Rotavirus NSP1 Subverts the Antiviral Oligoadenylate Synthetase-RNase L Pathway by Inducing RNase L Degradation

Figures

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  FIG 1. Importance of VP3 PDE and NSP1 to efficient rotavirus replication. (A) Domains of SA11 VP3 and NSP1. VP3: pink, kinase-like (KL); green, N7-methyltransferase (N7-MTase); magenta, 2’-O-methyltransferase (2’-O-MTase); lavender, guanylyltransferase (GTase); cyan, 2’-5′ phosphodiesterase (PDE). NSP1: yellow, RING domain; blue, IRF-binding domain (BD). Mutations in VP3 (H797R) and NSP1 (deletion of 479–496, ΔC17) are indicated. (B) Electrophoretic profiles of genomic dsRNA from wild type (rSA11) and mutant rotaviruses. (C) Immunoblot analysis of NSP1 and PCNA in mock- or virus-infected MA104 cells at 10 h pi. (D) Multistep growth curves of wild-type and mutant rotaviruses (MOI = 0.01) in MA104 cells. Virus titers were determined by plaque assay from two independent experiments. Statistical differences in the titers of rSA11 and rSA11-VP3H797R-NSP1ΔC17 viruses were evaluated using an unpaired t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (E) Plaques formed by wild-type and mutant rotaviruses on MA104 cells. (F) Plaques sizes formed by wild-type and mutant rotaviruses. Total of 60 plaques were measured by ImageJ software from three independent experiments. Black lines represent the…

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  FIG 2. ISG induction by rotaviruses with mutations in VP3 PDE and NSP1. (A) MA104 cells were infected with the indicated rotavirus strains (MOI = 6). Cells were harvested at 8 h pi. and analyzed by immunoblot assay for IFIT1, viperin, VP6, and PCNA. (B) Levels of IFIT1 and viperin were determined using ImageJ software. The data are representative of two independent experiments.

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  FIG 3. Impact of the JAK inhibitor, pyridone 6, on rotavirus replication. (A) MA104 cells were mock infected or infected with the double mutant virus rSA11-VP3H797R-NSP1ΔC17 (MOI = 0.01). After virus adsorption, DMSO or pyridine 6 dissolved in DMSO was added to the medium of some cells. The final concentration of pyridine 6 in medium was 2 μM. Cells were harvested at 30 h pi. and analyzed by immunoblot assay for IFIT1, VP6, and PCNA. (B) MA104 cells were infected with indicated viruses (MOI = 0.01) and treated with DMSO or pyridone 6 dissolved in DMSO. Cells lysates were collected at 36 h pi. and titered by plaque assay. Statistical differences in titers were evaluated using an unpaired t test. ns, not significant.

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  FIG 4. Effect of VP3 PDE and NSP1 on ribosomal (r) RNA degradation in rotavirus-infected cells. (A) MA104 cells were mock infected or infected with the indicated rotavirus. Cellular RNA was recovered from cells harvested at 10 h pi. and analyzed by automated electrophoresis using an Agilent TapeStation 2200 system. RNA integrity number (RIN) was calculated using TapeStation software. (B) Same as (A), except infected cells were transfected with 500 ng of dsRNA at 5 h pi. (C) Same as (A), except cells were pretreated with rhesus monkey IFN-β beginning 24 h prior to infection. Immunoblot assay was used to detect VP6 and PCNA in cell lysates at 10 h pi. Results are representative of three independent experiments.

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  FIG 5. Depletion of RNase L during rotavirus infection. (A) MA104 cells were infected with the indicated rotavirus strain (MOI = 6) and harvested at 8 h pi. Immunoblot assay was used to detect RNase L, PCNA and rotavirus VP6 in cell lysates. (B) HT-29 cells were transfected with plasmids expressing the indicated OAS proteins and, 24 h later, infected with rSA11 (MOI = 5). Immunoblot assay was used to detect OAS1-3, VP6 and GAPDH in cell lysates. Results are representative of three independent experiments. Sizes of protein markers (kDa) are indicated.

