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PLoS One
2024 Oct 31;1910:e0312098. doi: 10.1371/journal.pone.0312098.
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The SARS-CoV-2 ORF6 protein inhibits nuclear export of mRNA and spliceosomal U snRNA.
Taniguchi I
.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19). SARS-CoV-2 infection suppresses host innate immunity and impairs cell viability. Among the viral proteins, ORF6 exhibits potent interferon (IFN) antagonistic activity and cellular toxicity. It also interacts with the RNA export factor RAE1, which bridges the nuclear pore complex and nuclear export receptors, suggesting an effect on RNA export. Using the Xenopus oocyte microinjection system, I found that ORF6 blocked the export of not only mRNA but also spliceosomal U snRNA. I further demonstrated that ORF6 affects the interaction between RAE1 and nuclear export receptors and inhibits the RNA binding of RAE1. These effects of ORF6 may cumulatively block the export of several classes of RNA. I also found that ORF6 binds RNA and forms oligomers. These findings provide insights into the suppression of innate immune responses and the reduction in cell viability caused by SARS-CoV-2 infection, contributing to the development of antiviral drugs targeting ORF6.
Fig 1. Interaction among full-length ORF6, RAE1, and NUP98 proteins in vitro.(A) Coomassie Brilliant Blue (CBB) staining of purified recombinant proteins. (B) A protein mixture containing T7-ORF6-His (ORF6) (1 μg) and RNase A (5 μg) was mixed with MBP-FLAG-His (MBP) or MBP-FLAG-RAE1-His (MBP-RAE1) (1 μg each) that was pre-bound to Amylose Resin. The pulled-down proteins were analyzed by SDS-PAGE and western blotting (WB). (C) A protein mixture containing FLAG-NUP98-His (NUP98) (60 ng) and RNase A (5 μg) was mixed with MBP-FLAG-His (MBP) or MBP-FLAG-RAE1-His (MBP-RAE1) (1 μg each) that was pre-bound to Amylose Resin. Pulled-down proteins were analyzed by SDS-PAGE and western blotting. (D) A protein mixture containing FLAG-RAE1-His (RAE1) (20 ng), FLAG-NUP98-His (NUP98) (60 ng), and RNase A (5 μg) was mixed with GST or GST-T7-ORF6-His (GST-ORF6) (1 μg each) that was pre-bound to glutathione sepharose. Pulled down proteins were analyzed by SDS-PAGE and WB.
Fig 2. Effects of ORF6 on RNA export in Xenopus oocytes.(A) and (B) Purified recombinant GST or GST-T7-ORF6-His (GST-ORF6) (50 fmol/oocyte) was injected into the cytoplasm of Xenopus oocytes. After 12 h of incubation, a mixture of in vitro-transcribed 32P-labeled RNAs containing DHFR mRNA, pre-ftz mRNA, U1ΔSm snRNA, U6Δss snRNA, and tRNAPhe was injected into the nucleus. U6Δss snRNA and tRNAPhe were uncapped, and the other RNAs were m7G-capped. RNA was extracted from nuclear (N) and cytoplasmic (C) fractions immediately (0 h; A, lanes 1 and 2) or at 3 h (3 h; A, lanes 3–6) after the injection, and then analyzed by 8% denaturing PAGE and autoradiography. Bands corresponding to the spliced product (spliced-ftz mRNA) and the lariat intron (intron) are indicated. It is likely that the cytoplasmic pre-ftz mRNA band was the result of leakage from tiny holes made by injection needles, rather than export factors. (C) Quantification of the export efficiency of DHFR mRNA, spliced-ftz mRNA, U1ΔSm, and tRNAPhe from three independent experiments performed as in (B) is shown. Values are means (SD).
Fig 3. Effects of ORF6 on protein interactions of RAE1 in vitro.(A) pcDNA3-FLAG-TAP was transfected into HEK293T cells. Whole-cell lysates containing T7-ORF6-His (0, 1, 3, and 10 μg; -, x1, x3, and x10, respectively) and RNase A (25 μg) were mixed with MBP-FLAG-His (MBP) or MBP-FLAG-RAE1-His (MBP-RAE1) (2 μg each) that was pre-bound to Amylose Resin. Pulled-down proteins were analyzed by SDS-PAGE and WB. (B) A protein mixture containing FLAG-RAE1-His (RAE1) (1 μg), FLAG-NUP98-His (NUP98) (0.5 μg), T7-ORF6-His (ORF6) (0, 2.5, 7.5, and 25 μg; -, x1, x3, and x10, respectively), and RNase A (50 μg) was mixed with GST (2.5 μg) or the GST-TAPΔN:p15 complex (2.5 μg TAPΔN) that was pre-bound to glutathione Sepharose. Pulled-down proteins were analyzed by SDS-PAGE and WB. Quantification of the relative pull-down efficiency from three independent experiments is shown. The efficiency of the buffer is set to 1. Values are means (SD).
Fig 4. Effects of ORF6 on the RNA binding of RAE1 in vitro.(A) A mixture of in vitro-transcribed 32P-labeled RNAs containing intronless ftz mRNA, U1ΔSm snRNA, and tRNAPhe was incubated with or without purified FLAG-RAE1-His (FLAG-RAE1) (2 μg) that was pre-bound to Protein A Sepharose via an anti-FLAG antibody in the absence or presence of T7-ORF6-His (T7-ORF6) (4 μg). Co-immunoprecipitated RNAs were analyzed by 8% denaturing PAGE and autoradiography. (B) Quantification of the relative pull-down efficiency from three independent experiments performed as in (A) is shown. Values are means (SD). (C) 32P-labeled RNA was incubated in the absence or presence of T7-ORF6-His (ORF6) (0.1, 0.3, 1 and 3 μM) on ice. The sample was analyzed by native PAGE and autoradiography. (D) Quantification from three independent experiments performed as in (C) is shown. Values are means (SD). (E) The equilibrium dissociation constant (KD) was calculated from the quantification in (D). (F) A protein mixture containing T7-ORF6-His (ORF6) (1 μg) and RNase A (5 μg) was pulled down by GST or GST-T7-ORF6-His (GST-ORF6) (1 μg each). Pulled-down proteins were separated by SDS-PAGE and detected by WB. Quantification of the pull-down efficiency of T7-ORF6-His from three independent experiments is shown. Values are means (SD).
