RNase L plays a role in the antiviral response to West Nile virus

Abstract

Alleles at the Flv locus determine disease outcome after a flavivirus infection in mice. Although comparable numbers of congenic resistant and susceptible mouse embryo fibroblasts (MEFs) are infected by the flavivirus West Nile virus (WNV), resistant MEFs produce approximately 100- to 150-fold lower titers than susceptible ones and flavivirus titers in the brains of resistant and susceptible animals can differ by >10,000-fold. The Flv locus was previously identified as the 2'-5' oligoadenylate synthetase 1b (Oas1b) gene. Oas gene expression is up-regulated by interferon (IFN), and after activation by double-stranded RNA, some mouse synthetases produce 2-5A, which activates latent RNase L to degrade viral and cellular RNAs. To determine whether the lower levels of intracellular flavivirus genomic RNA from resistant mice detected in cells at all times after infection were mediated by RNase L, RNase L activity levels in congenic resistant and susceptible cells were compared. Similar moderate levels of RNase L activation by transfected 2-5A were observed in both types of uninfected cells. After WNV infection, the mRNAs of IFN-beta and three Oas genes were up-regulated to similar levels in both types of cells. However, significant levels of RNase L activity were not detected until 72 h after WNV infection and the patterns of viral RNA cleavage products generated were similar in both types of cells. When RNase L activity was down-regulated in resistant cells via stable expression of a dominant negative RNase L mutant, approximately 5- to 10-times-higher yields of WNV were produced. Similarly, about approximately 5- to 10-times-higher virus yields were produced by susceptible C57BL/6 RNase L-/- cells compared to RNase L+/+ cells that were either left untreated or pretreated with IFN and/or poly(I) . poly(C). The data indicate that WNV genomic RNA is susceptible to RNase L cleavage and that RNase L plays a role in the cellular antiviral response to flaviviruses. The results suggest that RNase L activation is not a major component of the Oas1b-mediated flavivirus resistance phenotype.

FIG. 1.

Quantification of intracellular viral RNA in RV and He MEFs. Congenic flavivirus-resistant (RV) and susceptible (He) mouse MEFs were infected with WNV (strain Eg101) at an MOI of 1, and total cell RNA was extracted at the indicated times after infection. Plus-sense viral RNA was quantified by real-time RT-PCR with a reference viral genomic RNA of known concentration. The amounts of viral RNA were normalized by comparison to GAPDH mRNA levels.

FIG. 2.

Induction of IFN-β and Oas gene expression and STAT1 phosphorylation in WNV-infected RV and He MEFs. Total cell RNAs were purified from primary RV and He MEFs that had been either mock infected or infected with WNV at an MOI 10 for 12, 24, 48, or 72 h. The RNA was subjected to semiquantitative RT-PCR (A) or real-time RT-PCR (B). Real-time RT-PCR was performed with inventoried TaqMan Gene Expression Assays (Applied Biosystems) for the gene for IFN-β and for the endogenous control gene, GAPDH. (C) Western blot analysis of STAT1 and phosphorylated STAT1. Cell lysates were harvested at various times after infection (6 to 64 h) or after 1 h of mock infection (M) and detected with primary antibodies specific for the indicated proteins and horseradish peroxidase-conjugated secondary antibodies. Bands were detected via enhanced chemiluminescence. Actin was used as the loading control. Lanes M, mock. (D to F) Total cell RNAs were purified from primary RV and He MEFs that had been mock infected or WNV infected with an MOI of 10 for 12, 24, or 48 h. Q-RT-PCR analysis was performed with inventoried TaqMan gene expression assays (Applied Biosystems) for the Oas1a (D), Oas-like2 (E), and Oas1b (F) genes. The gene for GAPDH was used as an endogenous control. PI, postinfection; RQ, relative quantification.

FIG. 3.

Effects of poly(I) · poly(C) and IFN pretreatments on WNV growth in MEFs. After various pretreatments, primary RV and He MEFs and C57BL/6 RNase L^+/+ and RNase L^−/− MEF cell lines were infected with WNV at an MOI of 1. Samples of culture fluid were taken at the indicated times, and titers were determined by plaque assay on BHK cells to compare the kinetics of virus growth. (A and D) Cells were either left untreated or incubated with 1,000-IU/ml universal type I IFN for 4 h prior to infection. (B and E) Cells were left untreated or incubated with 10-μg/ml poly(I) · poly(C) for an hour prior to infection. (C and F) Cells were left untreated or incubated with 1,000-IU/ml universal type I IFN for 4 h and with poly(I) · poly(C) (10 μg/ml) for the last hour prior to infection. The virus titers at each time point are the averages of results of duplicate titrations from two experiments. Error bars indicate standard deviation and are shown where appropriate. Asterisks indicate titers below the detection limit of 10² PFU/ml.

