Abstract
N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in cells or HIV-1-infected individuals. Here we show that m6A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in human monocytic cells. Transfection of differentiated monocytic cells with HIV-1 RNA fragments containing a single m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered RNA m6A levels by manipulating the expression of the m6A erasers or pharmacological inhibition of m6A addition in virus-producing cells. RNA transfection and viral infection of differentiated monocytic cells demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies revealed that m6A of HIV-1 RNA escaped the RIG-I-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Moreover, RNA of peripheral blood mononuclear cells from HIV-1 viremic patients showed increased m6A levels that correlated with increased IFN-I mRNA expression compared to levels from HIV-1-suppressed patients on antiretroviral therapy. Together, our results suggest that RNA m6A modifications regulate viral replication and antiviral innate immune responses in HIV-1-infected individuals.
Author Summary HIV-1 is known as a weak inducer of antiviral cytokines including IFN-I, but it is unclear how HIV-1 evades innate immunity. Different types of RNA modifications including m6A within the HIV-1 genome modulate viral replication; however, the role of m6A modifications of HIV-1 RNA in regulating innate immune responses remains elusive. In this study, we found that HIV-1 RNA modified with m6A suppresses the expression of IFN-I in differentiated monocytic cells by avoiding innate immune detection of viral RNA mediated by RIG-I, an RNA sensor in host cells. We also observed significantly increased RNA m6A modifications of peripheral blood mononuclear cells from HIV-1 viremic patients compared to virally suppressed patients on combined antiretroviral therapy, suggesting a functional link between m6A modifications and antiretroviral treatment. Investigating the functions of m6A modifications of HIV-1 RNA in regulating innate immune sensing and IFN-I induction in monocytic cells can help understand the mechanisms of HIV-1 persistence.
Introduction
Transcriptional modification of RNA in cells plays a crucial role in its stability, transportation, processing and thus regulation of gene expression. There are more than 160 RNA modifications identified in eukaryotes [1]. Methylation at the N6 position of adenosine (m6A) is a post-transcriptional RNA modification in internal and untranslated regions (UTRs) of eukaryotic mRNAs, microRNAs, small nuclear RNAs and long noncoding RNAs, which is important for RNA localization, stability and protein translation [1–5]. This methylation is controlled by two types of protein factors in cells, comprised of the writer complex [(methyltransferase-like 3 (METTL3) and METTL14] to incorporate methylation, and the erasers [fat mass and obesity associated protein (FTO) and α-ketoglutarate dependent dioxygenase AlkB homolog 5 (ALKBH5)] to remove m6A modification [6–9]. RNA m6A modification has been discovered in several RNA and DNA viruses over the past 40 years, although its effects on the viral lifecycle remain not fully understood [10–15]. Recent advancement of RNA sequencing based strategies expanded the identification and characterization of m6A to several clinically significant human pathogens [16], including HIV-1 [17–19]. Increasing evidence suggests that m6A modification plays a major role in regulation of viral replication and gene expression [16] and the immune system [20].
In the early stage of virus infections, sensing viral nucleic acids in infected cells is a critical step to induce innate immune responses that can lead to production of antiviral cytokines, including IFN-I (mainly IFN-α and IFN-β) [21]. Genomic RNA of HIV-1 and other viruses can be detected by cytosolic sensors, including retinoic acid-induced gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) [21]. Detection of viral RNA by these sensors triggers activation of several cellular kinases, which phosphorylate interferon regulatory factors 3 and 7 (IRF3 and IRF7) to induce IFN-I expression [22, 23]. HIV-1 is a weak inducer of host innate immune responses [24, 25], and it evades immune recognition by direct targeting of immune pathways, interacting with cellular proteins, or masking the viral genome from the cytosolic sensors [24, 26, 27]. HIV-1 RNA can be sensed by both RIG-I and MDA5, whereas it has evolved multiple strategies to escape innate immune surveillance [23, 28]. A recent study showed that 2’-O-methylation in HIV-1 RNA prevents MDA5-mediated sensing in myeloid cells, and thereby reduces IFN-I induction [29]. However, the role of m6A in regulating innate immune responses to HIV-1 RNA and the underlying mechanisms have not been defined.
Our previous in vitro studies showed that HIV-1 infection or HIV-1 envelope protein treatment of CD4+ T cells significantly up-regulates m6A levels of cellular RNA independently of viral replication [30]. However, it remains unclear whether m6A levels and IFN-I expression in HIV-1-infected individuals can be altered by effective antiretroviral therapy (ART), which leads to undetectable viral load in the vast majority of treated HIV-1 patients [31]. To address these fundamental questions and to better understand the role of m6A in HIV-1 infection in vivo, we measured the levels of m6A and IFN-I expression in peripheral blood mononuclear cells (PBMCs) of healthy donors, HIV-1 viremic patients before ART, and HIV-1 patients on ART.
