Introduction
HIV-1 infection of astrocytes results in an initial productive, non-cytopathogenic infection that diminishes to a poorly productive, persistent infection [
1,
2]. Even though astrocyte infection is ‘restricted,’ their infection results in cellular dysfunction, contributing to the development of HIV-associated neurocognitive disorders (HAND) [
3]. Though astrocytes lack surface CD4 receptor, astrocytes get infected by a CD4-independent viral entry, through the mannose receptor that is expressed on the surface of astrocytes [
4-
6]. Infected astrocytes initiate productive infection of HIV-permissive blood-derived cells in co-culture experiments [
7,
8]. Although potent highly active antiretroviral therapy (HAART) suppresses plasma viremia to undetectable levels [
9], it is unable to eliminate the virus from quiescent reservoirs [
10-
12] and/or from sanctuaries like the brain [
13-
15]. The astrocytic reservoir protected from HAART is capable of initiating new infections upon treatment discontinuation and constitutes an important hindrance to HIV clearance from the central nervous system (CNS) [
7,
16,
17].
Deciphering the innate molecular mechanisms that restrict HIV replication and establish persistent reservoirs is essential for developing strategies to reactivate the reservoir. Overcoming the blocks in HIV replication can lead to the upregulation of the gene expression, making these cells available to immune surveillance and HAART targeting. Efficient replication is blocked in astrocytes at different steps of the HIV life cycle [
1,
8,
18]. Although several pre-transcriptional and post-transcriptional mechanisms are shown to contribute to HIV latency, whether SAMHD1 contributes to the viral restriction in astrocytes is not known [
19,
20].
Specific ‘intrinsic host restriction’ factors restrict virus replication in mammalian cells. Some cells constitutively express these factors, and in others, they are induced by interferons as part of the innate immune response. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC-3G) [
21], bone marrow stromal cell antigen 2 (BST-2) [
22], tripartite motif protein 5 alpha (TRIM-5α) [
23], and cellular miRNAs [
24] are examples of restriction factors targeting HIV-1. SAMHD1, a recently identified nuclear protein restriction factor, hydrolyzes cellular deoxynucleotide (dNTP) pools and hampers retrovirus reverse transcription [
25]. Due to the limited deoxynucleotide substrate availability, SAMHD1 inhibits HIV infection of myeloid cells and naïve CD4
+T cells [
26,
27]. However, SAMHD1-mediated restriction has not been explored in CNS cells. We hypothesized that increased expression of SAMHD1 restricts HIV infection in astrocytes. In this study, we compared the expression of SAMHD1 in astrocytes and microglia and correlated with the HIV replication.
Some studies have shown that SAMHD1 phosphorylation status correlates with the restriction activity but not its dNTPase activity [
28]. However, little is known about the post-transcriptional regulation of SAMHD1 expression. MicroRNAs have been implicated in the HIV gene expression and latency in CD4
+T cells [
29-
31]. It is not known whether miRNA modulation plays a role in HIV-1 restriction in astrocytes, and whether miRNA regulates SAMHD1 gene expression. Hence, in this report, we also examined SAMHD1 regulation by miRNA. Here, we report that astrocytes display higher SAMHD1 expression compared to microglia, which is in part accountable for HIV restriction in the astrocytes and miR-181a regulates SAMHD1.
Discussion
Astrocytes, which constitute 40% to 70% of total cells in the CNS [
1], perform key regulatory functions critical to brain function. The different CNS cell types are differentially infected with HIV; microglia being highly susceptible, astrocytes moderately restrictive, and neurons highly restrictive. Despite the lack of CD4 receptors, astrocytes become infected via CD4-independent mechanism [
6,
44]. In line with the earlier studies [
1,
2], we also found low level of HIV replication in astrocytes compared to microglia. After the initial productive phase, infection subsides to a persistent stage in astrocytes, which goes in hand with reports from other investigators [
1,
2,
32]. Infected astrocytes produce very low levels of virus even in the acute phase in contrast to infection of T-lymphocytes [
45,
46]. Our results with pseudotyped virus confirm that regardless of the entry routes, there are post-entry blocks to infection in astrocytes. Though the introduction of potent HAART has significantly controlled the viral replication in HIV patients, the current antiviral strategies target only actively replicating virus. Having said that, persistently infected brain astrocytes would not be susceptible to antiviral drugs, allowing the virus to persist in these reservoirs. Hence, the complete eradication of the virus from the host remains an impossible task. Several post-transcriptional and post-translational mechanisms [
19,
20], a number of host restriction factors [
21,
22], and certain cellular miRNAs [
24] have been reported to hinder retroviral replication in astrocytes contributing to HIV latency.
