Abstract
Viral protein R (Vpr) of human immunodeficiency virus, type 1 (HIV-1) is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the preintegration complex into the nucleus and the induction of G2 host cell cycle arrest. Our recent investigation of the conformational heterogeneity of the proline residues in the N terminus of Vpr suggested a functional interaction between Vpr and a host peptidylprolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Using surface plasmon resonance spectroscopy, Far Western blot, and pulldown experiments a physical interaction of Vpr with the major host PPIase cyclophilin A (CypA) is now demonstrated. The interaction domain involves the N-terminal region of Vpr including an essential role for proline in position 35. The CypA inhibitor cyclosporin A and non-immunosuppressive PPIase inhibitors such as NIM811 and sanglifehrin A block expression of Vpr without affecting pre- or post-translational events such as transcription, intracellular transport, or virus incorporation of Vpr. Similarly to CypA inhibition, Vpr expression is also reduced in HIV-1 infected CypA-/- knock-out T cells. This study thus shows that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr and that in the absence of CypA activity, the Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells.
Highlights
In addition to the genes encoding the structural and enzymatic proteins common to all retroviruses, the human immu
Biochemical Evidence for cyclophilin A (CypA)-viral protein R (Vpr) Interaction in Vitro and in Vivo—Our recent finding [19] of unusually large amounts of cis proline conformations about the imidic bond for two of the proline residues in the N terminus of Vpr suggested that Vpr folding may require a peptidyl-prolyl isomerase (PPIase) enzyme activity. Since both Vpr and CypA are present in HIV-1 virions, and CypA binds to HIV-1 CA and promotes virus infectivity, we investigated whether Vpr and CypA interact
CypA Inhibitors Block Vpr Expression—In view of the interaction of the N-terminal region of Vpr with CypA, we examined whether cyclosporin A (CsA), an inhibitor of CypA activity, altered the synthesis of Vpr in HIV-1-expressing cells
Summary
In addition to the genes encoding the structural and enzymatic proteins common to all retroviruses, the human immu-. Nodeficiency virus, type 1 (HIV-1) genome contains four accessory genes that serve to accelerate viral replication One of these gene products, the highly conserved 96-amino acid viral protein R (Vpr) has received considerable attention, and a number of biological functions have been attributed to its presence in various cellular and extracellular compartments. Regarding the second function of Vpr, which leads to G2 cell cycle arrest in HIV-1-infected and/or Vpr-transfected human cells, it was implicated that this activity provides an intracellular milieu conductive for enhanced viral replication by increasing HIV LTRdriven gene expression [4] This response has been linked recently [5] with the capacity of Vpr to alter the structure of the nuclear lamina, leading to transient, DNA-containing herniations of the nuclear envelope that intermittently rupture. Based on the cis/trans phenomenon that could be important for the folding of Vpr, it was projected that a potential interaction between Vpr and a host peptidyl-prolyl isomerase (PPIase) might regulate the interconversion of the imidic bond of these N-terminal proline residues of Vpr in vivo
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