Plus-strand RNA viruses hijack Musashi homolog 1 to shield viral RNA from cytoplasmic ribonuclease degradation

Figures

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  Fig 1. MSI1 is required for the replication of CAstV and PEDV by interacting with their 3′UTR. (A) The qRT-PCR and WB results demonstrated that CAstV δC protein increased the expression of MSI1 at a multiplicity of infection (MOI) of 0.1 at 8 hpi, 12 hpi, 18 hpi, and 24 hpi in LMH cells (avian leghorn male hepatoma cell line). (B) The qRT-PCR and WB results demonstrated that PEDV N protein increased the expression of MSI1 at an MOI of 0.1 at 12 hpi, 24 hpi, and 36 hpi in Vero cells. (C) The qRT-PCR and WB results showed that MSI1 overexpression promoted CAstV δC RNA and protein levels at 48 hpi, and MSI1 knockdown decreased CAstV RNA and protein levels at 48 hpi. (D) The qRT-PCR and WB results showed that MSI1 overexpression promoted PEDV N RNA and protein levels, and MSI1 knockdown decreased PEDV RNA and protein levels. (E) Bioinformatics analysis identified one putative MSI1 binding site at the CAstV and the PEDV 3ʹUTRs. (F) Luciferase reporter assays revealed that MSI1 significantly inhibited the activity of the CAstV 3′UTR reporter. (G) Luciferase reporter assays revealed that MSI1 significantly inhibited the activity of the PEDV 3′UTR reporter. Data are presented as mean ± SEM for…

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  Fig 2. MSI1 is required for the replication of ALV-J and REV by binding U3s. (A) MSI1 overexpression activated ALV-J replication in ALV-J infection at an MOI of 0.01 at 48 hpi, as detected using WB and qRT-PCR. (B) MSI1 knockdown inhibited ALV-J replication in ALV-J infection at an MOI of 0.01 at 48 hpi, as detected using WB and qRT-PCR. (C) MSI1 overexpression activated REV replication in REV mono-infection at an MOI of 0.01 at 48 hpi, as detected using WB and qRT-PCR. (D) MSI1 knockdown inhibited REV replication in REV infection at an MOI of 0.01 at 48 hpi, as detected using WB and qRT-PCR. qRT-PCR data are presented as mean ± SEM and were obtained from three independent experiments (n = 3); each experiment contained triplicates. (E) CLSM was used to observe the localization and expression of MSI1 in normal DF-1 cells, ALV-J-infected, and REV-infected DF-1 cells at 72 hpi, as detected with an anti-Flag antibody, alpha-tubulin monoclonal antibody. Nuclei are labeled with DAPI (4′-6′-diamidino-2-phenylindole). (F–H) ALV-J and REV synergistically increased the protein expressions of MSI1 in the nucleus at an MOI of 0.01 at 24 hpi (F), 48 hpi (G), and 72 hpi (H), as detected using WB. (I…

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  Fig 3. MSI1 targeted U3 regions of ALV-J and REV. (A) Strategy for constructing pmirGLO-ALV-J U3 and REV U3 vector plasmids with different U3 sequence regions. (B and C) MSI1 or negative control was co-transfected with pmirGLO-Control or ALV-J U3 vector plasmids into 293T cells. Dual-luciferase assay showed that MSI1 significantly inhibited luciferase activities of the ALV-J region 1 vector but not the other region vectors. (D and E) MSI1 or negative control was co-transfected with pmirGLO-Control or REV U3 vector plasmids into 293T cells. Dual-luciferase assay showed that MSI1 significantly inhibited luciferase activities of the REV region 1 vector but not the other region vectors. (F) Bioinformatics analysis identified one putative MSI1 binding site at the ALV-J U3 and the REV U3. (G) Luciferase reporter assays revealed that MSI1 significantly inhibited the activity of the REV U3 reporter. (H) Luciferase reporter assays revealed that MSI1 significantly inhibited the activity of the ALV-J U3 reporter. Data are presented as mean ± SEM for n = 3, with each experiment being performed in triplicates. **P ≤ 0.01 using Student’s t-test versus the negative control group. n.s., not significant.

