Alpha interferon enhances TRIM5alpha-mediated antiviral activities in human and rhesus monkey cells

Abstract

Dominant, constitutively expressed antiretroviral factors, including TRIM5alpha and APOBEC3 proteins, are distinguished from the conventional innate immune systems and are classified as intrinsic immunity factors. Here, we demonstrate that interferon alpha (IFN-alpha) treatment upregulates TRIM5alpha mRNA in rhesus monkey cells, which correlates with the enhanced TRIM5alpha-mediated pre- and postintegration blocks of human immunodeficiency virus replication. In human cells, IFN-alpha increases the levels of TRIM5alpha mRNA, resulting in enhanced antiviral activity against N-tropic murine leukemia virus infection. These observations indicate that the TRIM5alpha-mediated antiviral effects can be orchestrated by the conventional innate immune response. It is conceivable that TRIM5alpha plays an essential role in controlling both the initial retroviral exposure and the subsequent viral dissemination in vivo.

FIG. 1.

IFN-α increases the levels of TRIM5α_rh transcript in rhesus monkey cells. (A) FrhK4 cells were treated with IFN-α (100 U/ml) for the indicated periods of time. As a control, untreated FrhK4 cells were used. After treatment, total cellular RNA was isolated and TRIM5α_rh mRNA was detected by RT-PCR (upper panel), along with α-tubulin mRNA as a control (lower panel). (B) FrhK4 cells were treated with IFN-α (100 U/ml) for the indicated periods of time. Serially diluted RT products (equivalent to 0.5, 0.25, 0.125, 0.063, and 0.031 μg of total RNA samples) were used to detect the TRIM5α_rh-specific transcript. (C) Frhk4 cells were treated with various concentrations of IFN-α, and the TRIM5α_rh mRNA was detected by RT-PCR. Untreated cells were used as a control. (D) FrhK4 cells were treated with IFN-α (100 U/ml), heat-inactivated (h.i.) IFN-α (100 U/ml), IFN-γ (1,000 U/ml), and heat-inactivated IFN-γ (1,000 U/ml) for 6 h, and the TRIM5α_rh mRNA was detected by RT-PCR. (E) FrhK4 cells were transfected with double-stranded RNA poly(I:C) by using Oligofectamine (Invitrogen). As a positive control, Frhk4 cells were treated with IFN-α (100 U/ml) for 4 h. Total RNA was isolated from the treated or untreated cells (control).

FIG. 2.

IFN-α enhances the TRIM5α_rh-mediated blocks of HIV-1 replication. (A) FrhK4 cells were pretreated with 0, 4, 20, or 100 U/ml of IFN-α for 6 h and then infected with increasing amounts of GFP-expressing HIV-1 vector. Two days after infection, the GFP-positive cell populations were determined by flow cytometry (left panel). The relative HIV-1 vector infectivity in IFN-α-treated cells (mean ± standard deviation [SD]) is shown in the right panel. (B) FrhK4 cells (1.0 × 10⁵) were transfected with 0.2 μg of pNL4-3 along with 1.0 μg of either pBlueScriptII (control), a parental shRNA plasmid (pLKO1), or TRIM5α-specific shRNA constructs (pLKO-H3 and pLKO-H4). After overnight transfection, the culture supernatants were replaced with fresh growth media containing various concentrations of IFN-α. Two days after transfection, culture supernatants were harvested to determine HIV-1 production. Viral titers were shown as infectious units per milliliter in GHOST(3)R3/X4/R5 cells. (C) FrhK4 cells (1.0 × 10⁵) were transfected with 0.2 μg of pNL4-3 or pSIV_MAC1A11 along with 1.0 μg of pLKO1, pLKO-H3, or pLKO-H4. After overnight transfection, the culture supernatants were replaced with fresh growth media containing 100 U/ml of IFN-α. Two days after transfection, culture supernatants were harvested and viral titers were determined in GHOST(3)R3/X4/R5 cells (mean ± SD). (D) FrhK4 cells (1.0 × 10⁵) were transfected with 1.0 μg of pNL4-3. After overnight transfection, the culture supernatants were replaced with fresh growth media containing 100 U/ml of IFN-α. Two days after IFN-α treatment, culture supernatants were harvested to determine the viral titers and p24 concentrations (mean ± SD).

FIG. 3.

IFN-α upregulates TRIM5α_hu mRNA and enhances postentry restriction against N-MLV in human cells. (A) TE671 and HeLa cells were treated with 100 U/ml of IFN-α for 4 h. A 1.0-μg amount of total RNA was used to synthesize the first-strand DNA, and one-tenth of the RT products were used to detect TRIM5α_hu and α-tubulin transcripts. Serially diluted RT products (equivalent to 0.1, 0.05, 0.025, 0.0125, 0.0063, and 0.0031 μg of total RNA samples) were used to detect the TRIM5α_hu-specific transcript. (B) Activated PBL from a healthy donor were treated the same as described forTE671 and HeLa cells in panel A. (C) Activated PBL from a healthy donor were infected with HIV-1 NL4-3 at a multiplicity of infection of 0.5 for 48 h. Heat-inactivated (h.i.) virus (NL4-3, preincubated at 56°C for 1 h) was also used as a noninfectious control. The TRIM5α_rh-specific transcript was detected by RT-PCR. (D) TE671 cells were pretreated with 0, 20, or 100 U/ml of IFN-α for 4 h and then infected with GFP-expressing N-MLV and HIV-1 vectors. Three days after infection, the GFP-positive cell populations were determined by flow cytometry (mean ± SD). (E) TE671 cells (1.0 × 10⁵) were transfected with 0.2 μg of pNL4-3 or p89.6 by using Fugene6 (Roche). After overnight transfection, the culture supernatants were replaced with fresh growth media containing 0, 20, or 100 U/ml of IFN-α. Two days after transfection, culture supernatants were harvested to determine HIV-1 production. Viral titers were determined in GHOST(3)R3/X4/R5 cells (mean ± SD).