Here we show that m6A modifications of HIV-1 RNA reduce viral RNA sensing and the induction of IFN-I in differentiated monocytic cells. We found that m6A-defective HIV-1 RNA induced IFN-I expression through RIG-I-mediated pathway, suggesting that m6A is an immune evasion strategy of HIV-1. In contrast to in vitro results, we also observed significantly increased levels of m6A RNA modifications and IFN-I expression in PBMCs from HIV-1 viremic patients compared to patients on ART. These results implicate that RNA m6A modifications can contribute to regulation of viral replication, innate immune responses, and ART in HIV-1-infected individuals.
Results
A single m6A modification of HIV-1 RNA oligos inhibits IFN-I induction in U937 cells
To examine the effect of m6A modification of HIV-1 RNA on IFN-I induction, we designed two different RNA oligos corresponding to two fragments of HIV-1 genome each with or without a single m6A modification [32] for transfection experiments. We have reported that these two m6A modifications in the 5’ untranslated regions (UTR) of HIV-1 genome are important for HIV-1 RNA binding to the m6A reader proteins (YTH domain family proteins 1-3) in vitro and viral replication in cells [32]. The m6A-modified RNA oligos 1 and 2 (both 42 mer) contained a single m6A-modified adenosine in the conserved GGACU motif of the HIV-1 (NL4-3 strain) genome [32]. The m6A modification of the oligos was confirmed by immunoblotting with equal amounts of RNAs using m6A-specific antibodies (Fig. 1A and 1D). To mimic cellular responses to viral RNA in non-dividing macrophages, we differentiated monocytic U937 cells with phorbol 12-myristate 13-acetate (PMA) before transfection with the RNA oligos. Compared to unmethylated control (Ctrl) RNA, m6A-modified RNA oligo 1 induced 3-to 4-fold lower (P < 0.005) levels of IFN-α and IFN-β mRNA in transfected cells (Fig. 1B and 1C). Similar results were obtained with transfection of oligo 2, although the effects were less significant compared to oligo 1 (Fig. 1E and 1F). These results indicate that m6A modification of the 5’ UTR of HIV-1 RNA fragments inhibits IFN-I induction in differentiated U937 cells.
Inhibition of m6A modifications of HIV-1 RNA by FTO increases IFN-I induction
The m6A erasers (FTO and ALKBH5) orchestrate cellular mRNA functions by removing m6A modifications on mRNA [2]. To investigate whether m6A modifications of HIV-1 genomic RNA could suppress IFN-I induction in cells, purified RNA from HIV-1 virions was demethylated with recombinant FTO in vitro, resulting in a 10-fold decrease in m6A level relative to control HIV-1 RNAs (Fig. 2A). Transfection of m6A-reduced HIV-1 RNA into U937 cells induced 3-fold higher IFN-α and IFN-β expression (P < 0.0005) compared to control RNAs (Fig. 2B and 2C), suggesting that m6A modification of HIV-1 genomic RNA suppresses IFN-I induction in myeloid cells.
To determine the effect of m6A of HIV-1 RNA on IFN-I induction during viral infection, HIV-1 containing lower levels of m6A in viral RNA was generated by overexpression of the eraser FTO in HIV-1-producing HEK293T cells. Compared to the vector control, FTO overexpression in HEK293T cells had no significant effect on the expression of HIV-1 Gag and capsid (CA, or p24) proteins (Fig. 2D). HIV-1 derived from FTO-overexpressed HEK293T cells (m6A-lower HIV-1) showed 10-fold lower m6A levels of viral RNA compared to viruses derived from control cells (Fig. 2E). When PMA-differentiated U937 cells were transfected with RNA of m6A-lower HIV-1, a 2-fold increase (P < 0.05) of IFN-α and IFN-β expression was observed compared to control HIV-1 RNA (Fig. 2F and 2G). As a positive control, poly(I:C) induced approximately 190-fold increases of IFN-α and IFN-β expression in transfected U937 cells (Fig. 2F and 2G). Moreover, differentiated U937 cells infected with m6A-lower HIV-1 expressed 2-fold higher (P < 0.05) IFN-α and IFN-β relative to control HIV-1 (Fig. 2H and 2I). Thus, HIV-1 containing reduced RNA m6A modifications induces higher IFN-I expression in differentiated U937 cells.