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  FIG 6. Replication of wild-type and NSP1 mutant viruses. HT-29 cells were infected with the indicated viruses (MOI = 1). Lysates were prepared from cells at 24 h pi., and virus titers in lysates were determined by plaque assay. Statistical differences in the titers of rSA11 and rSA11-NSP1-35TAA viruses were evaluated using an unpaired t test. ***, P ≤ 0.001.

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  FIG 7. SA11 NSP1 causes depletion of RNase L. (A) HT-29 cells were mock infected or infected with the indicated rotavirus (MOI = 5). Proteins in cell lysates were detected by immunoblot assay using antibodies specific to RNase L, NSP1, VP6, and PCNA. (B) HEK 293T cells were mock transfected or transfected with a plasmid expressing FLAG-tagged human RNase L alone or with an empty pHTN vector or a pHTN vector expressing Halo-tagged SA11 NSP1. Proteins in cell lysates were collected at 24 h pt. and analyzed by immunoblot assay using antibodies for HaloTag, FLAG, and PCNA. Blots are representative of the results of three independent experiments.

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  FIG 8. Neddylation inhibitor MLN4924 partially rescues RNase L. (A) HT-29 cells were treated with the indicated inhibitors and mock infected or infected with rSA11 (MOI = 5). Cells were harvested at 8 h pi. and analyzed by immunoblot assay for RNase L, IRF3, VP6, and PCNA. UT, untreated; ChlQ, chloroquine. (B) HEK 293T cells were treated with MLN4924 dissolved in DMSO or DMSO alone. At 1 h posttreatment, cells were transfected with a plasmid expressing FLAG-tagged human RNase L alone or with an empty pHTN vector or a pHTN vector expressing Halo-tagged SA11 NSP1. Cells were harvested at 24 h pt. and analyzed by immunoblot assay using antibodies specific for HaloTag, FLAG tag, and PCNA. Blots are representative of the results of three independent experiments.

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  FIG 9. NSP1 interacts with RNase L. (A) HEK 293T cells were treated with MLN4924, and 1 h later, cotransfected with a plasmid expressing FLAG-tagged RNase L and a plasmid expressing Halo-tagged SA11 NSP1 or an empty vector. Lysates prepared from the cells at 24 h pt. were incubated with magnetic beads conjugated with mouse anti-FLAG antibody to immunoprecipitate complexes containing FLAG-tagged proteins. The beads were split into two equal aliquots, with one aliquot treated with RNase A and RNase T1 cocktail. Proteins bounds to beads and input fractions were analyzed by immunoblot assay using antibodies to FLAG, HaloTag, and PCNA. (B) Reciprocal pulldown assay of (A) performed with anti-HaloTag resin instead of magnetic beads conjugated to anti-FLAG antibody. Blots are representative of the results of two independent experiments.

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  FIG 10. Regions of NSP1 contributing to RNase L degradation. (A) Schematic of pHTN plasmids encoding mutant Halo-tagged SA11 NSP1 proteins. (B) HEK 293T cells were cotransfected with a plasmid encoding FLAG-tagged RNase L and a plasmid encoding a Halo-tagged NSP1 protein. Cell lysates were prepared at 24 h pt. and analyzed by immunoblot assay for HaloTag, FLAG, and PCNA. (C) Schematic of pT7 plasmids encoding wild-type or mutant NSP1, used in recovering wild-type and mutant rotaviruses. (D) HT-29 cells were infected with the indicated rotavirus (MOI = 5). Cells were harvested at 8 h pi. and analyzed by immunoblot assay for RNase L, NSP1, VP6, and PCNA. Blots are representative of three independent experiments.

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  FIG 11. Rotavirus strain type and loss of RNase L. Human HT-29 cells were infected with the indicated rotavirus strain (MOI = 5). Cells were harvested at 8 h pi. and analyzed by immunoblot assay with antibodies against RNase L, VP6, and PCNA. The data are representative of three independent experiments. SRF, SOF, SNF, SKF, and SDF strains are SA11-L2 reassortants with NSP1 segments derived from RRV, OSU, K9, KU, and DS-1.

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  FIG 12. Strategies used by SA11 rotavirus to evade the OAS-RNase L pathway. The three mechanisms are degradation of 2-5A by VP3 PDE, inhibition of the IFN-signaling pathway by NSP1, and induction of RNase L degradation by NSP1.