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  Fig 4. MSI1 prevents the degradation of exogenous ALV-J U3 and exogenous REV U3 by cellular ribonucleases. (A and B) CLSM was used to observe the localization and expression of MSI1, ALV-J U3, REV U3, and alpha-tubulin in normal, ALV-J-infected, and REV-infected DF-1 cells at 72 hpi, as detected with anti-Flag antibody, alpha-tubulin monoclonal antibody, ALV-J U3 FISH probe, and REV U3 FISH probe. Nuclei are labeled with DAPI. (C) The qRT-PCR results also demonstrated significant upregulations in the expression of viral U3s following MSI1 overexpression. (D) RNase activity fluorometric assay indicated that cellular RNase activity remained unchanged, irrespective of the overexpression or interference of MSI1. (E) qRT-PCR results showed that the expression of exogenous ALV-J U3 and REV U3 significantly decreased upon overexpression of RNase A. The data are presented as the mean ± SEM for n = 3, with each experiment being performed in triplicates. **P ≤ 0.01 using Student‘s t-test versus the negative control group. n.s., not significant.

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  Fig 5. ALV-J, REV, CAstV, and PEDV hijack MSI1 to shield the cellular ribonuclease degradation. (A) The RNase activity fluorometric assay indicated that cellular RNase activity remained unaltered, regardless of ALV-J or REV infection. (B) qRT-PCR analysis revealed that transfection with Flag-RNase A led to a decrease in ALV-J U3 RNA levels at 48 hpi, while co-transfection with Flag-MSI1 conferred protection to ALV-J U3 RNA against degradation by RNase A. (C–E) WB and qRT-PCR analysis revealed that the transfection of Flag-RNase A resulted in decreases in both mRNA and protein levels of ALV-J (C), CAstV (D), and PEDV (E) at 48 hpi, while co-transfection with Flag-MSI1 was found to confer protection to these viruses against degradation by RNase A. The data are presented as the mean ± SEM for n = 3, with each experiment being performed in triplicates. **P ≤ 0.01 using Student‘s t-test versus the negative control group.

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  Fig 6. U3-binding regions of MSI1. (A) WB analysis demonstrated that RRM1 and RRM2 were successfully transfected into CEFs. (B and C) qRT-PCR results showed that only overexpression of MSI1 RRM1 increased ALV-J and REV replication in co-infection at 48 hpi. qRT-PCR data are presented as mean ± SEM and were obtained from three independent experiments (n = 3); each experiment contained triplicates. (D) Strategy for constructing MSI1 mutants with different RRM1 regions. The original backbone of MSI1 is shown. MSI1 mutants, including MS1 mut1 (F33L, G35R, G36A, L37R, S38R, and W39L), MS1 mut2 (L60R and M62R), MSI1 mut3 (R71K, G72A, F73L, and F75L), and MSI1 mut4 (K98R, D101A, K103R, V104R, A105G, and F106L) were constructed using the Fast site-directed mutagenesis kit. Mutations of amino acid sequences are shown in the same color. (E) All MSI mutants were successfully transfected into DF-1 cells co-infected with ALV-J and REV, as detected using WB with the anti-FLAG antibody at 48 hpi. (F and G) Among the four mutants, only MSI1 mut1 reversed the increase in ALV-J and REV replication as detected using qRT-PCR at 48 hpi. Data are presented as mean ± SEM for n = 3, with each experiment being…

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  Fig 7. Schematic of the molecular mechanisms by which multiple plus-strand RNA viruses hijack MSI1 to evade cellular ribonuclease degradation. Multiple plus-strand RNA viruses, including ALV-J, REV, CAstV, and PEDV, utilize specific regions such as U3 or 3′UTR to interact with MSI1, thereby protecting their viral RNA from host ribonuclease activity in the cytoplasm. This figure was created using Figdraw.