HIV-1 Vpr Induces Interferon-Stimulated Genes in Human Monocyte-Derived Macrophages

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.


Introduction
Antigen-presenting cells (APCs) are critical for both innate and adaptive immunity. Professional APCs such as macrophages play an integral role in the immune pathogenesis of the human immunodeficiency virus type 1 (HIV-1) [1]. HIV-1 is a member of the lentivirus family and is the etiologic agent of acquired immunodeficiency syndrome (AIDS). It interacts with host cells through multiple signaling pathways to establish the disease [2]. The infection involves complex mechanisms through which HIV-1 overcomes the host immune responses and causes reprogramming of the host transcriptome and proteome [3][4][5].
A striking feature of Vpr is its unique potential to promote viral productivity in monocytes/macrophages and in a small population of CD4 + T-cells [22][23][24][25][26]. Although Vpr is thought to play an important role in HIV-1-infected human macrophages [1,3,6,11,21,23], little is known about how it disrupts the expression profile of host cellular genes. In this study, we analyzed the effect of Vpr on the expression profiles of host cellular genes in human monocyte-derived macrophages (MDMs), with the idea that such an analysis would provide useful information about the involvement of genes not yet identified through biochemical approaches. Human MDMs were generated from peripheral blood mononuclear cells (PBMCs) and infected with a recombinant adenovirus expressing Vpr, and analyzed by cDNA microarray. HIV-1 Vpr protein induced interferon (IFN)-stimulated genes (ISGs) such as IRF7, and caused phosphorylation of STAT1 at tyrosine 701 in human MDMs. These findings enhance the current understanding of HIV-1 replication and pathogenesis in human macrophages.

Expression of Vpr and ZsGreen1 in human MDMs
To better understand the role of HIV-1 Vpr protein in human MDMs, a recombinant adenovirus expressing ZsGreen1 and FLAG-tagged Vpr, Ad-Vpr, was generated. As a control, a recombinant adenovirus expressing ZsGreen1, Ad-Zs, was used. A schematic diagram of both recombinant adenoviruses is shown in Figure 1A. To examine whether Vpr induces cell-cycle arrest at the G2 phase, HeLa cells were infected with Ad-Vpr or Ad-Zs at a multiplicity of infection (MOI) of 50. At 48 h post-infection, cells were harvested for analysis of DNA content and stained with propidium iodide (PI). The DNA content of ZsGreen1-positive cells was analyzed by flow cytometry, which revealed a dramatic increase in the proportion of cells in the G2 phase of the cell cycle in cells infected with Ad-Vpr (21.22% and 70.37% were in the G1 and the G2+M phases, respectively, and the G2+M: G1 ratio was 3.32) compared to cells infected with the control Ad-Zs (54.06% and 23.87% were in the G1 and G2/M phases, respectively, and the G2+M: G1 ratio was 0.44) ( Figure 1B). These results indicate that the recombinant adenovirus expressing FLAG-Vpr induces G2 cell-cycle arrest.
Purified and titrated Ad-Vpr and Ad-Zs were next used to infect MDMs derived from peripheral blood monocytes from two normal healthy donors ( Figure 2). PBMCs were isolated from heparinized whole blood from two healthy donors by standard density gradient centrifugation with Ficoll-Paque. PBMCs were harvested from the interface and CD14 + cells were separated by high-gradient magnetic sorting using MACS beads. The isolated CD14 + cells were differentiated into MDMs for 7 days, and then infected with the Ad-Vpr or Ad-Zs at a MOI of 100. After 48 h, the cells were either observed under a fluorescence microscope or lysed and analyzed for the expression of Vpr and ZsGreen1 protein by Western blotting. Fluorescence microscopy showed that ZsGreen1 was expressed in both Ad-Vpr-and Ad-Zs-infected MDMs compared to mock-infected controls, which remained ZsGreen1-negative ( Figure 2A). As shown in Figure 2B, a 26 kDa band representing ZsGreen1 and a 14 kDa band representing Vpr was detected; these apparent molecular masses are consistent with their respective predicted sequences. Further, there was no difference in ZsGreen1 expression between the two populations of MDMs ( Figure 2B). These results confirm the suitability of the adenovirus-infected MDMs for downstream assays.