Inhibition of m6A modifications of HIV-1 RNA by ALKBH5 increases IFN-I induction
To confirm the results of FTO treatment and overexpression, we also examined the effect of another m6A eraser ALKBH5 on HIV-1 RNA-mediated IFN-I induction in PMA-differentiated U937 cells. ALKBH5 overexpression in HIV-1-producing HEK293T cells had no significant effect on the expression of HIV-1 Gag and CA (Fig. 3A). The m6A modification of HIV-1 RNA generated from ALKBH5-overexpressed HEK293T cells showed a 2-fold decrease compared to HIV-1 RNA from control cells (Fig. 3B). IFN-α and IFN-β levels in U937 cells transfected with HIV-1 RNA from ALKBH5-overexpressed HEK293T cells were 1.8-fold higher (P < 0.05) compared to that from control cells (Fig. 3C and 3D). Furthermore, infection of U937 cells with HIV-1 from ALKBH5-overexpressed HEK293T cells induced 2-fold higher IFN-α and IFN-β expression (P < 0.0005) compared to HIV-1 from control HEK293T cells (Fig. 3E and 3F). Thus, inhibition of m6A modifications of HIV-1 RNA by eraser overexpression in virus-producing cells increases IFN-I induction in differentiated U937 cells.
Knockout (KO) of erasers increases m6A levels in HIV-1 RNA and reduces IFN-I induction
To validate the results from eraser overexpression, we constructed FTO-KO and ALKBH5-KO HEK293T cell lines by the CRISPR-Cas9 method. Next, these cell lines were transfected to generate HIV-1 with increased m6A of viral RNA. Western blotting results showed that FTO and ALKBH5 were completely silenced and HIV-1 Gag protein expression was not significantly affected by FTO and ALKBH5 knockout (Fig. 4A). HIV-1 RNA from FTO-KO and ALKBH5-KO cells showed 7- and 25-fold higher m6A levels, respectively, relative to that from control (Con-KO) cells (Fig. 4B). Transfection of PMA-differentiated U937 cells with HIV-1 RNA derived from FTO-KO or ALKBH5-KO cells showed a 3-4-fold decrease (P < 0.05) in IFN-I expression compared to that from Con-KO cells (Fig. 4C and 4D). Moreover, infection of PMA-differentiated U937 cells with HIV-1 from FTO-KO or ALKBH5-KO cells induced approximately 2-fold less IFN-I expression (P <0.005) compared to Con-KO cells (Fig. 4E and 4F). Thus, increasing m6A levels in HIV-1 RNA by eraser KO in virus-producing cells reduces IFN-I induction in differentiated monocytic cells.
m6A-defective HIV-1 RNA induces IFN-I expression through IRF3 and IRF7 phosphorylation
We next investigated pharmacological inhibition of m6A modification using 3-deazaadenosine (DAA), an inhibitor of S-Adenosylhomocysteine (SAH) hydrolase that can catalyze the reversible hydrolysis of SAH to adenosine and homocysteine [33]. DAA causes SAH accumulation thereby elevating the ratio of SAH to S-adenosylmethionine (SAM), a substrate of m6A modification, and subsequent inhibition of SAM-dependent methyltransferases [33]. DAA-treatment of HEK293T cells did not affect HIV-1 production and release, but reduced m6A level in HIV-1 RNA 7-fold compared to control cells (Fig. 5A and 5B). Transfection of PMA-differentiated U937 cells with purified RNA from HIV-1 produced from DAA-treated HEK293T cells (DAA-HIV-1) induced 15-fold and 2.3-fold higher IFN-α and IFN-β expression (P < 0.0005), respectively (Fig. 5C and 5D). Moreover, infection of PMA-differentiated U937 cells with DAA-HIV-1 induced a 2-3-fold increase in IFN-I expression (P < 0.0005) compared to viruses from control HEK293T cells (Fig. 5E and 5F). These data further validate that m6A of HIV-1 RNA suppresses IFN-I induction in differentiated monocytic cells.
Because IFN-I expression is predominately driven by IRF3 and IRF7 after their activation by phosphorylation upon virus infections [21, 34], we tested whether DAA-HIV affected phosphorylation of IRF3 and IRF7. Compared to mock-infected U937 cells, control HIV-1 and DAA-HIV-1 induced strong phosphorylation of IRF3 and IRF7 in differentiated U937 cells at 4 h post-infection (Fig. 5G). Notably, phosphorylation of IRF3 and IRF7 was 1.7-fold and 1.2-fold higher in U937 cells infected with DAA-HIV-1 relative to control HIV-1, respectively (Fig. 5G). These results suggest that inhibition of HIV-1 RNA m6A modification triggers innate immune responses by inducing IRF3/7-mediated IFN-I expression in myeloid cells.
