TRAF6 and IRF7 control HIV replication in macrophages

Abstract

The innate immune system recognizes virus infection and evokes antiviral responses which include producing type I interferons (IFNs). The induction of IFN provides a crucial mechanism of antiviral defense by upregulating interferon-stimulated genes (ISGs) that restrict viral replication. ISGs inhibit the replication of many viruses by acting at different steps of their viral cycle. Specifically, IFN treatment prior to in vitro human immunodeficiency virus (HIV) infection stops or significantly delays HIV-1 production indicating that potent inhibitory factors are generated. We report that HIV-1 infection of primary human macrophages decreases tumor necrosis factor receptor-associated factor 6 (TRAF6) and virus-induced signaling adaptor (VISA) expression, which are both components of the IFN signaling pathway controlling viral replication. Knocking down the expression of TRAF6 in macrophages increased HIV-1 replication and augmented the expression of IRF7 but not IRF3. Suppressing VISA had no impact on viral replication. Overexpression of IRF7 resulted in enhanced viral replication while knocking down IRF7 expression in macrophages significantly reduced viral output. These findings are the first demonstration that TRAF6 can regulate HIV-1 production and furthermore that expression of IRF7 promotes HIV-1 replication.

Figure 1. Type I IFNα2 inhibits HIV-1 replication in primary macrophages.

A) Gene expression of TAT spliced at 2, 4, 8, 24, 120 and 168 hours post-infection of macrophages. The macrophages were incubated in the absence or presence of IFNα2 (1000 UI/ml for 18 hours). The expression level was calculated by qRT-PCR assessments of triplicates and normalized to the level of 18 S. Results shown are a representative of 2 independent experiments. A.U. corresponds to arbitrary units. B) Cell-free culture supernatants were collected at 24, 72, 120 and 168 hours post-infection and analyzed for p24 content. Results shown represent 2 combined independent experiments.

Figure 2. HIV-1 modulates TRAF6 and VISA expression in IFNα2 pre-treated macrophages.

A) Intensity of the expression of the genes of interest obtained from the oligonucleotide array analysis (U133 Plus 2.0 array) at 2, 4, 8 and 24 hours post-infection of macrophages. Cells were treated with IFNα2 (1000 UI/ml for 18 hours) with or without HIV-1. B) Validation of the regulation of gene modulation by HIV-1 in IFNα2 pre-treated macrophages (differentiated with human serum) at 8 hours post-infection. Non-IFNα2 treated and non-HIV infected macrophages were used as negative control. An * denotes a significant difference (P<01, paired t-test) between IFNα2 pre-treated or not macrophages. The expression level was calculated by qRT-PCR assessments obtained from four donors and normalized to the level of 18S.

Figure 3. TRAF6 but not VISA controls the level of expression of IRF7 and HIV replication.

Primary human macrophages were subjected to siRNA knockdown for TRAF6 and VISA. The concentration of siRNA used was 200 nM. A) Percentages of inhibition of TRAF6 and VISA in the presence of specific siRNA targeting both gene individually [n = 3] compared to a control non-targeted siRNA [n = 3]. Level of expression was measured by qRT-PCR B) Macrophages were all infected with HIV-1 with and without an IFNα2 pretreatment, and treated with specific siRNA. The level of TAT spliced message measured 24 hours post-infection in TRAF6 and VISA conditions were compared to the negative control siRNA. C) Level of IRF7 and D) Level of IRF3 message expression in the absence of IFNα2 when TRAF6 and VISA are repressed by siRNA compared to the negative control siRNA. The qRT-PCR results were normalized to the level of 18S. An * denotes a significant difference (P<0.05, one sample t-test) compared to the negative control siRNA. Each symbol in a group represents one independent donor. E) Western blot for IRF3 and IRF7, 24 hours post-infection in macrophages with TRAF6 suppression compared to siRNA control. The densitometry of the bands was normalized to that of β-actin and shows a decrease in protein level of IRF3 and an increase of IRF7. The ratio is shown next to the respective panels. Results shown are representative of 3 independent experiments.

Figure 4. IRF7 expression modulates HIV-1 replication in primary macrophages.

A) Level of IRF7 gene expression in macrophages treated with siRNA targeting IRF7 (50 nM) [n = 3] compared to a control non-targeted siRNA [n = 3] resulted in a 55% reduction in IRF7 gene expression. The qRT-PCR results were normalized to the level of 18 S. B) Western blot for IRF7 protein treated as in panel A shows a decrease of 42% for IRF7 protein. The densitometry of the bands was normalized to that of β-actin. Results shown are representative of 2 independent experiments. C) Level of TAT spliced expression following inhibition (51%) by IRF7 as compared to control. The qRT-PCR results were normalized to the level of 18 S. An * denotes a significant difference (P<0.001, one sample t-test) compared to the negative control siRNA. Each symbol in a group represents one independent donor.

Figure 5. IRF7 overexpression increases HIV-1 replication in primary macrophages.

A) Western blot for IRF7 protein, 24 hours post-transfection of macrophages overexpressing IRF7 showed a 2 fold increase compared to empty vector control. The densitometry of the bands was normalized to that of β-actin. The ratio is shown next to the respective panels. Results shown are representative of 2 independent experiments. B) TAT spliced expression was augmented 14 fold in primary human macrophages overexpressing IRF7 compared to empty vector control. The qRT-PCR results were normalized to the level of 18 S. An * denotes a significant difference (P<0.05, one sample t-test) compared to the empty vector control. Each symbol in a group represents one independent donor.