Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses - PubMed (original) (raw)

Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

Yize Li et al. Proc Natl Acad Sci U S A. 2016.

Erratum in

Abstract

The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans--OAS1, OAS2, and OAS3--synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI):poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation.

Keywords: 2-5A; antiviral response; oligoadenylate synthetase 3; ribonuclease L; type I interferon.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Activation of RNase L by pIC in A549 cells requires OAS3 expression. (A) OAS1-, OAS2-, OAS3-, and RNase L-KO A549 cells were mock-treated or treated with IFN-β (1,000 U/mL) overnight. Cells were lysed, and proteins were analyzed by immunoblotting with antibodies against OAS1, OAS2, OAS3, RNase L, and GAPDH. (B and C) WT and KO cells were transfected with 0.25 μg/mL of pIC (B) or OAS3-KO cells were transfected with increasing doses of pIC (0–20 μg/mL) (C), and RNase L-KO and WT A549 cells were transfected with 20 μg/mL and 0.25 μg/mL of pIC, respectively. At 4 hpi cells were lysed, and RNA integrity was assessed with a Bioanalyzer. The positions of 18S and 28S rRNA are indicated.

Fig. S1.

Fig. S1.

Expression of OAS3 protein in OAS3-KO cells restores 2-5A production and RNase L activation. (A) OAS3-KO cells were transfected with p3XFlag-tagged OAS3 cDNA, and a cell line was generated by neomycin selection. Cell lysates were analyzed by electrophoresis followed by immunoblotting with anti-Flag antibody (Upper) and monoclonal antibody against human OAS3 (Lower). (B) OAS3-KO cells and OAS3-KO cells in which OAS3 expression was restored (as indicated) were transfected with pIC (1 μg/mL) or were mock-infected or were infected with IAVΔNS1 (MOI = 10) or VACVΔE3L (MOI = 10). Cells were lysed at 4 (pIC) or 24 (virus) hpi, and total RNA was isolated and monitored for integrity on a Bioanalyzer. (C) OAS3-KO cells and OAS3-KO cells in which OAS3 expression was restored (as indicated) were transfected with pIC (1 μg/mL) or were mock-infected or infected with IAVΔNS1 (MOI = 1) or VACVΔE3L (MOI = 1). Cells were lysed at 4 (pIC) or 24 (virus) hpi, and intracellular levels of 2-5A were determined by FRET assay. **P < 0.0001.

Fig. S2.

Fig. S2.

Viruses that cause minimal or no activation of RNase L as determined by monitoring rRNA integrity in A549 cells. Parental and RNase L-KO cells were infected at MOI = 20. Cells were lysed at 12 (LACV, VSV, SeV, EMCV), 21 (TMEV), or 60 (LCMV) hpi, and RNA integrity was analyzed on a Bioanalyzer. The positions of 18S and 28S rRNA are indicated.

Fig. 2.

Fig. 2.

Activation of RNase L during SINV infection of A549 cells requires OAS3 expression. (A) WT A549 and KO cells were infected with SINV (MOI = 1 pfu per cell), and at 24 hpi cells were lysed and RNA integrity was assessed. The positions of 18S and 28S rRNA are indicated. (B) Cells were infected with SINV (MOI = 1 pfu per cell), and at 24 hpi intracellular levels of 2-5A were quantified by FRET assay. (C) Cells were infected with SINV (MOI = 1 pfu per cell), and at the indicated time points infectious virus in the supernatant was titered by plaque assay on Vero cells. The data are from three biological replicates and are expressed as means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. S3.

Fig. S3.

Infections with SINV or IAVΔNS1 induce up-regulation of OAS1, OAS2, and OAS3 gene expression in A549 cells. (A) A549 cells (number of experiments, n = 3) were infected with SINV (MOI = 5). Cells were lysed at 2, 6, 12, and 24 hpi, and RNA was isolated. OAS1, OAS2, and OAS3 mRNAs were quantified by qRT-PCR and expressed as fold-increase over levels at 2 hpi. Data are expressed as mean ± SD. (B) WT or OAS3-KO A549 cells were infected with SINV (MOI = 1) or treated with 1,000 U/mL of IFN-α. Cells were lysed 24 hpi or post IFN treatment, and proteins were analyzed by Western immunoblot with antibodies against OAS1, OAS2, OAS3, and GAPDH. (C) WT A549 cells were infected by IAVΔNS1 (MOI = 1). Cells were lysed at 0, 4, 8, 12, and 24 hpi, and proteins were analyzed by Western immunoblots with antibodies against OAS1, OAS2, OAS3, and β-actin.

Fig. 3.

Fig. 3.

Activation of RNase L during WNV infection of A549 cells requires OAS3 expression. (A) WT A549 and KO cells were infected with WNV (MOI = 5 pfu per cell), and at 24 hpi RNA integrity was assessed. (B) WT A549 and KO cells were infected with WNV (MOI = 5 pfu per cell), and at 24 hpi 2-5A intracellular levels were quantified by FRET assay. (C) Cells were infected with WNV (MOI = 1 pfu per cell), and at the indicated time points infectious virus in the supernatant was titered by plaque assay. The data are from three biological replicates and are expressed as means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

Fig. 4.

