RIG-I activation inhibits HIV replication in macrophages

RIG-I signaling of macrophages against HIV can be beneficial in the treatment of HIV disease, where intracellular immune defense is compromised.

Abstract

The RIG-I signaling pathway is critical in the activation of the type I IFN-dependent antiviral innate-immune response. We thus examined whether RIG-I activation can inhibit HIV replication in macrophages. We showed that the stimulation of monocyte-derived macrophages with 5′ppp-dsRNA, a synthetic ligand for RIG-I, induced the expression of RIG-I, IFN-α/β, and several IRFs, key regulators of the IFN signaling pathway. In addition, RIG-I activation induced the expression of multiple intracellular HIV-restriction factors, including ISGs, several members of the APOBEC3 family, tetherin and CC chemokines, the ligands for HIV entry coreceptor (CCR5). The inductions of these factors were associated with the inhibition of HIV replication in macrophages stimulated by 5′ppp-dsRNA. These observations highlight the importance of RIG-I signaling in macrophage innate immunity against HIV, which can be beneficial for the treatment of HIV disease, where intracellular immune defense is compromised by the virus.

Introduction

Macrophages not only participate in the host anti-HIV-immune response but also are the target for HIV. HIV infection of macrophage plays a critical role in the HIV pathogenesis. The importance of macrophage in the pathogenesis of HIV infection is highlighted by its contribution to early-stage viral transmission, persistence, and virus dissemination throughout the body of the host [1]. It is believed that macrophages are an important virus reservoir and contribute to viral latency. Macrophages are resistant to the cytopathic effects of HIV and can support the virus replication and production for a long time [1–3]. As macrophage has the ability to cross the blood-tissue barrier and travel to tissues and organs, HIV-infected macrophages are important sources for the virus transmission [2].

Innate immunity is the first line of defense of viral infections, including HIV [4]. Viral infections result in induction of IFNs that are the key players in antiviral responses [5]. Induction of the IFN-based antiviral innate immunity depends on PRRs, including TLRs and the RIG-I-like receptors [6]. RIG-I is critical in the activation of the type I IFN-dependent antiviral innate-immune response. RIG-I can detect viral genomic RNA during negative-strand RNA virus infection [7] and trigger a type I IFN-mediated immune response that protects the host against viral infection [8]. A recent study showed that RIG-I could detect dimeric and monomeric forms of HIV viral RNA, resulting in the activation of the RIG-I signaling pathway [9]. Another study reported that genomic HIV secondary-structured RNA could induce innate-immune responses through the RIG-I-dependent signaling pathway in primary human PBMCs and macrophages [10]. However, HIV has the ability to inhibit RIG-I-mediated antiviral signaling through a protease-mediated sequestration of RIG-I [9]. In the present study, we examined whether the stimulation of macrophage with the RIG-I ligand 5′ppp-dsRNA can inhibit HIV infection of macrophages. We also examined the mechanisms involved in RIG-I-mediated anti-HIV activity in macrophages.

MATERIALS AND METHODS

Cell culture

Peripheral blood was purchased from the Center for AIDS Research at the University of Pennsylvania (Philadelphia, PA, USA). The protocol used to isolate blood from donors, purify the blood components, and distribute this material to the investigators was approved by the Institutional Review Board of the Center for AIDS Research. These blood samples were screened for all normal blood-borne pathogens and certified to be pathogen-free. Monocytes were purified from peripheral blood of three healthy adult donors, according to our technique described previously [11]. Freshly isolated monocytes were cultured in 48-well culture plates at a density of 2.5 × 10⁵ cells/well in DMEM containing 10% FCS. Macrophages refer to 7-day-cultured monocytes in vitro.

Reagents

LyoVec transfection reagent 5′ppp-dsRNA and 5′ppp-dsRNA control were purchased from Invivogen (San Diego, CA, USA). Mouse anti-HIV p24 mAb was obtained from the AIDS Research and Reference Reagent Program (NIH, Bethesda, MD, USA). Rabbit antibody against actin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

RIG-I ligand stimulation

Monocyte-derived macrophages were stimulated with RIG-I ligand (5′ppp-dsRNA, 1 μg/mL) using the LyoVec transfection reagent. Cells were collected for mRNA extraction 24 h poststimulation. As a negative control of the experiment, cells were stimulated with 5′ppp-dsRNA control.

