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. 2013 Aug;94(2):337-41.
doi: 10.1189/jlb.0313158. Epub 2013 Jun 6.

RIG-I Activation Inhibits HIV Replication in Macrophages

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Free PMC article

RIG-I Activation Inhibits HIV Replication in Macrophages

Yizhong Wang et al. J Leukoc Biol. .
Free PMC article

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.

Keywords: APOBEC3; interferon regulatory factors; interferon-stimulated genes; type I interferon.

Figures

Figure 1.
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).
Figure 2.
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).

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