Activation of RNase L during IAVΔNS1 infection of A549 cells requires OAS3 expression. (A) WT A549 and KO cells were infected with IAVΔNS1 (MOI = 10), and at 24 hpi RNA integrity was assessed. (B) Cells were infected with IAVΔNS1 (MOI = 1 pfu per cell), and at 24 hpi 2-5A intracellular levels were quantified by FRET assay. (C) Cells were infected with IAVΔNS1 (MOI = 1 pfu per cell), and at 24 hpi infectious virus in the supernatants was titered by plaque assays. The data are from three biological replicates and are expressed as means ± SD; **P < 0.01.

Fig. 5.

Fig. 5.

Activation of RNase L during VACVΔE3L infection of A549 cells requires OAS3 expression. (A) WT A549 cells and KO cells were infected with VACVΔE3L (MOI = 10 pfu per cell), and at 24 hpi RNA integrity was assessed. (B) Cells were infected with VACVΔE3L (MOI = 1 pfu per cell), and at 24 hpi 2-5A intracellular levels were quantified by FRET assay. (C) Cells were infected with VACVΔE3L (MOI = 1 pfu per cell), and at 24 hpi infectious virus in the supernatants was titered by plaque assays on BHK21 cells. The data are from three biological replicates and are expressed as means ± SD; **P < 0.01.

Fig. 6.

Fig. 6.

Activation of RNase L during WNV infection of HT1080 cells requires OAS3 expression. (A) OAS1-, OAS2-, OAS3-, and RNase L-KO HT1080 cells were mock-treated or were treated with IFN-α (2,000 U/mL) overnight. Proteins were analyzed by immunoblotting with antibodies against OAS1, OAS2, OAS3, and RNase L. (B) Cells were infected with WNV (MOI = 20 pfu per cell), and at 24 hpi RNA integrity was assessed. (C) Cells were infected WNV (MOI = 1 pfu per cell), and at the indicated time points infectious virus from the supernatant was titered by plaque assay. The data are from three biological replicates and are expressed as means ± SD; *P < 0.05, ***P < 0.001.

Fig. S4.

Fig. S4.

Activation of RNase L by pIC or VACVΔE3L in HT10810 or HME cells requires OAS3 expression. (A and B) HT1080 WT or KO cells were transfected with 500 ng/mL (A) or HME cells were transfected with1 μg/mL (B) of pIC, and at 4 h (HT1080 cells) or 3 h (HME cells) post transfection RNA was purified from cell lysates and analyzed on a Bioanalyzer. (C) WT or KO HME cells were infected with VACVΔE3L (MOI = 10). At 18 hpi RNA was purified from cell lysates and analyzed on a Bioanalyzer.

Fig. 7.

Fig. 7.

OAS3 binds to dsRNA with higher affinity than do OAS1 or OAS2 in intact cells. (A) A549 cells were transfected with empty vector (mock) or plasmids encoding Flag-tagged OAS1 p42, OAS1 p46, OAS2, or OAS3 and then were transfected with pIC-biotin. Streptavidin bead pull downs or proteins were carried out and were analyzed by immunoblotting with anti-Flag antibody. The blot was exposed for 5 or 20 min. Immunoblots without pull down show input levels of OAS proteins and GAPDH loading and transfer control. The additional, faster-migrating band in the p46 lanes is most likely a breakdown product of p46. (B) A549 cells were treated with IFN-β (1,000 U/mL) and then were transfected with pIC-biotin. Streptavidin bead pull down of pIC-biotin bound proteins was carried out and probed by immunoblotting with antibodies against OAS1, OAS2, and OAS3. Input levels of proteins are also shown. Thirty-fold more protein was used for pull down compared with input protein on the immunoblot.

Fig. S5.

Fig. S5.

IFN pretreatment reduces WNV titer in WT and OAS3-KO cells to the same level, independent of RNase L activation. (A) Cells were mock-treated or treated with 100 U of IFN-α overnight. Cells were transfected with 500 ng/mL of pIC or were infected with WNV (MOI = 20). Cells were lysed 4 h post transfection or 24 hpi, and RNA integrity was assessed on a Bioanalyzer. The position of 18S and 28S rRNA are indicated. (B) WT or OAS3-KO A549 cells were treated with 100 U of IFN-α overnight and then were infected with WNV (MOI = 1). At 36 hpi supernatants were harvested and titered for infectious virus. The data are pooled from three independent experiments carried out in duplicate. ***P < 0.001.

Fig. 8.

Fig. 8.

The OAS–RNase L pathway. Upon detection of viral dsRNA during infection with diverse human viruses (WNV, SINV, IAVΔNS1, VACVΔE3L), OAS3 produces 2-5A, which activates RNase L. RNase L degrades cellular and viral RNA, leading to the restriction of virus replication. [Modified with permission from ref. and with permission from ref. ;

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