Fig 1.
ALTO co-operates with STING to stimulate IFNβ expression.
A. HEK293 cells were transfected with plasmids carrying ALTO or an empty vector control (CO) and STINGR284S (or RFP control). Two days post-transfection, mRNA levels of IFNβ were measured using RT-qPCR. One of the values for transfection with vector and RFP was set as 1. B. HDF-inRFP and -inALTO cells were mock-treated or induced with Dox and treated with diABZI (or DMSO control) for 19 hours. mRNA levels were measured using RT-qPCR. The value from HDF-inRFP cells mock- and DMSO-treated was set as 1. Error bars indicate the standard deviation from three independent samples. ***p<0.001.
Fig 2.
ALTO stimulates TBK1S172 autophosphorylation and STING stabilization.
A. HDF-inALTO or -inRFP cells were mock-treated or induced with Dox for 24 hours, then stimulated with diABZI (or DMSO control) for 16 hours. Whole cell lysates were resolved by SDS/PAGE and immunoblotted with the indicated antibodies. B. HDF-inALTO or -inRFP cells were mock-treated or induced with Dox for 7 hours, then stimulated with diABZI (or DMSO control) for an additional 19 hours. Cells were fixed and immune-stained for STING, and counterstained with DAPI. Scale bar, 20 μm. C. HDF-inALTO or -inRFP were mock-treated or induced with Dox for 40 hours, then stimulated with diABZI (or DMSO control) for the indicated times. Whole cell lysates were resolved by SDS/PAGE and immunoblotted with indicated antibodies. Red asterisks indicate phosphorylated STING.
Fig 3.
ALTO interacts with STING and TBK1.
A. HDF-inALTO cells were induced with 0.01μg/mL Dox for 40 hours. Whole cell lysates were incubated with normal rabbit IgG or anti-ALTO antibody and immunoprecipitated with Protein G agarose. Co-immunoprecipitants were resolved by SDS/PAGE and immunoblotted with the indicated antibodies. B. Schematic diagram depicting the NanoBiT and BiFC structural complementation systems. NanoBiT reversibly reconstitutes NanoLuciferase and produces luminescence in live cells, whereas BiFC irreversibly reconstitutes Venus to produce GFP fluorescence. C. HEK293 cells were transfected with pairs of plasmids carrying NanoBiT-tagged ALTO, STING, and TBK1 or the Small BiT negative control construct. At 20 hours (ALTO-TBK1) or 24 hours (ALTO-ALTO and ALTO-STING) post-transfection, NanoGlo reagent was added, and luminescence was measured after a 15-minute (ALTO-ALTO) or 2-hour (ALTO-STING and ALTO-TBK1) incubation. Points indicate replicate wells from a single experiment, bars indicate means, and error bars indicate standard deviations. L-protein and protein-S (and similar) indicate the named protein tagged with the LgBiT at its N-terminus or SmBiT at its C-terminus, respectively. ***p<0.001; **p<0.01; *p<0.05. D. U2OS cells were transfected with pairs of plasmids carrying FLAG-tagged Venus (N-terminal half or C-terminal half) fused ALTO constructs. At 48 hours post-transfection, cells were fixed and immunostained for FLAG, and counterstained with DAPI. E. U2OS cells were transfected with pairs of plasmids carrying FLAG-tagged Venus (N-terminal half or C-terminal half) fused ALTO and TBK1, respectively. At 36 hours post-transfection, cells were fixed and immunostained for FLAG, and counterstained with DAPI. F. U2OS cells were transfected with pairs of plasmids carrying FLAG-tagged Venus (N-terminal half or C-terminal half) fused ALTO and STING, respectively. At 24 hours post-transfection, cells were fixed and immunostained for FLAG, and counterstained with DAPI. Scale bar, 20 μm.
Fig 4.
ALTO recruits Src into complex with TBK1 to stimulate its autophosphorylation.
A. AlphaFold2 model of ALTO protein, with the Src-binding P-X-X-P motifs denoted in red. The table provides details for P-X-X-P motif sequences and positions. B. HDF-inALTO or -inRFP cells were mock-treated or induced with Dox for 10.5 hours, then stimulated with diABZI (or DMSO control) for 1.5 hours. Whole cell lysates were incubated with mouse anti-Src antibody and immunoprecipitated with Protein G agarose. Immunoprecipitants were resolved by SDS/PAGE and immunoblotted with the indicated antibodies.
Fig 5.
MCPyV rescues STING levels to stimulate IFN signaling.