Microarray analysis of MDMs infected with Ad-Vpr or Ad-Zs
To evaluate changes in the expression of host cellular genes in response to HIV-1 Vpr protein, Ad-Vpr-and Ad-Zs-infected macrophages were subjected to cDNA microarray analyses using a commercially available Affymetrix GeneChip oligonucleotide array (Human Genome U133 Plus 2.0), which interrogates more than 47,000 transcripts from 38,500 genes. This approach enabled us to monitor Vpr-induced changes in the global gene profile of the MDMs. Data analysis using GeneSpring GX software showed that Vpr modulated the expression of 557 genes in Donor 1 and 116 genes in Donor 2. Given that the array analyzes more than 47,000 gene transcripts, this is considered a minor change in the global host gene profiles ( Figure 3). Heat maps from both donors ( Figure 3) show that the global gene expression profiles were different in each donor, indicating that there is individual variability in the response to Vpr at the transcriptional level.
The differentially regulated genes were filtered to determine gene entities common to both donors. Out of 557 genes altered in response to Vpr in Donor 1 and 116 genes in Donor 2, only 66 genes were common to both ( Figure 4A). Gene ontology was ranked based on the corrected p-values. The ten most significant pathways common to both donors are shown in Figure 4B. HIV-1 Vpr significantly altered the expression profiles of cellular genes mainly involved in the innate immune response, type I IFN signaling, and cytokine-mediated signaling. A complete list of all 66 genes common to both donors is shown in the form of heat maps in Figure 4C.
Validation of the expression of host genes involved in the type 1 IFN pathway by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Validation of the results obtained by microarray analysis was performed by qRT-PCR evaluating the mRNA levels of selected up-regulated genes involved in the immune response. Genes were selected for confirmation either because they were known to be induced in response to type I IFN and reportedly involved in the innate immune antiviral response [27,28] or because they were  common to both donors. The transcriptional levels of 15 genes were measured in Donor 1 by qRT-PCR with the primers listed in Table 2, using GAPDH as an internal control. In general, there was a strong correlation between the microarray data and the qRT-PCR data at 48 h post-infection; the two techniques yielded very similar expression profiles for all 15 genes in Donor 1 ( Figure 6 and Table 1). However, there were some discrepancies, e.g., the qRT-PCR results showed a slightly higher increase than the microarray analysis for IFI27. Similarly, the expression levels of IFIT1, IFI44L, MX1, and RSAD2 (which encodes the viperin protein) were higher in the microarray data compared to their respective relative expression levels in the qRT-PCR data. On the other hand, in Donor 2 except for IRF7 (4-fold) and MX1 (5-fold) higher expression levels of APOBEC3A (233-fold), ISG20 (132fold), IFIT2 (97-fold), IFIT1 (51-fold), ISG15 (38-fold), IFI27 (47fold), IFI44L (36-fold), TNFSF10 (TRAIL) (29-fold), RSAD2 (22fold), MX2 (17-fold), IFIT3 (16-fold), DDX58 (12-fold) and STAT1 (5-fold) were observed by qRT-PCR compared to their respective microarray data ( Figure 6 and Table 1). These inconsistencies were probably due to the differences in transcripts variants or due to the intrinsic differences between the two techniques, notably in the normalization methods. For microarray experiments, the normalization was based on a large number of genes, whereas in the qRT-PCR experiments, a single housekeeping gene was used as an internal control against which the results were normalized. Overall, the qRT-PCR results were in agreement with the array data i.e. differential up-regulation giving us strong confidence in the interpretation of the gene expression data obtained through microarray.
Next, to demonstrate whether similar results could be obtained in other healthy donors, the transcriptional levels of these 15 genes were measured in MDMs derived from three additional healthy donors (Donors 3-5) by qRT-PCR. As shown in Figure 6, the expression profiles of the three additional donors were generally consistent with the data obtained from Donor 1. However, the expression levels of the IFI27 and IFI44L genes, which were upregulated approximately 7-fold and 40-fold, respectively, in the presence of Vpr in MDMs derived from Donor 1, were only slightly up-regulated in Donors 3 and 4. These results indicated that the activation of the type I IFN pathway was common to all the tested healthy donors.