Infection of macrophages with HIV Bal strain

HIV Bal strain was obtained from the AIDS Research and Reference Reagent Program (NIH). Macrophages were infected with equal amounts of cell-free HIV Bal (p24, 20 ng/10⁶ cells) for 2 h at 37°C after 24 h of stimulation, with or without 5′ppp-dsRNA. The cells were then washed three times with DMEM to remove unabsorbed virus, and fresh media were added to the cell cultures. The final wash was tested for HIV RT activity and shown to be free of residual inocula. Untreated cells served as a control. Culture SN was collected for HIV RT activity assay at Day 8 postinfection. Cell samples were collected for HIV gag gene and p24 protein expression at Day 12 postinfection.

HIV RT assay

HIV RT activity was determined based on the technique of Willey et al. [12] with modifications [13]. In brief, 10 μl culture SN from macrophages infected with or without HIV was added to a cocktail containing poly(A), oligo(dT) (Amersham Biosciences, Piscataway, NJ, USA), MgCl₂, and [³²P]deoxythymidine triphosphate (Amersham Biosciences) and incubated for 20 h at 37°C. Then, 30 μl of the cocktail was spotted onto DE81 paper (Whatman International, Maidstone, UK), dried, and washed five times with 2× saline-sodium citrate buffer and once with 95% ethanol. The filter paper was then air-dried. Radioactivity was counted in a liquid scintillation counter (PerkinElmer Life Sciences, Boston, MA, USA).

RNA extraction and real-time RT-PCR

Total RNA from macrophages was extracted with Tri Reagent (Molecular Research Center, Cincinnati, OH, USA), as described previously [14]. Total RNA (1 μg) was subjected to RT using the RT system (Promega, Madison, WI, USA) with random primers for 1 h at 42°C. The reaction was terminated by incubating the reaction mixture at 99°C for 5 min, and the mixture was then kept at 4°C. The resulting cDNA was then used as a template for real-time PCR quantification. Real-time PCR was performed with one-tenth of the cDNA with the iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA), as described previously [15]. The amplified products were visualized and analyzed using the software MyiQ provided with the thermocycler (iCycler iQ real-time PCR detection system; Bio-Rad Laboratories). The oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA), and sequences will be available upon request. The cDNA was amplified by PCR, and the products were measured using SYBR Green I (Bio-Rad Laboratories). The data were normalized to GAPDH and presented as the change in induction relative to that of untreated control cells.

Statistical analysis

Student's t-test was used to evaluate the significance of difference between groups, and multiple comparisons were performed by regression analysis and ANOVA. P values of <0.05 were considered significant. All data are presented as mean ± sd. Statistical analyses were performed with SPSS 11.5 for Windows. Statistical significance was defined as P < 0.05.

RESULTS AND DISCUSSION

As a PRR, RIG-I plays an important role in host innate immunity against viral infections. RIG-I recognizes viral RNA and activates the type I IFN-dependent antiviral innate-immune response [8]. Although RIG-I operates independently of the TLRs [16], RIG-I signaling culminates in the induction of the IFN-α/β, which inhibits viral replication without killing infected cells [17]. It has been demonstrated that the activation of RIG-I signaling could inhibit a number of viruses, including hepatitis C virus [18, 19], ebolavirus [20], and influenza virus [21]. Recent studies [9, 10] indicated that RIG-I is involved in control of HIV replication, as RIG-I could sense secondary-structured RNA of HIV, resulting in the activation of innate-immune responses [10]. However, RIG-I-dependent antiviral signaling could be inhibited by HIV infection [9]. Thus, to activate RIG-I by its ligand represents a promising approach for the treatment of HIV infection. To evaluate the effect of RIG-I activation on HIV replication in macrophages, we stimulated macrophages with 5′ppp-dsRNA or 5′ppp-dsRNA control before or after infection of HIV Bal strain. As shown in Fig. 1A and B, cells that were pretreated with 5′ppp-dsRNA and then infected with HIV Bal had a significant decrease in RT activity and gag gene expression. RIG-I activation-mediated inhibition of HIV replication was also confirmed by diminished HIV p24 protein expression in macrophages stimulated with 5′ppp-dsRNA (Fig. 1C). Morphologically, HIV Bal-infected macrophage cultures without 5′ppp-dsRNA stimulation demonstrated characteristic giant syncytium formation, where 5′ppp-dsRNA-treated macrophages failed to develop HIV-induced giant syncytia (Fig. 1D). We next examined whether the stimulation with 5′ppp-dsRNA during or after HIV infection could inhibit the virus replication. Similarly, cells stimulated with 5′ppp-dsRNA and infected with HIV Bal, simultaneously or 8 h after HIV Bal infection, had lower levels of HIV replication than the unstimulated and infected cells (Fig. 1E and F).