SAMHD1, a recently identified nuclear protein restriction factor, is highly expressed in HIV-1 non-permissive cells, whereas it is absent from HIV-1-sensitive T-cell lines such as Jurkat, SupT1, human peripheral blood acute lymphoid leukemia, and U937 [
27]. Since SAMHD1 has not been studied in CNS cells, our aim in this study was to explore the role of SAMHD1 in CNS cells. And we found that astrocytes and microglia express differential endogenous levels of SAMHD1, suggesting that possibly the higher level of SAMHD1 contribute towards HIV restriction in astrocytes. Ours is the first study presenting the role of SAMHD1 in astrocytes.
We found higher reverse transcriptase activity in microglia compared to astrocytes, suggesting low reverse transcriptase activity may be responsible for the restriction of HIV infection observed in astrocytes. Our results also substantiate that decrease in viral replication in astrocytes was in correlation with the increased expression of SAMHD1 and depletion of cellular SAMHD1 in astrocytes is sufficient to alleviate the restriction to HIV infection. This suggests that replication kinetics of the virus follows an inverse relation with SAMHD1 and confirms that SAMHD1 is significantly regulated over the course of infection in astrocytes. Interestingly, SAMHD1 silencing did not show any significant change in the infection in microglia. Our results support the role of SAMHD1 as a restriction factor that facilitates in HIV-1 restriction in astrocytes. Since the role of SAMHD1 is to deplete dNTPs required for RT, we feel that SAMHD1-mediated repression of HIV-1 replication in astrocyte is also due to its ability to repress RT. This goes in hand with the previous reports of SAMHD1 responsible for suboptimal reverse transcription of viral RNA in T cells and DCs [
25]. However, SAMHD1 may not be the only restriction factor playing a role in astrocytes. The possibility of additional SAMHD1-dependent/independent mechanisms cannot be excluded.
Complete eradication of HIV is possible only if HAART regimens totally stop all new infections of susceptible cells, along with flushing out existing reservoirs and blocking formation of additional long-lived viral reservoirs or reactivating reservoirs in chronically infected patients on HAART [
47,
48]. To reactivate viral replication, it is necessary to understand the regulatory mechanisms involved in establishing persistent reservoirs. We found that reactivation of viral replication by HDAC inhibitor, TSA, is accompanied by decrease in SAMHD1 expression. This was in contrast to a recent report showing increase in SAMHD1 expression in CD4-T cells with TSA exposure [
49]. De Silva
et al. indicated in their study that HDAC inhibition did not significantly change the SAMHD1 protein expression [
28]. Though we are not sure the reason for this difference, this suggests that SAMHD1 may be subjected to post-transcriptional regulation. Even though, the restricted HIV-1 replication in myeloid cells, resting T-cells and macrophages has been linked to SAMHD1 expression, little is known about its regulatory mechanism. So, in our next set of experiments, we focused our attention on the molecular mechanism of SAMHD1 regulation.