RIG-I, but not MDA5, contributes to m6A modification of HIV-1 RNA induced IFN-I expression
To characterize the cellular sensing mechanisms of m6A-defective HIV-1 RNA, RIG-I and MDA5 in U937 cells were silenced by KO and shRNA, respectively. RIG-I-KO U937 cells were constructed and undetectable RIG-I expression was confirmed (Fig. 6A). To test whether these cells responded to RNA stimulation, poly(I:C) was transfected into cells and IFN-I expression was measured. Compared to untransfected cells (mock), poly(I:C) transfection induced high levels of IFN-I expression in RIG-I-KO and control U937 cells (Fig. 6B). As expected, the induction of IFN-I by poly(I:C) was significantly reduced by 2-fold in RIG-I-KO U937 cells (P < 0.005) compared to control cells (Fig. 6B), confirming that RIG-I acted as an RNA sensor to induce IFN-I expression in these cells. In control U937 cells, transfection of single m6A-modified HIV-1 RNA oligos induced lower IFN-I expression (P < 0.0001) compared to unmethylated RNA oligo counterparts (Fig. 6C and 6D). However, in RIG-I-silenced U937 cells, transfection of m6A-modified HIV-1 RNA oligos had no effect on IFN-I expression relative to unmethylated control oligos (Fig. 6C and 6D), suggesting a pivotal role of RIG-I in sensing m6A-defective HIV-1 RNA.
Furthermore, we examined the potential role of MDA5 in sensing m6A-defective HIV-1 RNA in monocytic cells. MDA5 expression was substantially reduced in differentiated U937 cells with MDA5 knockdown (shMDA5) compared to vector control (shCon) cells (Fig. 7A). As a positive control, poly(I:C) transfection induced high levels of IFN-I expression in both shCon and shMDA5 U937 cells. As expected, poly(I:C) transfection into shMDA5 U937 cells significantly decreased IFN-I levels relative to shCon cells (Fig. 7B). These cells were then examined for their ability to induce IFN-I expression by HIV-1 5’ UTR RNA oligos with or without single m6A modification [32]. Compared to unmethylated HIV-1 RNA oligos, transfection of m6A-modified HIV-1 RNA oligos reduced IFN-I expression in both shCon and shMDA5 U937 cells (Fig. 7C and 7D), suggesting that MDA5 is not a specific cellular sensor to detect m6A-defective HIV-1 RNA in differentiated monocytic cells.
HIV-1 infected patients have higher level of m6A modification in the RNA of PBMCs
To explore the significance of m6A modifications in HIV-1-infected individuals and investigate the effect of ART on m6A levels, we measured the levels of m6A and IFN-I mRNA in immune cells from HIV-1 viremic patients in comparison with healthy control donors and HIV-1 patients on ART. We obtained PBMCs from healthy control donors (n=9), HIV-1 viremic patients (n=6) with different viral load pre-therapy, and HIV-1-infected individuals on ART (n=16) whose viral load was undetectable for a minimum of 6 months (<20 copies/mL) (supplemental Table S1). The average m6A level in total RNA of PBMCs from HIV-1 viremic patients was significantly higher (P < 0.005) compared to that from patients on ART (Fig. 8A and Supplemental Fig. S1A), suggesting an inverse correlation between viral load and m6A level of patient PBMCs. A visible, but not statistically significant increase (P = 0.27) in cellular RNA m6A level was observed in viremic patients compared to healthy individuals (Fig. 8A and Fig. S1A). This observation is also consistent with our previous results showing that HIV-1 infection or treatment of cells with HIV-1 envelope proteins (Env) upregulated m6A levels in primary CD4+ T-cells in vitro [30]. It is possible that Env shedding from HIV-1 viremic patients could upregulate m6A levels in PBMCs.
Next, the levels of IFN-I mRNA in PMBCs were measured to analyze potential correlations with the RNA m6A levels. Compared to PBMCs from healthy donors, there was a trend of increase in IFN-α expression, and a significant increase in IFN-β expression in PBMCs from the viremic patients (Fig. 8B and 8C). Compared to PBMCs from the viremic patients, a significant decrease in both IFN-α and IFN-β expression (P < 0.005) was observed in patients on ART (Fig. 7B and 7C), suggesting that HIV-1 suppression by ART reduces innate immune responses to viral infection. Consistent with our results, a previous study [35] also reported similar results of decreased IFN-α expression in HIV-1 patients on ART compared to patients without ART.