Figure 1. RIG-I activation suppresses HIV infection of macrophages.

(A–C) Effect of 5′ppp-dsRNA stimulation on HIV Bal infection of macrophages. Seven-day-cultured macrophages were stimulated with 5′ppp-dsRNA (1 μg/mL) for 24 h prior to HIV Bal infection. Culture SN was collected at Day 8 postinfection, and cells were collected at Day 12 postinfection. SN was subjected to RT assay (A), total RNA from cells was subjected to HIV gag gene expression by real-time PCR (B), and total protein extracted from cells was subjected to HIV p24 protein expression by Western blot (C). Representative blots from three independent experiments were shown. Densitometry analysis of the blot was performed using ImageJ 1.44 software (NIH) and plotted into graphs from data collected from triplicate experiments. (D) Effect of 5′ppp-dsRNA stimulation on HIV-induced syncytium formation in macrophages. The morphology of unstimulated and uninfected, unstimulated and HIV-infected, vehicle-stimulated and HIV-infected, 5′ppp-dsRNA control-stimulated and HIV-infected, and 5′ppp-dsRNA-stimulated and HIV-infected macrophages was observed and photographed under a light microscope (original magnification, ×200) at Day 8 postinfection. Arrows indicate giant syncytium formation. Five fields were examined in each well of triplicate cultures. One representative experiment is shown. (E and F) Suppression of HIV replication in macrophages by RIG-I activation under three conditions. Seven-day-cultured macrophages were cultured in media conditioned with or without 5′ ppp-dsRNA stimualtion for 24 h prior to HIV infection or simultaneously or 8 h postinfection. SN was collected at Day 8 postinfection, and cells were collected at Day 12 postinfection. SN was subjected to RT assay (E), and total RNA from cells was subjected to HIV gag gene expression (F) by real-time PCR. The data are expressed as RNA levels relative (percent) to the control (without stimulation, which is defined as 100%). The results shown are mean ± sd of triplicate cultures, representative of three experiments (5′ppp-dsRNA vs. 5′ppp-dsRNA control; *P<0.05; **P<0.01).

We next examined whether 5′ppp-dsRNA can trigger the RIG-I signaling pathway, leading to IFN production in macrophages. We observed increased expression of RIG-I and IFN-α/β expression in 5′ppp-dsRNA-stimualted macrophages (Fig. 2A). Although RIG-I activation of macrophages induced the expression of type I IFNs, we did not observe the induction of IFN-λ in 5′ppp-dsRNA-stimulated macrophages. This finding was unexpected, as it has been shown that RIG-I signaling could induce type III IFN expression [19, 22]. This discrepancy could be a result of the cell types used in different studies [19, 22]. Nevertheless, it would be interesting to investigate the mechanism(s) involved in the differential regulation of IFN-λ in different cell types. To investigate the mechanism for the effect of RIG-I activation on IFN-α/β expression, we examined whether RIG-I activation could induce the expression of IRFs, showing that 5′ppp-dsRNA stimulation selectively induced the expression of IRF-1, IRF-7, and IRF-9 in macrophages (Fig. 2B). It is well-known that the action of IFN on virus-infected cells is to elicit an antiviral state, which is characterized by the induction of ISGs [5]. We found that RIG-I signaling of macrophages induced ISGs (ISG15, ISG56, MxA, OAS-1, OAS-2, and Viperin) expression (Fig. 2C). These ISGs have been shown to inhibit viruses [23–25], including HIV [26–28].