The miRNAs induce mRNA cleavage or translation repression by targeting complementary or partly complementary sequence in the 3′-untranscribed region (UTR) of target mRNAs [
50]. Certain cellular miRNAs have a physiological role in controlling HIV-1 replication and in the maintenance of HIV-1 latency in the peripheral cells [
29-
31]. We found six miRNAs (miR-124, −150, −155, −181a-d, −490, and −496) to have binding site on SAMHD1, suggesting that SAMHD1 may be regulated by these miRNAs. miR-181 is reported to be very strongly expressed in the brain [
41-
43], downregulation of which contributes to accelerated HIV-associated dementia [
51], and miR-155 is reportedly absent from latently infected cells [
30]. Hence, we concentrated our further experiments on these two miRNAs. In our efforts to verify the role of these miRNAs in the regulation of SAMHD1 and HIV, we saw that modulation of these miRNAs had an impact on the expression of SAMHD1 and also on HIV-1 replication. Combination of these two miRNA inhibitors resulted in a substantial increase in SAMHD1 expression. Interestingly, miR146a (non-targeting miRNA) inhibition resulted in a slight increase, which was unexpected. This may be because, miR146a controls HIV replication through some other means (other than SAMHD1). The restriction in the astrocytes was alleviated miRNA overexpression, which was mediated by the change in SAMHD1 expression. Viral miR-K12-11, an orthologue of cellular miR-155, has been reported to target SAMHD1 [
52], which goes in hand with our finding. To the best of our knowledge, ours is the first study showing miR181a regulation of SAMHD1 in astrocytes.
In summary, the importance of our study is that it provides a proof of concept that SAMHD1 facilitates HIV-1 restriction in astrocytes and miR-181a/miR-155 regulates SAMHD1 expression. Overexpression of these cellular miRNAs or suppression of SAMHD1 removed the inhibition of HIV-1 replication resulting in increased viral production from the astrocytes. As a result, this may allow persistently infected astrocytes to be exposed to immune surveillance or for the action of HAART. At this time, this concept is of theoretical nature, as we did not have any in vivo data. Future in vivo experiments are needed to evaluate if optimal combinations of miRNA or SAMHD1 siRNA delivered to astrocytes along with HAART could reactivate the HIV replication in persistently latent cells.
Material and methods
Cells and reagents
The primary astrocytes (HA #1800) and microglia (HM #1900) types are isolated from normal human brain and are commercially available (Sciencell Research Laboratories, Carlsbad, CA, USA). Cells were grown in the specified media as recommended by the manufacturer. Depending upon the experiments, cells were cultured at a concentration of 5 × 105 cells/ml in six-well plates (for transfection experiment) or 1 × 106 cells/ml and allow them to reach at least 70% confluency before any further treatment.
HIV infection
Microglia and astrocytes (1 × 106 cells/ml) were infected with HIV-1Bal, (NIH AIDS Research and Reference Reagent Program; catalog no. 510) at a concentration of HIV-1 p24 100 ng/106 cells for 24 h. Cells were washed to remove unbound virus and replenished with fresh media.
SAMHD1 mRNA quantification
For SAMHD1 mRNA quantification, total cellular RNA was extracted using the RNeasy mini kit (Qiagen, Limburg, the Netherlands) according to the manufacturer’s guidelines. One hundred nanograms of the total RNA from each cell type was used as a template for first-strand cDNA synthesis using high-capacity reverse transcriptase kit (Applied Biosystems, Waltham, MA, USA, #4368814). SAMHD1 mRNA was quantitated by SYBER Green-based quantitative real-time PCR (Stratagene-3000; Stratagene, La Jolla, CA, USA) analysis was performed using the specific primers. Quantification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was carried out for normalization. Relative expression of mRNA species was calculated using the comparative C
T method.