Enhanced m6A levels in viremic patients could not be attributed to inherent differences in the levels of m6A writers and erasers, because there was no significant change in the expression of METTL3, FTO and ALKBH5 in PBMCs from the three groups (Fig. 8D and Fig. S1B). However, a slight increased level of METTL14 expression was observed in PBMCs from patients with ART compared to viremic patients (Fig. 8D and Fig. S1B). Together, these results suggest that HIV-1 infection upregulates the m6A level of cellular RNA in PBMCs from viremic patients without altering the expression of the writers and erasers.
Discussion
HIV-1 genomic RNA contains 10-14 sites of m6A modifications in the 5’-, 3’-UTR and several coding regions [17–19]. Recent studies indicate that m6A modification has important effects on HIV-1 replication, gene expression, and host responses to viral infection [30, 32, 36].
It has been shown that cellular enzymes involved in RNA m6A modifications negatively regulate the innate immune response to infection of human cytomegalovirus, influenza A virus, adenovirus, or vesicular stomatitis virus by targeting the IFN-I pathway [37, 38]. A recent study showed that m6A modifications of human metapneumovirus RNA mimic the host RNA to avoid RIG-I-mediated innate immune sensing, and thereby reduce the production of IFN-I and enhance viral replication [39]. However, it remains unknown whether m6A modifications of HIV-1 RNA have any impact on innate immune responses.
In this study, we show that m6A modifications of HIV-1 RNA act as a negative regulator of IFN-I induction by avoiding RIG-I-mediated RNA sensing in PMA-differentiated U937 cells. We observed that two different HIV-1 RNA oligos of the HIV-1 5’-UTR containing a single m6A-modification significantly reduced IFN-I induction relative to their unmodified RNA counterparts. The different inhibitory effects on IFN-I induction by two m6A-modified RNA oligos compared to their unmodified counterparts might be due to different sequences or conformation of the RNA fragments [32]. We also demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, but HIV-1 RNA with increased m6A modifications had opposite effects. Our results suggest that HIV-1 genomic RNA and viral transcripts are masked by m6A modifications to avoid RIG-I-mediated sensing and IFN-I induction during viral infection. Thus, HIV-1 has likely evolved an immune evasion strategy through m6A modification of viral RNA (Fig. S2).
Several RNA modifications, such as N-1-methylpseudouridine, 5-methylcytidine (m5C), 5-hydroxymethylcytidine, 5-methoxycytidine, and 2’ fluoro-deoxyribose, have significant impact on RIG-I- and MDA5-mediated RNA sensing [40]. In addition to m6A modification, HIV-1 genomic RNA contains eight types of epitranscriptomic modifications that are higher than the average cellular mRNA, with m5C and 2’-O-methyl modifications being most prevalent [41]. It is possible that HIV-1 RNA exploits multiple epitranscriptomic modifications to avoid innate sensing as mechanisms of immune evasion. This possibility may explain how HIV-1 is able to avoid innate immune responses to establish persistent and latent infection even in patients on combined ART [31].
The IFN-I gene itself is m6A-modified and targets its destabilization for the maintenance of homeostatic state in mouse and humans [38]. Rubio et al. showed that, following human cytomegalovirus infection, depletion of METTL14 or increase in ALKBH5 proteins leads to decrease level of m6A in IFN-β gene and stabilizes and elevates the IFN-I response [37]. In this study, we observed increased m6A levels in cellular RNA of PBMCs from HIV-1 viremic patients compared to HIV-1 suppressed patients on ART. However, we did not observe significant changes in the levels of m6A writers and erasers in PMBCs from healthy donor, HIV-1 viremic patients, and HIV-1 patients on ART. These results are consistent with our previous data showing increased m6A levels in HIV-1 infected primary CD4+ T-cells in the absence of altered expression of m6A writers or erasers [30]. It is possible that HIV-1 may modulate the activity or localization of writers or eraser, thereby upregulating m6A levels in HIV-1 infected cells. It remains to be established whether m6A modification of HIV-1 RNA regulate innate immune responses in primary CD4+ T-cells or macrophages.
We found that m6A-modified HIV-1 reduces the activation of IRF3 and IRF7 through RIG-I-mediated signaling to suppress IFN-I induction. However, it remains unclear how m6A modifications of HIV-1 RNA reduces phosphorylation of IRF3 and IRF7 during early stage of HIV-1 infection. Previous studies suggest that HIV-1 proteins can target several cellular RNA and DNA sensors including RIG-I to surpass the IFN-I response [42–45]. Moreover, HIV-1 can also target downstream proteins in the IFN-I pathway including IRF3 and IRF7 to contribute to chronic and persistent infection [46–50]. For example, HIV-1 Vpr protein mediates degradation of IRF3 to avoid the innate antiviral immune response [51].
