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. 2021 Aug 2;40(18):e105658. doi: 10.15252/embj.2020105658

Figure 7. PPxPxY‐containing host proteins modulate EBOV infectivity, viral transcription, and VP30 phosphorylation.

Figure 7

  1. HeLa cells were transfected with scrambled siRNA (SCR si) or siRNAs‐targeting hnRNP L, hnRNPUL1 or PEG10, 48 h post‐transfection, cells were challenged with EBOV‐EGFP at an MOI of 0.5. 24 h post‐infection, cells were fixed, stained for nuclei with Hoechst dye, and imaged. Scale bar is 200 µm. The graph (right) depicts relative number of infected cells as determined by calculating the ratio of number of infected cells to nuclei and shown relative to SCR siRNA. The data show a representative result of two independent experiments with the mean ± SD calculated from three independently treated wells. Statistical significance was calculated relative to the values obtained in SCR siRNA‐treated cells using ANOVA with Dunnett’s multiple comparisons test. ***P < 0.0001, ***P < 0.001, **P < 0.01
  2. Levels of transcription and replication in the MG assay upon over‐expression of the indicated host factors. MG assays were performed with 50 and 500 ng of host protein expression plasmid. RNA was isolated and analyzed by qRT–PCR to measure the levels of viral mRNA/cRNA and vRNA. The sample with vector control was set to 100%, and mean ± S.D. values from two independent experiments are shown (each experiment was performed in duplicate, n = 2). **P < 0.005, *P < 0.05. Statistical significance was calculated relative to vector control using ANOVA with Tukey’s multiple comparisons test.
  3. Immunoblots to assess VP30 and pVP30 levels in lysates from MG assay upon knockdown of RBBP6, hnRNP L, or hnRNPUL1.
  4. Immunoblots to assess the levels of VP30, pVP30, and NP in EBOV‐infected lysates upon knockdown of NPC1 or RBBP6. HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6. 48 h post‐transfection cells were infected with EBOV at MOI = 0.1. At 24 h post‐infection, cells were lysed in TRIzol. Protein was extracted from the TRIzol reagent and analyzed by Western blotting to determine levels of VP30, pSer29 VP30, and NP.
  5. Western blots detecting pVP30, VP30 and NP levels in EBOV‐infected HeLa cells upon hnRNP L, hnRNPUL1, or PEG10 knockdown.
  6. Proposed model for inhibition of EBOV replication by host factors containing PxPPPPxY motifs. Host proteins present at varying endogenous levels disrupt VP30–NP interaction thereby preventing VP30 dephosphorylation by NP‐recruited host phosphatase PP2A B56, and therefore inhibit virus transcription.