Confirmation of protein expression by Western blotting
Finally, Western blotting was performed to examine the effect of HIV-1 Vpr on the protein expression levels of IRF7, STAT1, ISG15, ISG20, APOBEC3A, and TRAIL in MDMs. Cell lysates were prepared from Ad-Vpr, Ad-Zs, or mock-infected MDMs and subjected to Western blotting using specific antibodies. bactin was used as a loading control. Consistent with the microarray data and the qRT-PCR results, STAT1, ISG15, ISG20, IRF7, and TRAIL were up-regulated in Ad-Vpr-infected macrophages compared to Ad-Zs-or mock-infected controls ( Figure 7); however, APOBEC3A, which was originally shown to be up-regulated at the transcriptional level by both microarray and real-time PCR, was not induced at the protein level compared to controls, as measured by Western blotting (Figure 7). Why the APOBEC3A gene transcript failed to express its gene product is not clear; however, differential regulation of gene transcription does not ensure a corresponding change in gene product levels. Taken together, these results clearly indicate that HIV-1 Vpr protein leads to the activation of the type I IFN pathway and the subsequent up-regulation of various ISGs in human MDMs.

Discussion
The data presented herein are the first analysis of the changes in gene transcription that occur following in vitro infection of human MDMs with an adenovirus expressing HIV-1 Vpr protein.
Although some previous studies have shown that HIV-1 infection leads to the activation of innate immunity and thus the induction of various ISGs in human MDMs [5,[29][30][31][32][33][34][35][36][37], the specific role of Vpr in the induction of ISGs in human MDMs has not been documented.
In this study, by utilizing an Affymetrix oligonucleotide microarray, we demonstrated that the majority of the genes differentially regulated by Ad-Vpr in both donors were involved in the immune response, indicating the important role played by HIV-1 Vpr protein in human MDMs. A large number of genes from this group is predicted to be activated during the innate immune response ( Table 1) as part of the host defense response to clear viral infections [27,28,38]. We observed an increase in the levels of various ISGs such as MX1, IFI44L, DDX58, RSAD2, and several of the IFITs, which have been shown to play an important role against HIV-1 infection in MDMs [31]. MX2 has recently been reported to be an IFN-induced inhibitor of HIV-1 infection in human monocytoid cell lines [39]. Since the differential expression levels of the MX1, MX2, IFIT1, IFIT2, IFIT3, IFIT27, IFI44L, DDX58, and RSAD2 genes obtained through microarray strongly correlated with the real-time PCR data, it is reasonable to speculate that the expression of these proteins may be up-regulated following HIV-1 infection in human MDMs.
Real-time PCR data and Western blot analysis confirmed the activation of IRF7 by HIV-1 Vpr in human MDMs (Figures 6 and  7). IRF7 is the master regulator of type I IFN-dependent immune responses [40] and plays an important role in HIV-1 pathogenesis [34]. IRF7 promotes autocrine and paracrine activation of STAT1 and plays a critical role in virus-mediated induction of IFN-a [41]. It is known that type I IFNs activate the Janus kinases (JAKs) and the STAT transcription factors, which ultimately leads to the expression of target genes [42,43]. The STAT1 gene encodes a 91-kDa protein which is activated by both type I and type II IFNs [44]. This important transcription factor is phosphorylated by the JAKs in response to proinflammatory and regulatory factors [38]. It has been shown that the STAT1 pathway plays an important role in the pathogenesis of HIV-1 infection [45,46]; indeed, activation of the STAT1 pathway by HIV-1 Vpr is demonstrated in this study. We further showed that in the presence of Vpr protein the level of STAT1 phosphorylation at tyrosine 701 is much higher than the control recombinant adenovirus (Figure 7). The exact mechanism through which Vpr leads to the phosphorylation of STAT1 at tyrosine 701 is not known and requires further study.