Durbin et al. showed that a RIG-I-activating RNA ligand, the 106-nucleotide polyU/UC sequence derived from the 3’ UTR of hepatitis C virus with m6A modification bound RIG-I with low affinity and did not trigger the conversion to the activated RIG-I conformer and thus has an immunosuppressive potential [40]. Our data indicated that m6A-defective HIV-1 RNA enhanced RIG-I-mediated RNA sensing and IFN-I induction in cells. Further studies are needed to examine whether the m6A-modified HIV-1 RNA binds RIG-I with a low affinity, which might be the possible cause of reduced IFN-I induction during viral infection.
In summary, our study uncovered a previously unidentified strategy of how HIV-1 RNA escapes the host antiviral innate immune system through m6A modifications of its RNA genome. HIV-1 RNA m6A modifications can act as an immune suppressor of RIG-I-mediated viral RNA sensing. Our findings suggest that pharmacological reduction in m6A modification of HIV-1 RNA may enhance IFN-I-mediated innate antiviral immune responses, thereby inhibiting viral replication.
Materials and Methods
Cell culture
HEK293T cell line was a kind gift from Vineet KewalRamani (National Cancer Institute, USA) and maintained in complete Dulbecco’s modified Eagle’s medium (DMEM) as described [32]. U937 cell line was obtained from the American Type Culture Collection (ATCC) and maintained in complete RPMI-1640 medium as described [52]. All the cell lines were maintained at 37 °C in 5% CO2 and tested negative for mycoplasma contamination using a universal mycoplasma detection kit (ATCC 30-1012K) as described [53].
Plasmids and HIV-1 RNA oligos
The HIV-1 proviral DNA construct pNL4-3 was used to generate viral stocks as described [19]. For over-expression of the m6A erasers, the corresponding control vectors, pCMV6-FTO, pCMV-ALKBH5 were described [9, 54]. For knockout of eraser genes, CRISPR-Cas9 vectors containing sgControl, sgFTO, and sgALKBH5 were used as described [38]. For RIG-I knockout, pCR-BluntII-Topo-sgRIGI-1 and 2 vectors were described [55], which were kindly provided by Dr. Stacy Horner (Duke University, USA), and the plasmid hCas9 (catalog no. 41815, Addgene) was described [56]. For MDA5 and RIG-I knockdown, shControl, shMDA5 and shRIG-I plasmids [29] were kindly provided by Dr. Yamina Bennasser (Université de Montpellier, France). Four RNA oligo sequences are from the 5’ UTR of HIV-1 genomic RNA (NL4-3 strain) with or without a single m6A site [32], which were commercially synthesized (Integrated DNA Technologies). The sequences and the location of the m6A sites in the conserved GGACU motifs of the HIV-1 genome were described [32] and are listed below: RNA oligo 1 (nt. 235-281, the m6A-modified adenosine is nt. 241): 5’-CGCAGGACUCGGCUUGCUGGAGACGGCAAGAGGCGAGGGGCG-3’. To eliminate RNA dimerization in our previous RNA binding assays [32], the original dimer initiation sequence of HIV-1 (AAGCGCGC) in oligo 1 was replaced with the underlined nucleotides GAG. RNA oligo 2 (nt. 176-217, the m6A-modified adenosine is nt. 197): 5’-AGCAGUGGCGCCCGAACAGGGACUUGAAAGCGAAAGUAAAGC-3’.
Generation of U937 cells with MDA5 knockdown or RIG-I knockout, and HEK293T cells with FTO or ALKBH5 knockout
For MDA5 knockdown U937 cell line construction, HEK293T cells were transfected with shControl or shMDA5, together with pMD2.G and psPAX2 plasmids by polyethyleneimine (PEI) [53]. At 48 h post-transfection, lentiviruses were harvested and purified to infect U937 cells for 48 h and then the U937 cells were selected in RPMI-1640 media with 1 μg/mL puromycin. To generate RIG-I knockout cells, pCR-BluntII-Topo-sgRIGI-I or pCR-BluntII-ToposgRIGI-2, along with hCas9, which has neomycin (G148) resistance, were transfected into U937 cells by TransIT mRNA transfection kit (mirus, USA) for 48 h according to the manufacturer’s protocol. Then, G418 (1 mg/mL) was added to transfected cells for 8 days to select RIG-I knockout U937 cells, which were confirmed by Western blotting. For Control, FTO, and ALKBH5 knockout HEK293T cell generation, HEK293T cells were transfected with corresponding single guide RNAs (sgRNAs), together with pMD2.G and psPAX2 plasmids. At 48 h post-transfection, lentiviruses were collected to infect fresh HEK293T cells for 48 h. Then, the single clones were selected by 1 μg/mL puromycin in 96 well plates. The KO cells were confirmed by DNA sequencing and for specific protein expression by Western blotting.
