CAPRIN1 Is Required for Control of Viral Replication Complexes by Interferon Gamma

Figures

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  FIG 1. Human GBP1 localizes with the MNV replication complex and is important for IFNG-mediated control of MNV replication. (A) Immunofluorescence assay (IFA) for the localization of GBPs to the MNV RC in HeLa cells expressing individual GBPs. The cells were transfected with a plasmid expressing MNV ORF1 for 6 h followed by treatment without or with IFNG at 100 U/mL for an additional 18 h. Quantitation of IFA for the localization of GBPs (1–7) on to the MNV RC is shown (B) Representative Western blot of HeLa cells from (A); expressing COVA (cytoplasmic ovalbumin; used as a control) and individual GBPs. (C) IFA for the localization of GBPs to the MNV RC. HeLa cells expressing individual GBPs were transfected with a plasmid expressing MNV ORF1 for 6 h followed by treatment without or with IFNG at 100 U/mL for an additional 18 h. Representative images shown here. Zoomed images shown only for cells showing colocalization. Scale bar, 5 μm. (D) As human cells do not express MNV receptor, MNV vRNA were transfected into the WT, ATG16L1−/− and GBP1−/− HeLa cells and MNV production was measured by TCID50. (E) Representative Western blot of HeLa cells for (D); WT, ATG16L1−/− and GBP1−/−. Cells…

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  FIG 2. GBP1 is the necessary and sufficient human GBP for IFNG inhibition of MNV replication. (A) Quantitation of IFA for the localization of GBP1 mutants on to the MNV RC. HeLa cells expressing GBP1 and mutants were transfected with a plasmid expressing MNV ORF1 for 6 h followed by treatment with IFNG at 100 U/mL for an additional 18 h. After incubation, the cells were fixed and stained with anti-Propol antibody and anti HA antibody for GBP1 and its mutants (B) Representative Western blot of HeLa cells to confirm the protein reconstitution for (A and C). (C) MNV production in the WT and GBP1−/− HeLa cells expressing either COVA (control) or WT/mutants GBP1. (D) Representative Western blot of WT, ATG16L1−/− and GBP1-7−/− HAP1 cells expressing either COVA (control) or GBP1 for (E). Cells were untreated or treated with 100 U/mL IFNG for 24 h (E) MNV production in the WT, ATG16L1−/− and GBP1-7−/− HAP1 cells expressing either COVA (control) or GBP1. For all MNV production measurement in HeLa cells, each group of cells were untreated or treated with the 100 U/mL of IFNG for 24 h and then transfected with 50 ng MNV viral RNA. At 24 hpt, cells were harvested to titer infectious virus by…

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  FIG 3. ATG16L1 localizes to the RCs formed by MNV protein expression and WIPI2 binding region of ATG16L1 (208-230) is required for IFNG-mediated inhibition of MNV replication. (A) IFA for the localization of ATG16L1 to viral RC (via anti-Propol) in HeLa cells. The cells were transfected with MNV ORF1 and pDRFP-ATG16L1 plasmid and incubated for 24 h. After incubation, the cells were fixed and stained with anti-Propol antibody. (B) IFA for the localization of ATG16L1 to viral RC-like compartment formed by NS4 in HeLa Cells. The cells were transfected with MNV NS4-EGFP and pDRFP-ATG16L1 plasmid and incubated for 24 h. After incubation, the cells were fixed. Representative images (A and B, left) and Mander’s coefficients from 2 experiments (A and B, right) are shown. (C) Schematic overview of wild type and mutants ATG16L1 indicating functional regions and mutation. (D and E) MNV production in Atg16l1f/f control BMDM (D) and Atg16l1f/f+LysMcre BMDM (E). BMDM cells were untransduced (-, control) or transduced with control (EGFP) or WT/mutants ATG16L1s described in (C). After completing BMDM differentiation (25 days), cells were untreated or treated with 100 U/mL IFNG for 24 h and then…

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  FIG 4. Wipi2 is not required for IFNG-mediated control of MNV replication. (A) MNV production in the WT and Wipi2−/− BV2 cells. The cells were untreated or treated with 100 U/mL of IFNG for 24 h and then infected with 0.05 MOI of MNV. At 24 hpi, cells were harvested to titer infectious virus by TCID50. (B) A representative Western blot data of cells described in Fig. 4A. Data were analyzed using unpaired t test. Ns, not significant; *, P < 0.05; **, P < 0.01. Dashed line indicates the limit of detection (LD).

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  FIG 5. CAPRIN1 is required for LC3 targeting of RC and IFNG inhibition of MNV replication. (A-C) qRT-PCR of selected genes in control and shRNA-mediated gene knockdown (KD) in BV2 cells. BV2 cells were transduced with lentivirus expressing shRNA against Caprin 1 (A), Vim (B), or DHx9 (C) and then selected with puromycin (3 μg/mL) for 4 days. After selection, mRNA expression level from each KD cell lines were measured by qRT-PCR. Results are normalized to an average of control WT BV2 cells. (D) MNV production in untreated or IFNG treated WT and selected gene KD BV2 cells. WT and KD BV2 cells were untreated or treated with 100 U/mL of IFNG for 24 h and then infected with 0.05 MOI of MNV. At 24 hpi, cells and supernatants were harvested to titer infectious virus by TCID50. (E) MNV production in CAPRIN1 reconstituted BV2 cells. WT, CAPRIN1 KD, and CAPRIN1 KD expressing CAPRIN1 construct BV2 cells were untreated or treated with 100 U/mL of IFNG for 24 h and then infected with 0.05 MOI of MNV. At 24 hpi, cells and supernatants were harvested to titer infectious virus by TCID50. Representative Western blot of CAPRIN1 KD and reconstituted BV2 cells is shown (E, right). (F) Representative…

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  FIG 6. Model of RC recognition and inhibition by TAg. Viral RCs are affiliated with WIPI2 and CAPRIN1, which are bound by ATG16L1/AGT5/ATG12 to conjugate LC3 to RCs. INFG treatment induces the relocalization of hGBP1/mGBP2 to LC3-containing RCs, which promotes their destruction.