Enteroviruses Antagonize the Host Restriction Factor ABL2 via Proteasomal Degradation to Facilitate Viral Replication

Figures

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  Fig. 1. Enterovirus infection downregulates ABL2 protein expression. A HeLa cells were infected with CVB3 at a MOI of 1. Cell lysates were collected at 7, 10, and 12 hpi from mock-infected and CVB3-infected cells, and ABL2 expression was analyzed by Western blotting. GAPDH was used as the sample loading control. B, C HeLa cells were infected with CVB3 at a MOI of 5 (B) or 10 (C). Cell lysates were collected at 3, 5, and 7 hpi from mock-infected and infected cells, and ABL2 expression was analyzed by Western blotting. D HL-1 cells were infected with CVB3 at a MOI of 50. Cell lysates were collected at 12, 16, and 24 hpi from mock-infected and infected cells, and ABL2 expression was analyzed by Western blotting. E, F HeLa cells were infected with CVB5 at a MOI of 1 (E) or 5 (F). Cell lysates were collected at 12 and 24 hpi from mock-infected and infected cells, and the expression of ABL2 was analyzed by Western blotting. G, H RD cells were infected with EV-A71 (G) or CVA16 (H) at a MOI of 5. Cell lysates were collected at 5 and 7 hpi from mock-infected and infected cells, and the expression of ABL2 was analyzed by Western blotting.

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  Fig. 2. Enteroviral non-structural protein 2B induces ABL2 degradation in a dose- and proteasome-dependent manner. A HeLa cells were transfected with 2 μg of empty vector or plasmids encoding FLAG-tagged CVB3 non-structural proteins (2A, 2B, 2C, 3A, 3C, or 3D). Cell lysates were collected 48 h post-transfection and analyzed by Western blotting for ABL2 expression. B HeLa cells were transfected with increasing doses of FLAG-2B plasmid (0.5, 1, or 2 μg). Cell lysates were collected 48 h post-transfection and after which the expression of ABL2 was analyzed by western blotting. C qRT-PCR analysis of ABL2 mRNA levels in HeLa cells transfected with 2 μg of FLAG-2B or pcDNA3.1 empty vector (Vec). D Western blot analysis of ABL2 protein levels in HeLa cells transfected with 2 μg of FLAG-2B for 48 h and then treated with Z-VAD-FMK (50 μM), CQ (50 μM), or MG132 (20 μM) for 6 h. MG132 rescued ABL2 protein levels, indicating proteasomal degradation. E Predicted potential ubiquitination sites on ABL2 based on the PhosphoSitePlus database. F HeLa cells were co-transfected with wild-type ABL2 or K331R mutant and FLAG-2B plasmid. Cell lysates were collected 48 h after transfection, and the expression of…

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  Fig. 3. ABL2 inhibits enterovirus replication. Stable HeLa cell lines overexpressing ABL2 (oeABL2), silencing ABL2 (shABL2), and their respective negative controls (NC and shNC) were generated by lentiviral transduction. A, D NC and oeABL2 cells were infected with CVB3 (A) or EV-A71 (D). Cell lysates were collected at the indicated time points post-infection, and VP1 expression was analyzed by western blotting. B, C shNC and shABL2 cells were infected with CVB3 (B) or CVB5 (C). Cell lysates were collected at the indicated time points post-infection, and VP1 expression was analyzed by western blotting. E, F Viral titers in culture supernatants of CVB3-infected control, oeABL2 (E), and shABL2 (F) cells at 7 hpi were determined by standard plaque assay. G Viral titers in culture supernatants of NC and oeABL2 cells infected with EV-A71 for 7 h were similarly quantified by plaque assay. H, I Control and oeABL2 (H) or shABL2 (I) cells were transfected with a CVB3-eGFP replicon, and eGFP fluorescence was visualized by fluorescence microscopy at 24 h post-transfection. ∗∗P < 0.01, ∗∗∗P < 0.001 (biological repeats with the mean ± SD, n = 3).

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  Fig. 4. ABL2 affects the early stage of enterovirus replication. A NC and shABL2 cells were infected with CVB3 (MOI = 5) by adsorption at 4 °C for 1 h to allow binding. Unbound virus was removed by washing with cold PBS, and cell-associated viral RNA was quantified by qRT-PCR. B For the internalization assay, cells were bound with the virus as described in (A), and then shifted to 37 °C for 2 h to allow entry. External virus was removed by acid washing, and internalized viral RNA was quantified by qRT-PCR. C, D Control and shABL2 (C) or oeABL2 (D) cells were infected with CVB3 (MOI = 5) for the indicated times. Total RNA was extracted, and CVB3 RNA levels were quantified by qRT-PCR. E NC and oeABL2 cells were infected with CVB5 (MOI = 5) for the indicated times. Total RNA was extracted, and CVB5 RNA levels were quantified by qRT-PCR. F NC and oeABL2 cells were infected with EV-A71 (MOI = 5) for the indicated times. Total RNA was extracted, and EV-A71 RNA levels were quantified by qRT-PCR. NS, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (biological repeats with the mean ± SD, n = 3).