Dot immunoblotting of m6A modification in RNA
RNA was extracted from purified and concentrated HIV-1 stocks by using TRIzol (Invitrogen) or RNA purification kit (Qiagen). The synthesized RNA oligos were directly used for dot-blot assays as described [30]. Briefly, HIV-1 RNA or RNA oligos (diluted to 100 μL using 1 mM EDTA) were mixed with 60 μL of 20× saline-sodium citrate (SSC) buffer (3 M NaCl, 0.3 M trisodium citrate) and 40 μL of 37% formaldehyde (Invitrogen) and incubated at 65 °C for 30 min. Nitrocellulose membrane (162-0115, Bio-Rad) or nylon membranes (11209299001, Roche) were pre-soaked with 10X SSC for 5 min and assembled in dot-blot apparatus (Bio-Rad) with vacuum-on. Equal amounts of RNA were transferred to nitrocellulose or nylon membranes, then membranes were washed twice with 200 μL of 10× SSC buffer. Nylon membranes were washed once with TBST buffer (20 mM Tris, 0.9% NaCl, and 0.05% Tween 20) for 5 min and stained with methylene blue staining (MB119, Molecular Research Center) for 2-5 sec followed by two or three washes with ddH2O. Nitrocellulose membranes were blocked with 5% milk in TBST buffer and used to detect m6A levels by probing with m6A specific antibodies (Synaptic Systems; 202 003). Images were taken by Amersham Biosciences Imager 600 (GE Healthcare) and analyzed by ImageJ software (National Institutes of Health). Densitometry quantification of relative RNA m6A levels was normalized to MB staining as described [30].
In vitro FTO demethylation of HIV-1 RNA m6A
Demethylation of HIV-1 RNA m6A was performed with recombinant FTO treatment of purified HIV-1 RNA. Briefly, 500 ng HIV-1 RNA were used for FTO in vitro treatment in 100 μL reaction buffer containing 50 mM HEPES buffer (pH7.0), 75 μM (NH4)2Fe (SO4)2•6H2O, 2 mM L-ascorbic acid, 300 μM L-ascorbic acid, 200U RNAsin, 5 μg/mL BSA, and 0.2 nmol FTO protein. The reaction was performed at 37 °C for 1 hr and then stopped by adding 5 mM EDTA. Finally, RNA samples were denatured at 70 °C for 2 min and quickly put into ice for m6A detection.
HIV-1 production, p24 quantification, U937 cells transfection and HIV-1 infection assays
HIV-1 stocks were generated by transfection of HEK293T cells with the proviral DNA pNL4-3 using PEI as described [53]. Cell culture medium was exchanged at 6-8 h post-transfection with supernatants and was harvested at 48 h. The cell culture media containing viruses were filtered (0.45 μm) and purified by 25% sucrose using an SW28 rotor (Beckman Coulter) at 141,000g for 90 min. The pellet was resuspended with PBS and digested with DNase I (Turbo, Invitrogen) for 30 min at 37 °C. To extract HIV-1 genome RNA, concentrated HIV-1 virions were lysed by Trizol (Invitrogen) and RNA was purified by phenolic-chloroform sedimentation and isopropanol precipitation. For transfection, cells were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 24 h and changed with fresh RPMI-1640 media for another 24 h. PMA-differentiated U937 cells were then transfected with TransIT mRNA transfection kits (Mirus) according to the manufacturer protocol. At 16 h post-transfection, cells were harvest for RT-qPCR analysis. For infection assays, HIV-1 p24 levels were quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-p24-coated plates (The AIDS and Cancer Virus Program, NCI-Frederick, MD) as described [30]. PMA-differentiated U937 cells were infected by equal amounts of HIV-1 (250 pg of p24) for 16 h and then cells were collected for Western blotting or RT-qPCR analysis.
