Vpr-GFP virion particle identifies HIV-infected targets and preserves HIV-1Vpr function in macrophages and T-cells

Human immunodeficiency virus type 1 (HIV-1) is known for its ability to inf ect immune cells, including T-cells and macrophages. The 96-amino acid Vpr, a virion-associated protein, is essential for viral replication in monocyt es/macrophages and increases viral replication in primary and established T -cell lines. The Vpr protein regulates a number of host cellular events, in cluding proliferation, differentiation, apoptosis, cytokine production, and NF-KB-mediated transcription. Most of these functions have been analyzed u sing either endogenous Vpr protein or cells transfected with a Vpr expressi on plasmid, We developed a lentiviral vector complemented with a Vpr expres sion plasmid that results in viral particles packaged with Vpr protein. To facilitate identification of the target cells infected with the particles c ontaining Vpr, we fused green fluorescent protein (GFP) with the Vpr open r eading frame and analyzed the biology of this novel particle, Vpr itself is expressed as a 14-kDa protein; however, in vitro translation of the pVpr-G FP plasmid resulted in the expression of 39-kDa fusion protein. The fusion molecule exhibited the same activity in arresting the cell cycle in Gz as d oes the wildtype Vpr molecule. Subcellular localization of Vpr and Vpr-GFP by immunofluoresence in human and murine cell lines indicated that Vpr by i tself or with the reporter GFP showed a perinuclear staining pattern. Repli cation kinetics showed no significant difference between Vpr-GFP and native complemented pseudovirus replication in a single-round infectivity assay, A flow cytometry analysis of peripheral blood lymphocytes and macrophages i nfected with Vpr-GFP-packaged virions and selected by GFP showed 56.7 % inf ectivity for lymphocytes and 83.6 % infectivity for macrophages. Additional analysis of CD24 (HSA)-positive cells showed infection of CD4+ cells, macr ophages, and, importantly, dendritic cells, This system will allow us to id entify specific cell populations including antigen-presenting cells, and al low quantitative analysis of the precise effect of Vpr on both target and b ystander cells ill vitro as well as in vivo.