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  Fig. 5. ABL2 interacts with RAC1 and downregulates RAC1 protein levels. A Co-immunoprecipitation (co-IP) and immunoblotting analysis of the interaction between ABL2 and RAC1. HeLa cells were lysed and subjected to immunoprecipitation with anti-ABL2, anti-RAC1, or control IgG antibodies, followed by immunoblotting with the indicated antibodies. B HeLa cells were immunostained with anti-ABL2 (red) and anti-RAC1 (green) antibodies. Nuclei were counterstained with DAPI (blue). Images are representative of three independent experiments (Scale bar = 20 μm). C, D Control and oeABL2 (C) or shABL2 (D) cells were infected with CVB3 (MOI = 5) for the indicated times. Cell lysates were analyzed by Western blotting to assess the impact of ABL2 modulation on RAC1 protein levels. E, F NC and oeABL2 cells were infected with CVB5 (MOI = 5) (E) or EV-A71 (MOI = 5) (F) for the indicated times. Cell lysates were analyzed by Western blotting to determine the effect of ABL2 overexpression on RAC1 expression. NS, not significant, ∗∗P < 0.01, ∗∗∗P < 0.001 (biological repeats with the mean ± SD, n = 3).

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  Fig. 6. RAC1 promotes CVB3 replication and activates the PI3K/AKT pathway. HeLa cells were transfected with 2 μg of pcDNA3.1-RAC1 (oeRAC1), 50 nM siRAC1, or their respective negative controls (pcDNA3.1 empty vector and scrambled siRNA). At 36 h post-transfection, cells were mock-infected or infected with CVB3 at an MOI of 5 for the indicated times. A, B, G, H Cell lysates were subjected to Western blot analysis using antibodies against RAC1, CVB3 capsid protein VP1, phosphorylated PI3K (p-PI3K), phosphorylated AKT (p-AKT), total PI3K, and total AKT. GAPDH served as the loading control. (A, G) RAC1-overexpressing cells. (B, H) RAC1-silenced cells. C, D Viral titers in culture supernatants were determined by standard plaque assay at 7 hpi. E, F HeLa cells were co-transfected with a CVB3-eGFP replicon and either a RAC1-expressing plasmid (E), siRAC1 (F), or their respective controls. eGFP fluorescence was visualized by fluorescence microscopy at 24 h post-transfection. ∗P < 0.05, ∗∗∗P < 0.001 (biological repeats with the mean ± SD, n = 3).

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  Fig. 7. ABL2 regulates CVB3 replication through RAC1 and the PI3K/AKT pathway. A, B Control and oeABL2 (A) or shABL2 (B) cells were infected with CVB3 (MOI = 5) for the indicated times. Cell lysates were analyzed by Western blotting for the expression of ABL2, p-PI3K, p-AKT, and total PI3K and AKT. C NC and oeABL2 cells were transfected with RAC1-expression plasmid (oeRAC1) or pcDNA3.1 empty vector (Vec). At 48 h post-transfection, cells were mock-infected or infected with CVB3 (MOI = 5) for 7 h. Cell lysates were analyzed by Western blotting with the indicated antibodies. The numbers below the bars correspond to the lanes in the Western blot above. D shNC and shABL2 cells were transfected with RAC1-expression plasmid (oeRAC1) or pcDNA3.1 empty vector (Vec). At 48 h post-transfection, cells were mock-infected or infected with CVB3 (MOI = 5) for 7 h. Cell lysates were analyzed by Western blotting with the indicated antibodies. The numbers below the bars correspond to the lanes in the Western blot above. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (biological repeats with the mean ± SD, n = 3).

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  Fig. 8. Schematic representation of the antiviral action of ABL2 and the mechanism by which enteroviruses degrade ABL2.

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  Supplementary Figure S1. HeLa cells were cotransfected with FLAG-2B . IP and immunoblot analysis of ABL2 and FLAG-2B were shown.

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  Supplementary Figure S2. Stable HeLa cell lines overexpressing (A) or silencing (B) ABL2 were generated using lentiviral transduction. ABL2 expression levels were verified by western blotting.

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  Supplementary Figure S3. HeLa cells were infected with CVB3 (MOI = 5) for the indicated times. Cell lysates were analyzed by western blotting to assess RAC1 protein expression.