Figure 2. RIG-I activation induces viral restriction factor expression.

Seven-day-cultured macrophages were stimulated with 5′ppp-dsRNA (1 μg/mL) for 24 h. Total RNA extracted from cells was subjected to the real-time RT-PCR for the mRNA levels of viral restriction factors. (A) RIG-I activation induces RIG-I and type-I IFN expression. (B) RIG-I activation enhances IRF (IRF-1, -7, and -9) expression. (C) RIG-I activation induces ISG (ISG15, ISG56, MxA, OAS-1, OAS-2, and Viperin) expression. (D) RIG-I activation up-regulates APOBEC3 family member (A3B, A3F, A3G, and A3H) expression. The data are expressed as mRNA levels relative (fold) to the control (without stimulation, which is defined as 1). The results shown are mean ± sd of triplicate cultures, representative of three experiments (5′ppp-dsRNA vs. 5′ppp-dsRNA control; * P<0.05; **P<0.01).

In addition to the induction of the ISGs, RIG-I signaling activates the expression of some members (A3B, A3F, A3G, and A3H) of the APOBEC3 family (Fig. 2D). These members are cellular cytidine deaminases that have the ability to inhibit the mobility of HIV [29, 30]. It has been shown that A3G, A3F, and A3H have the ability to restrict HIV replication in CD4⁺ T cells and macrophages [31–34]. A3G can edit the newly synthesized viral DNA or have an inhibitory effect through lethal editing of nascent reverse transcripts of the HIV life cycle [35–37]. A3F encodes an antiretroviral protein that is selectively packaged into HIV virions and profoundly inhibits HIV infectivity [34]. Thus, the induction of these members of the APOBEC3 family in macrophages provides an additional mechanism for RIG-I activation-mediated anti-HIV replication. In addition, we demonstrated that 5′ppp-dsRNA stimulation of macrophages induced the expression of CC-chemokines (MIP-1α, MIP-1β, and RANTES; data not shown), the natural ligands for the HIV coreceptor (CCR5). These CC-chemokines block the entry of HIV strains that use the CCR5 coreceptor by competitive inhibition [38, 39]. Furthermore, tetherin, which has been identified as an IFN-α-inducible cellular factor [40], was induced in macrophages stimulated with 5′ppp-dsRNA (data not shown). Tetherin is a transmembrane protein that specifically restricts HIV by preventing its release from infected cells [40]. These findings provide sound mechanisms for the RIG-I-mediated anti-HIV effect in macrophages.

Taken together, our study provides compelling experimental evidence that RIG-I activation by 5′ppp-dsRNA significantly suppresses HIV infection and replication in macrophages through multiple antiviral mechanisms at cellular and molecular levels. Although additional mechanism(s) might also be involved, the induction of type I IFNs and IFN-inducible antiviral factors should account for much of the RIG-I activation-mediated anti-HIV activity. There are at least two distinct components involved in type I IFN-dependent anti-HIV activities: (1) the induction of extracellular factors, CC chemokines that block HIV entry into macrophages; (2) the activation of intracellular viral restriction factors, such as members of the APOBEC3 family, ISGs, and tetherin. Each of these antiviral factors plays an important role in restriction of HIV replication, as each directly restricts HIV at different steps of the viral replication cycle. Therefore, the activation of the RIG-I signaling pathway may represent a promising, novel strategy for treatment of HIV infection. This approach is likely to be effective, as it induces multiple cellular antiviral factors that block HIV replication at different steps, which is unlikely for the virus to develop resistance. Obviously, future studies are necessary to confirm our in vitro findings and develop RIG-I agonist-based therapy for people infected with HIV.

AUTHORSHIP

Y.W. and W.H. conceived of and designed the experiments. Y.W. and J.L. performed the experiments. Y.W. analyzed the data. X.W. and Y.Z. contributed reagents/materials/analysis tools. Y.W. and W.H. wrote the paper.

DISCLOSURES

The authors declare that there is no conflict of interest.