HIV-1 Vpr induces interferon-stimulated genes in human monocyte-derived macrophages

Abstract

Macrophages act as reservoirs of human immunodeficiency virus type 1 (HIV-1) and play an important role in its transmission to other cells. HIV-1 Vpr is a multi-functional protein involved in HIV-1 replication and pathogenesis; however, its exact role in HIV-1-infected human macrophages remains poorly understood. In this study, we used a microarray approach to explore the effects of HIV-1 Vpr on the transcriptional profile of human monocyte-derived macrophages (MDMs). More than 500 genes, mainly those involved in the innate immune response, the type I interferon pathway, cytokine production, and signal transduction, were differentially regulated (fold change >2.0) after infection with a recombinant adenovirus expressing HIV-1 Vpr protein. The differential expression profiles of select interferon-stimulated genes (ISGs) and genes involved in the innate immune response, including STAT1, IRF7, MX1, MX2, ISG15, ISG20, IFIT1, IFIT2, IFIT3, IFI27, IFI44L, APOBEC3A, DDX58 (RIG-I), TNFSF10 (TRAIL), and RSAD2 (viperin) were confirmed by real-time quantitative PCR and were consistent with the microarray data. In addition, at the post-translational level, HIV-1 Vpr induced the phosphorylation of STAT1 at tyrosine 701 in human MDMs. These results demonstrate that HIV-1 Vpr leads to the induction of ISGs and expand the current understanding of the function of Vpr and its role in HIV-1 immune pathogenesis.

Figure 1. Schematic diagram of the Ad-Vpr and Ad-Zs vectors and analysis of their functional expression.

(A) Recombinant adenovirus vectors expressing either FLAG-Vpr and ZsGreen1 or ZsGreen1 were generated using the Adeno-X™ expression system, as described in Materials and Methods. The transgene cassettes that replace the deleted E1 region contain a cytomegalovirus (CMV) promoter driving the expression of FLAG-Vpr and ZsGreen1 or ZsGreen1 protein, followed by an SV40 polyadenylation signal. The solid triangles indicate the regions deleted in the recombinant adenovirus (rAd) backbone. ITR: Inverted terminal repeats. (B) HeLa cells were infected with Ad-Vpr or Ad-Zs at MOI 50. At 48 h post-infection, cells were fixed and stained with propidium iodide for the analysis of DNA content. ZsGreen1-positive cells were analyzed by flow cytometry using Cell Quest for acquisition and ModFit LT. Arrowheads indicate peaks representing cells in the G1 and G2+M phases. The G2+M: G1 ratio is indicated in the upper right of each graph.

Figure 2. Expression analyses of HIV-1 Vpr protein in human monocyte-derived macrophages (MDMs).

(A) Peripheral blood mononuclear cells (PBMCs) were isolated from two healthy donors through leukophoresis, cultured in vitro, and differentiated into MDMs as described in Materials and Methods. At day 7, the MDMs were infected with either Ad-Vpr or Ad-Zs, or were left untreated as mock-infected controls (left). At 48 h post-infection, the cells from Donor 1 were visualized by fluorescence (FL) and bright field phase contrast (BF) microscopy. (B) The cells from the two donors (upper panel, Donor 1; lower panel, Donor 2) were lysed and subjected to Western blot analyses using Vpr, ZsGreen1, and β-actin antibodies.

Figure 3. Differential expression profiling of cellular genes after infection with Ad-Vpr in human monocyte-derived macrophages (MDMs).

Heat map of hierarchical gene clustering showing all genes that were either up- or down-regulated (>2-fold change) upon Ad-Vpr infection in MDMs from both donors. The color represents the normalized expression of genes in MDMs infected with Ad-Vpr or Ad-Zs (see color key). Gene up-regulation is denoted in red and gene down-regulation is denoted in blue.

Figure 4. Gene ontology of differentially expressed genes after infection of human monocyte-derived macrophages (MDMs) with Ad-Vpr.

(A) Venn diagram representing the number of differentially expressed cellular genes (>2-fold change in both donors) after infection of human MDMs with Ad-Vpr. (B) The top ten genes ontology classified by corrected p-value, and (C) heat map of hierarchical gene clustering of the 66 differentially regulated in both donors. Gene up-regulation is denoted in red and gene down-regulation is denoted in blue.

Figure 5. Differential expression profiling of cellular genes involved in the immune response and the type I interferon pathway after infection with Ad-Vpr in human monocyte-derived macrophages (MDMs) from Donor 1.

Heat map showing genes related to the immune response (left: GO: 0006955) and the type I interferon signaling (right: GO: 0060337) that were either up- or down-regulated (>2-fold change) upon Ad-Vpr infection of MDMs from Donor 1. The color coding represents the normalized expression of genes in MDMs infected with Ad-Vpr or Ad-Zs (see color key). Gene up-regulation is denoted in red and gene down-regulation is denoted in blue.

Figure 6. Validation of microarray data by qRT-PCR.

Peripheral blood mononuclear cells (PBMCs) isolated from Donor 1, Donor 2 and three other healthy donors (Donors 3–5) through leukophoresis were cultured in vitro and differentiated into human MDMs as described in Materials and Methods. At day 7, the MDMs were infected with Ad-Vpr or Ad-Zs. At 48 h post-infection, RNA was extracted and subjected to qRT-PCR to amplify the selected genes using specific primers. Relative mRNA levels of the indicated genes are shown. Values are expressed as the fold change in Ad-Vpr-infected cells compared to Ad-Zs-infected cells and normalized to the expression of a housekeeping gene (GAPDH). The results represent the mean ± standard deviation (SD) of three samples from one experiment (P<0.05).

Figure 7. Validation of differentially expressed genes at the protein level.

Human monocyte-derived macrophages (MDMs) were infected with Ad-Vpr or Ad-Zs, or mock-infected as a control. At 48 h post-infection, the cells were washed, lysed, and subjected to Western blot analyses with the indicated antibodies. A β-actin antibody was used as a loading control.