A. Normal HDFs mock-infected or infected with MCPyV were stimulated with diABZI (or DMSO control) at the time FBS was added (2 days post-infection). At 5 days post-infection, cells were harvested, and mRNA levels were quantified by RT-qPCR. The value for mock-infected and DMSO-treated HDFs was set as 1. Error bars indicate standard deviation from 3 replicates. B. Normal HDFs were mock-infected or infected with MCPyV, then stimulated with diABZI (or DMSO control) at the time FBS was added (2 days post-infection). Whole cell lysates were collected at 5 days post-infection, resolved by SDS/PAGE, and immunoblotted with the indicated antibodies. C. Normal HDFs were mock-infected or infected with MCPyV, then stimulated with diABZI (or DMSO control) at the time FBS was added (2 days post-infection). Cells were fixed at 5 days post-infection and immunostained for LT and STING, and counterstained with DAPI. Scale bar, 20 μm.
Fig 6.
MCPyV ALTO suppresses viral infection activity.
A. Normal HDFs were mock-infected or infected with 6x108 copies of MCPyV per 48-well. Whole cell lysates were collected at the indicated time points post-infection. Lysates were resolved by SDS/PAGE and immunoblotted with the indicated antibodies. B. Normal HDFs were infected with the indicated titers of WT or ALTOnull MCPyV per 96-well. Cells were harvested and DNA extracted at 5 days post-infection. Viral replication was quantified by qPCR using the NCRR-specific primers and normalized to genomic GAPDH levels. Bars indicate mean; error bars indicate standard deviation from 3 replicates. **p<0.01; *p<0.05. C. HDF-inALTO cells were infected with the indicated titers of ALTOnull MCPyV per 96-well. Cells were mock-treated or induced with Dox at 2- and 4-days post-infection. Cells were harvested and DNAs were extracted at 5 days post-infection. Viral replication was quantified by qPCR using the NCRR-specific primers and normalized to genomic GAPDH levels. Bars indicate the mean; error bars indicate the standard deviation from 3 replicates. ***p<0.001. D. HDF-inALTO cells were infected with ALTOnull MCPyV. Cells were mock-treated or induced with Dox at 2- and 4-days post-infection. Cells were fixed on day 5 post-infection and stained for LT and either ALTO or MX1, and counterstained with DAPI. Scale bar, 50 μm.
Fig 7.
ALTO suppresses MCPyV infection at the single-cell level.
A. Normal HDFs were either mock-infected or infected with 6x108 viral genome equivalents of MCPyV virions per well in a 48-well plate. Cells were fixed on day 4 post-infection, immunostained for ALTO and LT, and counterstained with DAPI. Scale bar: 20 μm. B. Normal HDFs were either mock-infected or infected with 6x108 viral genome equivalents of WT or ALTOnull MCPyV virions per well in a 48-well plate. Cells were fixed on day 5 post-infection, immunostained for ALTO, subjected to FISH using an MCPyV probe, and counterstained with DAPI. Scale bar: 20 μm. C. The FISH signals within individual cells treated as in B were quantified using ImageJ. Shown are the quantification results for the FISH signal in individual ALTO-positive and ALTO-negative HDFs infected with WT MCPyV, as well as HDFs infected with ALTOnull MCPyV. Each type of cell is indicated with color-coded arrows. More than 40 cells were analyzed for each sample group to achieve a statistically meaningful sample size. Central diamonds represent the median values of the data groups. ***p<0.001; **p<0.01; n.s. = not significant.
Fig 8.
TBK1 inhibition reactivates MCPyV infection activity.
A. HDF-inALTO cells were treated with the indicated TBK1 inhibitor (or DMSO control) for one day, then treated with 0.05 μg/mL Dox for two additional days. Whole cell lysates were resolved by SDS/PAGE and immunoblotted with the indicated antibodies. B. HDF-inALTO cells were infected with ALTOnull MCPyV. Beginning 2 days post-infection, the cells were treated with the TBK1 inhibitor GSK8612 (or DMSO control) at the indicated doses, then treated with 0.05 μg/mL Dox at 3 days post-infection. At 5 days post-infection, cells were harvested, and DNA extracted. Viral replication was quantified by qPCR using the NCRR-specific primers and normalized to genomic GAPDH. The value from one group of uninduced cells treated with DMSO was set as 1. Bars indicate the mean from three replicates; error bars indicate the standard deviation. Percentages reflect relative replication in Dox-induced cells compared to their respective uninduced controls. ***p<0.001; **p<0.01; *p<0.05. C. ALTO stimulates STING-TBK1 signaling to negatively regulate MCPyV infection. The MCPyV protein ALTO recruits Src via its PXXP motifs (red) to phosphorylate TBK1, leading to STING stabilization and activation. MCPyV replication also stimulates the activation of the STING downstream signaling pathway. Activated STING induces an enhanced downstream ISG response via IRF3 and NF-κB. This antiviral response provides a negative feedback mechanism to control MCPyV propagation and persistence.