FIG. 4.

Assay of intracellular RNase L activity in RV and He MEFs. RNase L produces characteristic rRNA cleavage products in the presence of 2-5A. (A) RV and He MEF cell lines were transfected with 2-5A at a concentration of 2, 5, or 10 μM for 4 to 5 h with Lipofectamine (Invitrogen). Total cell RNA was then isolated, separated on RNA chips (Agilent Technologies), and analyzed with an Agilent Bioanalyzer 2100. The positions of major RNase L-mediated rRNA cleavage products are indicated on the right. (B) Assay of RNase L activation after WNV infection. Total cell RNAs extracted from RV and He MEFs infected with WNV at an MOI of 1 for 24, 48, or 72 h were separated on RNA chips and analyzed. (C) Northern blot analysis of total cellular RNA from He and RV cells infected with WNV at an MOI of 1 for 48 or 72 h. Total cell RNA (5 μg) was separated on a 1% denaturing agarose gel and transferred to a Hybond-XL membrane. The blot was hybridized to a ³²P-labeled DNA probe specific for the 3′ UTR of the WNV genome RNA.

FIG. 5.

Cleavage of HCV and WNV RNAs by purified recombinant RNase L. (A) ³²P-labeled HCV (8 kb) or WNV (9.4 kb) RNA was incubated for 0 to 10 min in reaction mixtures containing purified human RNase L (10 nM). Lanes 1 to 5, HCV RNA; lanes 6 to 11, HCV RNA plus 25 nM 2-5A; lanes 12 to 17, WNV RNA plus 25 nM 2-5A. The radiolabeled RNAs were then fractioned by electrophoresis on 1% agarose gels and visualized by phosphorimaging. (B) Graphic depiction of the kinetics of disappearance of genome-sized RNA. The relative amounts of radioactivity in the viral RNA bands in each lane in panel C were quantified with MacBAS software on an image analyzer. The values obtained were expressed as photostimulated luminescence (PSL) minus the background (B) per square millimeter and plotted versus time of incubation.

FIG. 6.

Effect of suppressing or knocking out endogenous RNase L activity on the cleavage of rRNA and WNV RNA. (A) Clones of RV MEFs stably expressing truncated RNase L were generated as described in Materials and Methods. To detect those clones that expressed both RNase L proteins, covalent cross-linking of ³²P-labeled 2-5A to endogenous and truncated (dominant negative) RNase L in cell extracts was used. Lane 1, extract from untransfected RV MEFs (RU). Lanes 2 to 7, extracts from six G418-selected clones of RNase L mutant-transfected RV MEFs (RT/ZB1). Lane 8, 2 μg of crude extract from insect cells expressing human RNase L from a baculovirus vector (16). (B) rRNA cleavage assay of total RNA extracted from untransfected RV cells (RU) or RV cells transfected with the empty expression vector pcDNAI/Neo (RT/neo) or an RV clone expressing mutant RNase L (RT/ZB1-83). 2-5A (10 μM) was transfected into cells with Lipofectamine 2000 for 4 h. Total RNA was then extracted, separated, and analyzed with RNA chips (Agilent). (C) rRNA cleavage assay of total RNA isolated from RT/neo, RT/ZB1-83, C57BL/6 RNase L^+/+, or C57BL/6 RNase L^−/− MEFs. Cells were infected with WNV at an MOI of 1 for 72 h. (D and E) Northern blot analysis of total cellular RNAs from C57BL/6 RNase L^+/+ and RNase L^−/− MEFs infected for 48 or 72 h with WNV at an MOI of 1. RNA (5 μg) was separated on a 1% denaturing agarose gel (D) or by 5% urea-PAGE (E) and then transferred to a Hybond-XL membrane. The blots were hybridized to a ³²P-labeled DNA probe specific for the 3′ UTR of the WNV genome RNA. vRNA, viral RNA.

FIG. 7.

Effect of suppressing endogenous RNase L activity on the growth of WNV in MEFs. Cells were infected with WNV at an MOI of 5 (A), an MOI of 1 (B), or an MOI of 0.05 (C), and virus growth kinetics were compared. Samples of culture fluid were harvested at the indicated times, and titers were determined by plaque assay on BHK cells. ST/neo, susceptible He MEFs stably transfected with the empty expression vector pcDNAI/Neo. RT/neo, resistant RV MEFs stably transfected with the empty expression vector pcDNAI/Neo. RT/ZB1-83, resistant RV MEFs stably expressing dominant negative mutant RNase L from pDNAI-ZB1. The virus titers at each time point are the averages of results of duplicate titrations from two experiments. Error bars indicate standard deviation and are shown where appropriate. The asterisk indicates titers below the detection limit of 10² PFU/ml.