Abstract
Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.
Highlights
Human immunodeficiency virus type 1 (HIV-1)1 Vpr is a 96-amino acid non-structural protein that is associated with virus particles and can accumulate at the nuclear envelope and in the nucleus of infected cells [1,2,3,4]
To further characterize the respective molecular determinants of Vpr and uracil-DNA glycosylase (UNG) involved in the interaction, we developed an in vitro binding assay using recombinant UNG expressed in E. coli in fusion with the glutathione S-transferase (GST-UNG)
Because it has been reported that Vpr binding is related to the presence of a WXXF motif found within the C-terminal part of UNG2, a GST-UNGW231A/F234G mutant was included as a control of specificity in this in vitro binding assay
Summary
Retroviral Vectors and Expression Plasmids—Most of the retroviral vectors and yeast and mammalian expression plasmids used in this study have been described previously [5, 11] except plasmids for the expression in bacteria of wild type (WT) and mutated forms of UNG2 fused to the glutathione S-transferase (GST) and for expression in mammalian cells of UNG fused to the C terminus of the wild type or W54R Vpr proteins. Cell lysates from 6 ϫ 106 HeLa cells expressing HA-Vpr (WT or W54R) were prepared and incubated with purified GST or GST-UNG proteins as previously described [4]. We cotransfected (Superfect, Qiagen) a vpr-defective HIV-1 vector and Vpr and UNG expression plasmids derived from pAS1B [11] into 293T cells. Infected peripheral blood mononuclear cells (PBMCs) and monocytes-derived macrophages (MDMs) were not placed under drug selection, and after purification of proviral DNA with the lac repressor protein, the vector cassette containing the mutation target was PCR amplified before analysis of mutant frequencies in E. coli. After overnight incubation at 37 °C, the cells were washed twice and placed in either RPMI, 10% fetal calf serum (macrophages, resting T-cells) or medium supplemented with 10 units/ml interleukin-2 (stimulated PBMCs/stimulated T-cells). Amounts of CAp24 produced were determined by enzyme-linked immunosorbent assay
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