Cytosolic sulfotransferase 1A1 regulates HIV-1 minus-strand DNA elongation in primary human monocyte-derived macrophages

Abstract

Background: The cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases, which sulfonate proteins, glycoproteins, glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs), which sulfonate various small molecules including hormones, neurotransmitters, and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions, cell signaling, and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism, namely reverse transcription.

Methods: MDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions, MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels, and qPCR was used to measure viral RNA and DNA products.

Results: We demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs, and through siRNA knockdown experiments, we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription.

Conclusions: These studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules, raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics.

Figures

Fig. 1

SULT1A1 is highly expressed in primary human monocyte-derived macrophages (MDMs). a The cellular sulfonation pathway. The first step of the cellular sulfonation pathway involves import through a sulfate transporter of a sulfate ion that is then used as a substrate by either 3′-phosphoadenosine-5′phosphosulfate (PAPS) synthetase enzymes PAPSS1 or PAPSS2. These proteins catalyze two enzymatic steps to generate PAPS, the high-energy universal sulfonate-donor from sulfate and two molcules of ATP. PAPS can be transported across the Golgi membrane and used by the Golgi sulfotransferases to generate sulfonated proteins, glycoproteins, glycoproteins, glycolipids, and proteoglycans. Alternatively, PAPS can be used by cytosolic sulfotransferases (SULTS) to sulfonate small molecules such as hormones, neurotransmitters, and xenobiotics. b Human CD4+ T cells and CD14+ monocytes were isolated from donor PBMCs by magnetic bead isolation. Resting CD4+ T cells were lysed directly after separation, and the remaining CD4+ T cells were activated using CD3/CD28 beads for three days. Monocytes were cultured for 7 days in the presence of 20 ng/ml M-CSF, were lysed, subjected to gel electrophoresis, and immunoblotting was performed to detect SULT1A1 or the loading control Ku86 protein

Fig. 2

SULT1A1 knockdown is associated with decreased viral gene expression following infection of MDMs with VSV-G pseudotyped HIV-1 and SIV vectors. a Schematic showing experimental timeline. Briefly, CD14+ monocytes were isolated from human donor PBMCs using positive selection with magnetic beads. Monocytes were differentiated into MDMs and on day 7 were electroporated with siRNA and plated at 1.5 × 104 MDMs per well in a 48 well plate. After 96 h, protein knockdown was confirmed by immunoblot and cells were infected with 100 μl (corresponding to 164 ng p24 HIV-1 and 234 ng p27 SIV viral vectors) of the indicated virus. Luciferase and cell viability measurements were determined at 24 h post-infection. b Representative immunoblot showing SULT1A1 knockdown 96 h post transfection with SULT1A1 siRNAs (1–3) and AllStars Negative siRNA Control (ASN), Qiagen. SULT1A1 expression is compared to endogenous Ku86 used as a loading control. Results from one representative donor are shown. SULT1A1 expression was generally decreased by 70–80 % with siRNA treatment compared to the control ASN siRNA (as shown in Fig. 2c, middle panel). c HIV-1 luciferase reporter expression (left panel), SULT1A1 protein expression (middle panel), and cell viability of MDMs (right panel) were measured 24 h after infection with the VSV-G pseudotyped NL43-Luc HIV-1 vector. Results shown are from 6 donors assayed twice. All values were compared to ASN control. d SIV-1 luciferase reporter expression (left panel), SULT1A1 protein expression (middle panel), and cell viability (right panel) for MDMs 24 h post infection with VSV-G-pseudotyped SIVagm-Luc. Mean and SD shown, *** p < 0.0005 ** p < 0.005 * p < 0.05 one sample t test. Results shown are from 6 donors assayed twice. Samples with <60 % SULT1A1 knockdown and/or <65 % cell viability were not used in the analysis. All values were compared to ASN control

Fig. 3

SULT1A1 knockdown is associated with decreased viral gene expression following infection of MDMs with a replication-competent HIV-1 vector. a The same method was used as in Fig. 2a, however 100 μl (corresponding to 66.3 ng p24) HIV-1 Env + JM1186-Rluc virus was added to the cells at day 11 and luciferase and cell viability was assayed at 72 h post-infection. b HIV-1 luciferase reporter expression, SULT1A1 protein expression, and cell viability of MDMs 24 h post infection with HIV-1 Env + JM1186-RLuc.. Mean and SD shown, *** p < 0.0005 ** p < 0.005 * p < 0.05 one sample t test. Results shown are from 6 donors assayed twice. Samples with <60 % SULT1A1 knockdown and/or <65 % cell viability were not used in the analysis. All values were compared to ASN control

Fig. 4

SULT1A1 regulates HIV-1 reverse transcription. a MDMs were treated with control siRNA or SULT1A1-specific siRNA and subesequently challenged with VSV-G pseudotyped NL43-Luc HIV-1 vector. DNA was isolated 24 h post-infection and qPCR was performed using primers that detect early RT DNA products or late RT DNA products compared to the cellular PBGD gene as an endogenous control. The levels of early and late RT products were normalized to the ASN siRNA control. Results shown are from MDMs derived from 6 donors tested twice. b MDMs were transfected with SULT1A1 siRNA 2 or ASN siRNA and total cellular RNA was isolated 24 h post infection with VSV-G pseudotyped NL43-Luc HIV-1 vector. qPCR using primers specific for HIV-1 multiply spliced mRNA (MS RNA, forward and reverse primers span the first and second exons of Tat/Rev, respectively) was performed, and relative MS RNA (normalized to GAPDH) was then normalized to ASN siRNA control. Results shown are from MDMs derived from 6 donors tested twice. c Representative immunoblot showing HIV-1 Vpu and Vif protein levels compared to endogenous Ku86 or GAPDH loading control, respectively, from protein lysate collected 48 h post infection with VSV-G pseudotyped NL43-Luc HIV-1 vector pre-treated with either ASN siRNA control or SULT1A1 siRNA 2. d Quantitative immunoblot analysis using Image Studio software of Vpu and Vif as shown in Fig. 4c. Mean and SD shown, *** p < 0.0005 ** p < 0.005 * p < 0.05 one sample t test. Results shown are from 6 donors assayed twice. Samples with <60 % SULT1A1 knockdown and/or <65 % cell viability were not used in the analysis. All values were compared to ASN control

Fig. 5

SULT1A1 influences the kinetics of minus-strand DNA elongation. a The step during HIV-1 reverse transcription that SULT1A1 regulates was investigated by quantitative real-time PCR analysis with the indicated primer sets. The stages of reverse transcription are shown: 1. Viral genomic RNA; 2. Minus-strand DNA initiation; 3. Minus-strand DNA transfer; 4. Minus-strand DNA elongation and plus-strand DNA initiation; 5 and 6. Plus-strand DNA transfer; 7. Plus-strand DNA elongation. b MDMs were treated with control or SULT1A1-specific siRNA and challenged with VSV-G pseudotyped NL43-Luc HIV-1 vector. Total cellular DNA was isolated at 8, 16, or 24 h post infection. Quantitative real-time PCR was performed to measure the relative abundance of DNA products corresponding to specific steps during the process of reverse transcription using primers indicated in Fig. 5a. Results shown are from MDMs derived from 6 donors assayed once. Mean and SD shown, *** p < 0.0005 ** p < 0.005, one sample t test