CDKL5 regulates p62-mediated selective autophagy and confers protection against neurotropic viruses
Josephine W. Thinwa, … , Tiffany A. Reese, Michael U. Shiloh
Josephine W. Thinwa, … , Tiffany A. Reese, Michael U. Shiloh
Published November 2, 2023
Citation Information: J Clin Invest. 2024;134(1):e168544. https://doi.org/10.1172/JCI168544.
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Research Article Infectious disease Virology

CDKL5 regulates p62-mediated selective autophagy and confers protection against neurotropic viruses

Abstract

Virophagy, the selective autophagosomal engulfment and lysosomal degradation of viral components, is crucial for neuronal cell survival and antiviral immunity. However, the mechanisms leading to viral antigen recognition and capture by autophagic machinery remain poorly understood. Here, we identified cyclin-dependent kinase–like 5 (CDKL5), known to function in neurodevelopment, as an essential regulator of virophagy. Loss-of-function mutations in CDKL5 are associated with a severe neurodevelopmental encephalopathy. We found that deletion of CDKL5 or expression of a clinically relevant pathogenic mutant of CDKL5 reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid protein aggregates and cellular cytotoxicity. Cdkl5-knockout mice displayed increased viral antigen accumulation and neuronal cell death after SINV infection and enhanced lethality after infection with several neurotropic viruses. Mechanistic studies demonstrated that CDKL5 directly binds the canonical selective autophagy receptor p62 and phosphorylates p62 at T269/S272 to promote its interaction with viral capsid aggregates. We found that CDKL5-mediated phosphorylation of p62 facilitated the formation of large p62 inclusion bodies that captured viral capsids to initiate capsid targeting to autophagic machinery. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear intracellular toxic viral protein aggregates during infection.

Authors

Josephine W. Thinwa, Zhongju Zou, Emily Parks, Salwa Sebti, Kelvin Hui, Yongjie Wei, Mohammad Goodarzi, Vibha Singh, Greg Urquhart, Jenna L. Jewell, Julie K. Pfeiffer, Beth Levine, Tiffany A. Reese, Michael U. Shiloh

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Figure 3

Loss of CDKL5 impairs viral antigen clearance and promotes virus-induced cell death.

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Loss of CDKL5 impairs viral antigen clearance and promotes virus-induced...
(A–C) WT and CDKL5-KO HeLa cells were mock infected or infected with SINV/mCherry-capsid (MOI = 10) for 24 hours. (A) Representative fluorescence micrographs of mCherry-capsid. Scale bar: 20 μm. (B) Representative flow cytometry plots of mCherry-capsid–positive cells and (C) quantification of 3 biological replicates. Bars represent mean ± SEM. P values were determined by unpaired, 2-tailed t test. (D and E) WT and CDKL5-KO HeLa cells reconstituted with EV, KD, or WT CDKL5 were infected with SINV/mCherry-capsid (MOI = 10, 24 hours) and (D) mCherry-capsid detected by Western blot using an anti–SINV capsid antibody. (E) Quantification of capsid/actin ratio from 4 independent experiments. P values determined by 1-way ANOVA with Dunnett’s test for multiple comparisons. (F and G) WT and CDKL5-KO HeLa cells infected with SINV/3×HA-capsid (MOI = 10, 8 hours) and stained with antibodies against LAMP1 and HA for capsid detection. (F) Representative immunofluorescence micrographs with (G) quantification of colocalization. Bars are mean ± SEM of percentage capsid+ puncta colocalized with LAMPI in 8 images (>50 cells). Arrowheads denote examples of capsid+/LAMP1+ puncta. P values were determined by unpaired, 2-tailed t test. (H) WT and CDKL5-KO HeLa cells were infected with SINV/mCherry-capsid (MOI = 10, 24 hours) and cell viability determined by CellTiter-Glo assay (see Supplemental Methods). Bars on graph represent mean ± SEM from 3 independent experiments. P values were determined by unpaired, 2-tailed t test. (I) High-MOI (MOI = 10) and (J) low-MOI multistep growth curve analysis (MOI = 0.01) of WT and CDKL5-KO HeLa cells infected with SINV/mCherry-capsid. Progeny viruses were quantified by plaque assays. Bars represent mean ± SD of 3 experimental replicates and P values were determined by 2-way ANOVA with Šidák’s multiple-comparison test. **P < 0.01; ***P < 0.001; ****P < 0.0001.