An analysis of cell death and cell cycle arrest during human immunodeficiency virus type I infection

Acquired immunodeficiency syndrome (AIDS) results from the dramatic loss of CD4+ T cells in human immunodeficiency virus type 1 (HIV-1) infected individuals. The mechanism(s) of this depletion remains unclear. Recent studies show that the initial depletion of CD4+ T cells is due primarily to HIV-1 cytopathicity in infected cells of the gastrointestinal tract. The 14 kD HIV-1 accessory protein viral protein R (Vpr) contributes importantly to HP/-1-induced necrosis, which is correlated with cell cycle arrest. Phosphorylation of Vpr serine 79 (S79) is required to activate G 2,M cell cycle blockade. However, the kinase responsible for phosphorylating Vpr remains unknown. Using bioinformatics tools, we found that serine 79 of 1/pr is part of a putative phosphorylation site recognized by the cAMP-dependent Protein Kinase, PICA. We show that PICA interacts with Vpr by immunoprecipitation and fluorescence resonance energy transfer (FRET) and directly phosphorylates S79. Inhibition of PKA activity during HIV-1 infection abolishes Vpr cell cycle arrest. In addition to the need for post-translational modification, the structure of Vpr exhibits two exposed hydrophobic patches that we hypothesize may be important protein interaction sites. A previous study showed that the patch along the third a helix of Vpr was important for both cell cycle arrest and cell death. In the work presented here, we show that the exposed hydrophobic amino acids along the first a helix are also important in cell cycle arrest and cell death using a structurally based mutagenesis approach. Although HIV-1 encodes proteins with direct cytopathic properties, infection also elicits elevated levels of tumor necrosis factor alpha (TNF), a potent pro-inflammatory cytokine capable of inducing cell death. We found that HIV-1 infected cells are highly sensitive to TNF-induced necrosis. In addition, we show that the HIV-1 accessory protein Vpu was necessary and sufficient for this increased sensitivity to TNF. Taken together these findings provide new insights into the processes that likely control the depletion of CD4+ T cells during HIV-1 infection.