Immunoblotting of SAMHD1
To assess SAMHD1 protein levels, cells were harvested after respective treatments and/or transfection (supernatant was collected for the detection of p24 and reverse transcriptase activity assay). Cells were washed twice with PBS and homogenized in cell lysis buffer (MPER mammalian protein extraction reagent, Thermo Scientific, Waltham, MA, USA) supplemented with protease inhibitor mixture (Thermo Scientific, Waltham, MA, USA) by incubating on ice for 15 min. The homogenates were centrifuged at 10,000 rpm for 10 min and the supernatant was used for further analysis. Protein quantification was carried out by using Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Thirty micrograms of whole cell lysate was electrophoretically separated on an SDS-polyacrylamide gel. Separated proteins were transferred to nitrocellulose paper for 90 min at 100 V, using a wet electroblotting system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Blots were blocked for 1 h in 5% non-fat dry milk in Tris-buffered saline-Tween (TBST) (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5), followed by incubation overnight with the primary antibody (1:500 SAMHD1 polyclonal antibody; # sc-86212, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) in 1% milk solution. After several washes in TBST, the membranes were incubated in TBST/2% non-fat dry milk, containing the donkey anti-goat coupled to horse radish peroxidase (1:50,000 dilution; #sc-2020, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at RT. After washes in TBST, immunoreactivity was visualized using Western blotting detection reagent (SuperSignal West Pico enhancer, Thermo Scientific, Waltham, MA, USA). Expression of GAPDH (monoclonal mouse, Sigma-Aldrich, St. Louis, MO, USA, 1:50,000) was used as loading control.
SAMHD1 siRNA transfection
SAMHD1-siRNA and scrambled siRNA were procured from Santa Cruz Biotechnology. Astrocytes (2 x 105) were seeded into antibiotic-free Dulbecco's Modified Eagle’s medium (DMEM) supplemented with 10% FBS 24 h before transfection. On day of transfection, 75 pmols siRNA duplex and 6 μl of siRNA transfection reagent (sc-29528) are diluted into 100 μl serum and antibiotic-free siRNA transfection medium (sc-36868) in separate tubes. Both the solutions are mixed gently and incubated for 30 min at room temperature. This mixture is diluted with 0.8 ml siRNA transfection medium, overlayed onto the cells and incubated at 37°C in a CO2 incubator. After 5 h, cells are supplemented with 1 ml of normal growth medium containing two times the normal serum and antibiotics concentration without removing the transfection mixture. Cells were left untreated on infected with HIV and incubated for another 72 h. A second round of transfection was performed 24 h after the first transfection, whenever it was needed. Cells were harvested for RNA or protein isolation, and supernatant collected for p24 and RTase measurements.
miRNA expression assay
Total RNA was isolated using the mir-Vana miRNA isolation (#:1560; Ambion), in accordance with the manufacturer’s protocol. RNA concentrations were determined and 2 ng of each RNA sample were used for cDNA synthesis using TaqMan MicroRNA reverse transcription kit. Quantitative real-time RT-PCR (qRT-PCR) was performed using specific primers for miR-155 and miR-181a (Applied Biosystems, Waltham, MA, USA). All reactions were analyzed using StepOne Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). miRNA levels were normalized to U6 snRNA control and the cycle threshold (Ct) values were calculated.
miRNA transfection
Cells in 12-well plates were transfected either with anti-miR inhibitor or miRNA mimic (Applied Biosystems, Waltham, MA, USA) in inhibition or in overexpression experiments respectively, using Lipofectamine according to the manufacturer’s instructions. Briefly, 5 ul Lipofectamine was added to 200 ml of serum free medium (DMEM + F10) per condition for 5 min followed by incubation with anti-miR or miR-mimic at a final concentration of 50 nM. The volume was adjusted to 1 ml with medium containing 10% FCS and added to the cells. Following transfection, cells were treated with HIV or left uninfected for 72 h after which the cells were harvested for RNA or protein isolation, and supernatant collected for p24 and RTase measurements.
HIV LTR
Following HIV infection/transfection, cells were collected, RNA isolated (RNAeasy mini kit; Qiagen, Limburg, the Netherlands) and qRT-PCR run to amplify a 180 bp fragment in the LTR-R/U5 region that represents early stages of reverse transcription of HIV-1 using the following primers: 5′-primer, 5′-TCTCTCTGGTTAGACCAGATCTG; 3′-primer, and 5′-ACTGCTAGAGATTTTCCACACTG.
