Kaposi’s sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies
Fig 10
NDP52 immunoprecipitates with the PB resident protein Pat1b.
A: HEK293T cells were transfected with plasmids expressing pcDNA-FLAG-NDP52 with either pLJM1-GFP (control), pLJM1-GFP-Dcp1a, or pLJM1-GFP-Pat1b for 48 h. Cells were harvested in lysis buffer and incubated with a FLAG mouse antibody (CST) overnight at 4°C. Immunoprecipitation was performed and samples were eluted from the magnetic beads through boiling in 4X Laemmli buffer (BioRad) with 10% v/v beta-mercaptoethanol. Samples were resolved by SDS-PAGE. A representative blot of three independent experiments is shown. B: HUVECs were infected with rKSHV.219 via spinoculation and infection was allowed to proceed for 96 h. Cells were harvested in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for Dcp1a or Pat1b. Samples were quantified by normalizing Dcp1a or Pat1b protein levels to the total protein in each lane using Image Lab (BioRad) and then to Mock. Results were plotted in GraphPad and an unpaired t-test was performed, ±SEM; n = 3, ** = P<0.01. C: Model of PB disassembly induced by KSHV/KapB. KapB increases the phosphorylation of Beclin at Serine 90 to enhance autophagic flux during KSHV latency while Torin induces autophagic flux via the inhibition of mTORC1/2 and dephosphorylation of the ULK complex. Enhanced activation of autophagic flux by either mechanism results in PB disassembly that is dependent on the selective autophagy receptor, NDP52. In our model, we predict that NDP52 recruits a PB component(s) to the nascent autophagosome. Our top candidate for this recruitment is the PB scaffolding protein, Pat1b, as NDP52 co-immunoprecipitated with Pat1b and Pat1b protein level decreases after KSHV infection and Torin treatment. Steady-state levels of Dcp1a also decreased in response to KapB expression, KSHV infection, or Torin treatment, suggesting that Dcp1a decreases may be mediated by its partial interaction with Pat1b despite observations that Dcp1a did not co-immunoprecipitate with NDP52. Autophagic degradation of Pat1b and Dcp1a, both key PB scaffolding proteins, leads to PB disassembly. Figure created with Biorender.