Extracellular 2'-5' oligoadenylate synthetase stimulates RNase L-independent antiviral activity: a novel mechanism of virus-induced innate immunity

Abstract

The 2'-5' oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, both in vitro and in vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells.

FIG. 1.

Antiviral protection of exogenous OAS in vitro and in vivo. (A to D) OAS1 in vitro. HepG2 (A) and Vero (B to D) cells were treated with recombinant porcine OAS1, Pop2p, heat-denatured OAS1, LPS, or IFN-α for 24 h and subsequently infected with EMCV. Cell survival was analyzed using the conversion of MTT to formazan in living cells, measuring the A₅₉₅. Data are presented as a percentage of a noninfected cell control. Error bars depict standard deviations of results of four independent experiments. Data represent results of multiple experiments with similar results. (E) OAS1 in vivo. On day 1, C57BL/6 mice (n = 6) were treated with OAS1, C5 mutant (R38E/K41E/K59E/R194E/R198E), or PBS (mock) 2 h prior to i.p. infection with 1 × 10³ PFU EMCV. Treatments were repeated on day 2, hearts were harvested on day 3, and viral titers were determined. Data were analyzed by a Mann-Whitney non-Gaussian one-tailed test with a 99% confidence interval. See also Fig. S1 and Table S1 in the supplemental material. EID₅₀, 50% egg infective dose.

FIG. 2.

Antiviral protection of OAS1 against EMCV, VSV, and HSV-2. Vero cells were treated with OAS1 WT, the OAS1 RNA binding site mutant, C5 (R38E/K41E/K59E/R194E/R198E), or IFN-α or left untreated (UT) as a control (n = 3). Cells were incubated for 24 h, washed extensively to remove access or unbound protein, and infected with the indicated virus at an MOI of 0.001. The infection was allowed to occur until substantial cytopathic effect was observed in the UT samples, and supernatants were collected by freeze-thawing and subsequent centrifugation. Supernatants were titrated on Vero cells in 96 wells to determine TCID₅₀/ml. Data were analyzed using a paired, one-tailed t test. The data are paired, as the individual replicates were titrated together. See also Table S2 in the supplemental material.

FIG. 3.

Antiviral protection of exogenous OAS1 is not mediated through IFN signaling. HT1080 cells were treated with Pop2p, OAS1WT, or IFN-α or left untreated as a control (UT) for 4 h or infected with SeV for 6 h. Cells were harvested, RNA purified, cDNA synthesized, and subjected to quantitative PCR using gene-specific primers for IFNB, ISG56, OASL, OAS1, and GAPDH as a reference. Data are presented relative to results for the untreated sample, and error bars depict standard deviations (n = 3).

FIG. 4.

Uptake of an OAS-EGFP fusion protein. Vero cells were treated with OAS1-EGFP (green) (upper panels) or C5-EGFP (middle panels) or left untreated (UT) (lower panels) for 4 h, trypsinated, and reseeded on coverslips. Supernatants were removed, cells were washed thoroughly with PBS and fixed, and the nuclei were stained with DAPI (blue). Blue, nucleus; green, OAS1-EGFP. Merge indicates merged results. Cells were visualized using confocal microscopy.

FIG. 5.

Antiviral activity of exogenous OAS1 is independent of the 2-5A activity and RNase L. HepG2 (A) and MEF (B) cells were treated with recombinant OAS1 or mutants for 24 h and subsequently infected with EMCV. Cell survival was analyzed using the conversion of MTT to formazan in living cells, measuring the A₅₉₅. Data are presented as a percentage of results for a noninfected cell control. Error bars depict standard deviations (n = 4). See also Table S3 in the supplemental material.