Reverse transcriptase activity
Reverse transcriptase activity of samples was assayed using a colorimetric reverse transcriptase assay (Roche Applied Science, Basel, Switzerland). Supernatants were collected at the end of infection, and the viruses were pelleted by ultra-centrifugation at 100,000 g for 120 min at 4°C. Virus pellets were resuspended and lysed by adding 40 μl lysis buffer, followed by addition of 20 μl of mixture containing template/primer hybrid and nucleotide, and incubated for 15 h at 37°C. Samples were then processed as per kit protocol for ELISA and absorbance read at 405 nm. Reactions were performed in triplicate, and experiments were performed thrice and the results averaged.
p24
p24 Ag concentration in the culture supernatants were assayed in an ELISA method using ZeptoMetrix RETRO-TEK ELISA kit (ZeptoMetrix Corporation, Buffalo, NY, USA) as per manufacturer’s recommendations.
TSA treatment
For the TSA (Cell Signaling, Danvers, MA, USA) treatment, after reaching confluency, the cells were with 50 nM TSA for 24 h and washed and proceeded with cell isolation for RT-PCR and Western blot.
Flow cytometry
Cells (1 × 106) were permeabilized with perm wash buffer and stained with fluorescein isothiocyanate (FITC)-conjugated SAMHD1 monoclonal ab (Abcam plc, Cambridge, MA, USA; #ab128107) at 4°C for 20 min, and washed. Samples were acquired using FACSCalibur (BD Biosciences, San Jose, CA, USA) with collection of 100,000 cells and analyzed using FlowJo software. SAMHD1 ab was conjugated to FITC by EasyLink FITC Conjugation Kit (Abcam plc, Cambridge, MA, USA; #ab102885) as per the kit protocol.
Plasmids, transfections, and luciferase assay
The following plasmids were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pHIV-CAT (cat#2619) from Dr. Gary Nabel and Dr. Neil Perkins; pSVTat (cat#294) from Dr. Alan Frankel; pNL4-3-Luc.R−E−(cat#3418) Dr. Nathaniel Landau, Aaron Diamond AIDS Research Center, The Rockefeller University; and pHEF-VSV-G from Dr. Lung-Ji Chang. Amphoteric HIV pseudoviruses containing the firefly luciferase gene were produced by transfecting 293 T cells with pHEF-VSV-G (10 μg) and pNL4-3. Luc.R-E- (10 μg) in 90-mm culture dishes. Briefly, plasmid DNA was diluted in Opti-MEM serum free media (Invitrogen, Carlsbad, CA, USA). Lipofectamine LTX and PLUS reagent (Invitrogen, Carlsbad, CA, USA) diluted in equal volume and added to diluted DNA at a 1:1 ratio. Mixture was incubated for 5 min to allow formation of DNA-liposome complexes. Culture supernatants containing the pseudoviruses were collected 72 h post-transfection and clarified by centrifugation at 400 g. Pseudoviruses were competent for single round of replication. Astrocytes infected with the HIV-luciferase pseudovirus (20 ng) were harvested 3 dpi, washed with PBS and lysed. Lysates were pre-cleared by centrifugation at 12,000 g for 2 min at 4°C. Luciferase concentration in whole cell lysates from cells was determined using a Luciferase assay system (Promega, Madison, WI, USA), according to the manufacturer’s instructions (Technical Bulletin #TB281). One hundred microliters of luciferase assay reagent were dispensed in a 96-well microplate (Costar, Corning, NY), 20 μl of cell lysate added to each well, mixed by pipetting, and read on a luminometer (Synergy HT, BioHIT, Sartorius AG, Goettingen, Germany).
Statistical analysis
Results are presented as mean ± SE of three independent experiments. Differences between treated and untreated cultures were compared by Student’s t-test. Comparisons of more than two groups were made using analysis of variance (ANOVA) (Kruskal-Wallis). Data were analyzed using Graphpad Prism version 5.0 software and statistical significance considered when P value ≤0.05.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SPK, MN, AR, and SKS conceived and designed the experiments. SPK, VSRA, PD, SC, and MC performed the experiments. SPK, AR, and VS analyzed the data. SPK and MN contributed the reagents/materials. SPK prepared the manuscript. All authors read and approved the final manuscript.