HIV-1 Vpr protein caused the up-regulation of various ISGs, such as ISG15 and ISG20 (Figures 6 and 7), which can inhibit    [27,28]. Previously, it has been shown that HIV-1 Vpr protein activates NF-kB [47], which might explain the up-regulation of various ISGs in our study. The ISGs act through a variety of mechanisms to render cells resistant to viral infection [27]. It has been shown that ISG15 is induced in HIV-1-infected MDMs [27], where it restricts and impedes HIV-1 replication by causing ISGylation of viral Gag protein and certain cellular factors [33]. Similarly, ISG20 has been shown to exhibit antiviral activity against HIV-1 [48]. Induction and activation of ISGs such as ISG15, ISG20, the IFITs, and viperin are thought to be the reason MDMs are relatively resistant to cell death and can act as long-term carriers of HIV-1 [31]. The observation that these genes were up-regulated in Ad-Vpr-infected MDMs in our study suggests that in HIV-1-infected macrophages, Vpr is responsible for the induction of these ISGs; thus due to these ISGs, macrophages are relatively resistant to Vpr-induced cell death. TRAIL protein is produced after HIV-1 infection in monocytes due to the IFNa/b-mediated activation of the STAT1 signaling cascade [49], and has been shown to cause apoptosis in several cell lines during HIV-1 infection. Although an initial increase in TRAIL protein was shown to kill HIV-1-infected macrophages  [50], the exact role of TRAIL-mediated apoptosis in the elimination of HIV-1-infected cells is not known. Here, we have shown that HIV-1 Vpr protein caused elevated levels of TRAIL protein in macrophages (Figures 6 and 7), which would presumably help to eliminate HIV-1-infected cells through TRAILmediated cell death [29,[50][51][52].  Our findings further demonstrated that HV-1 Vpr differentially regulated the expression levels of chemotactic cytokines such as CXCL1, CXCL5, CXCL7, CXCL9, CXCL10, and CXCL11 (Table 1). A previous report has shown that CXCL10 and CXCL11 are up-regulated in HIV-1-infected macrophages and play a key role in the recruitment and spread of HIV-1 to susceptible CD4 + T-cells [53]. Surprisingly, our microarray data also showed that CXCL10 and CXCL11 were up-regulated in MDMs, by 23-fold and 7-fold, respectively (Table 1). Whether HIV-1 Vpr has a role in HIV-1 dissemination and the mechanism through which Vpr leads to the differential regulation of these chemokines in MDMs is not known. However, the recruitment of susceptible T-cells by HIV-1-infected human macrophages and the role of CXCL10 and CXCL11 will be intriguing to investigate in future studies.
HIV-1 Vpr is essential for efficient infection of non-dividing cells such as macrophages. It has been shown that HIV-1 Vpr is expressed within infected cells and is packaged into HIV-1 virions. Although, the virion-associated Vpr is able to cause cell cycle arrest of CD4 + T cells in vivo [54], the induction of ISGs by this biologically active form of Vpr is not known. However, our recent studies have confirmed that the induction of ISGs in HIV-1 AD8 / Vpr + infected MDMs (Unpublished results) is similar to ISGs induced by Vpr in Ad-Vpr infected MDMs. Our data indicating that Vpr leads to the induction of ISGs and activation of innate immune responses is contrary to some of the previously published reports which showed that Vpr helps HIV-1 to escape the innate immune responses by either counteracting the UNG2, a host cellular intrinsic factor which inhibits HIV-1 replication [55][56][57] or by manipulating the cellular SLX4 complex which is a negative regulator of Type 1 IFN production [58]. Therefore, the complex role played by Vpr in escaping HIV-1 virions from innate immune responses or by activating innate immunity through inducing ISGs in HIV-1 infected macrophages must be investigated in future studies.
Our data confirmed that HIV-1 Vpr leads to the induction of ISGs in MDMs. However, our findings showed some donorspecific differences in the expression profiles of these ISGs, which might be due to differences in overall susceptibility and the host response to the HIV-1 Vpr infection. These differences and the number of donors used in the study should not be considered a limiting factor because the expression profiles of the selected genes in all donors were independently confirmed by qRT-PCR with reproducible and consistent readouts each time ( Figure 6). Furthermore, we cannot rule out the possibility that some of these ISGs are regulated by direct or indirect interactions of Vpr with cellular proteins related to the innate immune response, including cellular transcription factors such as NF-kB, AP-1, and Sp-1 [1,47].
In conclusion, our studies have identified IRF7, STAT1, ISG15, ISG20, and TRAIL as key up-regulated molecules in MDMs harboring HIV-1 Vpr. Based on previous published reports and our present data; we suggest a potential role for these genes in host defense against HIV-1 replication and infection. Future studies to elucidate the mechanisms through which Vpr up-regulates these molecules as well as their roles in HIV-1 pathogenesis will certainly improve our understanding of the replication and pathogenesis of the HIV-1.

Cell culture and preparation of human MDMs
Human cervical HeLa cells and human embryonic kidney HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma) and 100 units/ mL penicillin/streptomycin (Sigma). Plasmid transfection was performed using Lipofectamine 2000 (Life Technologies).

