 Vpr That Transduces Cells, Localizes to the Nucleus, and Induces G2 Cell Cycle Arrest

Abstract

Human immunodeficiency virus (HIV)Vpr contributes to nuclear import of the viral pre-integration complex and induces G 2 cell cycle arrest. We describe the production of synthetic Vpr that permitted the first studies on the structure and folding of the full-length protein. Vpr is unstructured at neutral pH, whereas under acidic conditions or upon addition of trifluorethanol it adopts ␣-helical structures. Vpr forms dimers in aqueous trifluorethanol, whereas oligomers exist in pure water.1 H NMR spectroscopy allows the signal assignment of N-and C-terminal amino acid residues; however, the central section of the molecule is obscured by self-association. These findings suggest that the in vivo folding of Vpr may require structure-stabilizing interacting factors such as previously described interacting cellular and viral proteins or nucleic acids. In biological studies we found that Vpr is efficiently taken up from the extracellular medium by cells in a process that occurs independent of other HIV-1 proteins and appears to be independent of cellular receptors. Following cellular uptake, Vpr is efficiently imported into the nucleus of transduced cells. Extracellular addition of Vpr induces G 2 cell cycle arrest in dividing cells. Together, these findings raise the possibility that circulating forms of Vpr observed in HIV-infected patients may exert biological effects on a broad range of host target cells.

Molecular mimicry in inducing DNA damage between HIV-1 Vpr and the anticancer agent, cisplatin

“…The necrotic region may result from the changes in cells via a combination of endogenous and Vpr-induced cytotoxicity. As Vpr has been shown to enter cells (Henklein et al, 2000;Sherman et al, 2001;Nakamura et al, 2002), it is possible that the necrotic regions may result from spreading of Vpr from the site of inoculation.…”

HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination

“…First, we introduced a plasmid DNA encoding Vpr or its control plasmid DNA, but it was not possible to assess the effects of Vpr correctly because transfection of plasmid DNA by itself influenced the rate of HR (data not shown). It has been well reported that Vpr enters cells and expresses its biological activity when added to the cell culture (Jenkins et al, 1998;Henklein et al, 2000;Huang et al, 2000;Taguchi et al, 2004). These observations encouraged us to perform the experiments by adding Vpr to cells exogenously with subsequent measurement of GFP-positive cells.…”

Structural Characterization of the HIV-1 Vpr N Terminus

“…The majority of amino acid residues of the inner core of the protein could not be assigned because of broadening and overlap of the NMR signals (20). We interpreted these phenomena as arising from the internal tendency of sVpr to undergo self-association in aqueous solution.…”

“…However, the design of effective Vpr antagonists requires more detailed knowledge of its dynamic structure and folding during heterologous and homologous protein-protein interactions. In our previous work (20) we have characterized the effect of the solution conditions on the folding and self-association of synthetic full-length Vpr (sVpr), and initial information on the secondary structure of sVpr was obtained by CD spectroscopy (20). In these studies we were able to demonstrate that both the secondary structure of sVpr and its folding, as well as its tendency to undergo protein-protein interaction, critically depend upon the solution parameters such as pH and hydrophobicity.…”

“…Furthermore, Vpr is highly conserved in HIV-1 and other primate lentiviruses, HIV-2 and SIV, which also encode an additional Vprrelated protein termed Vpx, which is believed to function synergistically with Vpr. Vpr of HIV-1 is reported to exhibit numerous biological activities including nuclear localization based on the presence of at least two nuclear localization signals (2,(5)(6)(7)(8)(9), ion channel formation (10), transcriptional activation of HIV and heterologous promoters (11)(12)(13)(14), co-activation of the glucocorticoid receptor (15,59), regulation of cell differentiation (16), induction of apoptosis (17,18), cell cycle arrest (16,19), and transduction through cell membranes (20). Although significant amounts of Vpr (ϳ0.15-fold molar ratio to viral core proteins (21)) are packaged into budding HIV-1 particles in a process dependent on interaction of Vpr with the C-terminal p6…”