Antibodies and immunoblotting
The antibodies used in this study were: anti-GAPDH (AHP1628, Bio-Rad), anti-FLAG (F1804, Sigma-Aldrich), anti-METTL3 (15073-1-AP, Proteintech Group), anti-METTL14 (HPA038002, Sigma-Aldrich), anti-FTO (ab124892, Abcam), anti-ALKBH5 (HPA007196, Sigma-Aldrich), anti-MDA5 (D74E4, Cell signaling), anti-RIG-I (D14G6, Cell signaling), anti-HIV-1 Gag (clone #24-2, the NIH AIDS Reagent Program), anti-IRF3 (124399, Abcam), anti-phospho-IRF3 (49475, Cell Signaling), anti-IRF7 (SC-9083, Santa Cruz), anti-phospho-IRF7 (5184, Cell Signaling) and anti-m6A polyclonal rabbit Ab (202003, Synaptic Systems). Cells were harvested and lysed in cell lysis buffer (Cell Signaling) supplemented with protease inhibitor cocktails (Sigma-Aldrich). Immunoblotting was performed as described [30]. Detection of GAPDH expression was used as a loading control.
Quantitative RT-PCR
Real-time quantitative RT-PCR (qRT-PCR) was performed as described [53] to assess the relative levels of IFN-α and IFN-β mRNA expression in cells induced by HIV-1 RNA transfection or HIV-1 infection. Following primers (IDT) were used:
IFN-α, F 5’-GTACTGCAGAATCTCTCCTTTCTCCT-3’
IFN-α, R 5’-GTGTCTAGATCTGACAACCTCCCAGG-3’
IFN-β, F 5’-AACTTTGACATCCCTGAGGAGATTAAGC-3’
IFN-β, R 5’-GACTATGGTCCAGGCACAGTGACTGTAC-3’
GAPDH, F 5’-GGAAGGTGAAGGTCGGAGTCAACGG-3’
GAPDH, R 5’-CTGTTGTCATACTTCTCATGGTTCAC-3’
Ethics statement
The study using human PBMCs from healthy control subjects and HIVpositive individuals has been approved by the Institutional Review Board of the University of Iowa. The study was conducted according to the Declaration of Helsinki guidelines.
PBMCs from healthy donors and HIV-1 patients
Healthy control subjects and HIV-positive individuals attending the University of Iowa HIV Clinic who were receiving ART and had HIV-1 viral load levels below the limit of detection (< 20 copies/mL) for over 6 months were invited to participate in these studies, and all provided written informed consent. HIV-1 viral load was determined using the COBAS® AmpliPrep/COBAS® TaqMan HIV-1 test (Roche). PBMCs were purified using BD Vacutainer® CPT™ Mononuclear cell preparation tubes (BD Biosciences) as recommended by the manufacturer. Cells were stored in 92% fetal calf serum, DMSO in liquid nitrogen until use. PBMCs were obtained with 9 healthy donors, 6 HIV-1 viremic patients, and 16 HIV-1 patients treated with ART (Supplemental Table 1). Both viral RNA and protein were isolated from these PBMCs at the same day using Ambion Paris RNA and protein extraction kit (ThermoFisher Scientific) and stored in −80 °C until use. The RNA was quantitated using the NanoDrop spectrophotometer (ThermoFisher Scientific) and was used for m6A dot-blot detection (200 ng) and IFN-α and IFN-β mRNA analyses as described [53]. The protein was quantitated using Pierce BCA reagent (ThermoFisher Scientific) and subjected to Western blot analysis of the m6A writers and erasers.
Statistical analyses
Data were analyzed using either Mann-Whitney’s t-test or the one-way analysis of variance (ANOVA) with Prism software and statistical significance was defined as P < 0.05. All experiments were repeated at least three times.
Author contributions
S.C., S.K., and N.T. performed experiments and contributed to manuscript preparation. J.W. and J.T.S. provided PBMCs from HIV-1 patients and uninfected individuals, and helped data analyses. L.H. and C.H. provided recombinant FTO and the in vitro m6A demethylation protocol. All authors analyzed data and contributed to experiment design. S.C. and S.K. drafted the manuscript. L.W. conceived the study, supervised the work, and revised the manuscript. All authors contributed to manuscript editing and revision.
Competing interests
The authors declare no competing interests.
Supplemental Fig. S1-S2 legends
Acknowledgments
This work was supported in part by National Institutes of Health grants R01AI150343 and R01AI141495 (to L. W.). We thank Alexis Hawkins for technical assistance in detection cellular m6A levels of PBMCs, and the Wu lab members for helpful discussions and suggestions. We also thank Michael Cahill for outstanding technical support. The authors appreciate generous reagents from Drs. Yamina Bennasser, Stacy Horner, Vineet KewalRamani, Noam Stern-Ginossar, and the NIH AIDS Reagent Program.