Generation of recombinant adenoviruses
Adenoviruses were constructed using the Adeno-X TM expression system (Clontech Laboratories). Briefly, wild-type (wt) Vpr from HIV-1 NL43 [59] (GenBank accession no. M 19921) was PCR-amplified with the FLAG tag incorporated using the primers GAAGCTAGCGACTACAAGGATGACGATGACAAAATGG-AACAAGCCCCAGAAGA (forward) and GCTCTAGACTAG-GATCTACTGGCTCCAT (reverse), and cloned into the pShut-tle2 vector at the NheI and XbaI restriction sites. Similarly, the ZsGreen1 gene was PCR-amplified with the FLAG tag incorporated using the primers TAATCTAGAGACTACAAGGAT-GACGATGACAAAGCCCCTCTCCCTCCCCCCCCCCTAA (forward) and TAGCGGCCGCTCAGGGCAAGGCGGAGCC-GGAG (reverse) using the pRetroX-IRES2-ZsGreen1 plasmid (Clontech Laboratories) as a template, and then cloned into the pShuttle vector just downstream of Vpr at the XbaI and NotI restriction sites. The integrity of the generated recombinant plasmids was confirmed by DNA sequencing. Then the entire cassette (flanked by unique I-CeuI and PI-SceI restriction sites) was excised and ligated into Adeno-X viral DNA using the Adeno-X expression system 1, according to the manufacturer' s instructions (Clontech Laboratories).
Adeno-X viral DNA containing the FLAG-Vpr or ZsGreen1 was linearized with PacI and transfected into HEK293 cells with Lipofectamine 2000 (Life Technologies). The recombinant adenoviruses were purified using the Adeno-X maxi purification kit (Clontech Laboratories) and titrated using the Adeno-X rapid titer kit (Clontech Laboratories), following the recommendations of the manufacturer. The virus stocks were stored at 280uC for future use.

RNA extraction
MDMs were transduced with Ad-Vpr or Ad-Zs at a MOI of 100. The cells were harvested for RNA extraction at 48 h posttransduction. MDMs were washed three times with ice-cold PBS, and total RNA was extracted using the RNeasy mini kit with DNase digestion, according to the manufacturer's instructions (QIAGEN). RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher) and stored at 280uC. For microarray analysis, the quality of the RNA was determined using the Agilent Bioanalyzer (Agilent Technologies).
Microarray and data analysis RNA samples were analyzed by microarray using the GeneChip Human Genome U133 2.0 plus array (Affymetrix). Microarray hybridization and fluorescence detection were performed as described in the Affymetrix GeneChip Expression Analysis Technical Manual. The. cel data files generated by the Affymetrix microarray hybridization platform were analyzed using Gene-Spring GX ver. 12.0 software (Agilent Technologies). Probe-level analysis was performed using the RMA algorithm. Microarray data have been deposited in NCBI's Gene Expression Omnibus and assigned the GEO Series accession number GSE56591. Fold changes in gene expression, hierarchical clustering, and gene ontology annotations were determined.
Real-time qRT-PCR analysis of differentially expressed genes Total RNA was prepared using the RNeasy mini kit as described above. RT-PCR was performed using specific primers and One-Step SYBR Green PCR mix (Takara), according to the manufacturer's manual. qRT-PCR was performed using a Prism 7500 sequence detection system (Applied Biosystems). Samples were run in triplicate and all data were normalized to GAPDH mRNA expression as an internal control.

Western blotting
Mock or virus-infected MDMs were washed with PBS and then lysed with CelLytic TM MT Cell Lysis reagent (Sigma) which was supplemented with a protease inhibitor cocktail (Roche Diagnostics) according to the manufacturer's instructions. Protein concentrations were determined with a BCA protein assay kit (Pierce) using bovine serum albumin as a standard. Proteins were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyl difluoride (PVDF; Millipore Corp.) membranes. The PVDF membranes were probed with the primary antibodies mentioned above followed by an anti-mouse HRP or anti-goat HRP or anti-rabbit HRP secondary antibody (Sigma), and signals were detected by enhanced chemiluminescence (GE Healthcare).

Analysis of the cell cycle
HeLa cells were infected with an adenoviral vector expressing Vpr or expressing only ZsGreen1, as a control. At 48 h postinfection, the cells were harvested and fixed with 1% formaldehyde followed by 70% ethanol. Fixed cells were incubated in PBS containing RNase A (50 mg/ml) at 37uC for 20 min and then stained with PI (40 mg/ml). For each sample, at least 7,000 cells were analyzed using a FACS Calibur instrument (Becton-Dickinson) with CELL Quest software (Becton-Dickinson). Ratios of the numbers of cells in the G1 and G2/M phases (G2+M: G1 ratios) were calculated using ModFit LT Software (Verity Software House).