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 Article has an altmetric score of 14

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 6

CDKL5 kinase activity facilitates p62 degradation and binding to ubiquitinated cargo during SINV infection.

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CDKL5 kinase activity facilitates p62 degradation and binding to ubiquit...
(A and B) CDKL5-KO HeLa cells reconstituted with EV, WT, or KD CDKL5–3×FLAG were infected with SINV/HA-capsid (MOI = 10, 8 hours). (A) Immunoblot with anti-HA, anti-p62, and anti-FLAG antibodies after CDKL5–3×FLAG immunoprecipitation with anti-FLAG antibody. (B) Quantification of p62 that coimmunoprecipitated with WT or KD CDKL5-FLAG normalized to FLAG. Bars are mean ± SEM from 3 independent experiments. (C and D) CDKL5-KO HeLa cells reconstituted and infected with SINV as in A were treated with either vehicle (DMSO) or BafA1 (100 μM) for 2 hours, followed by immunoblotting and quantification of p62 coimmunoprecipitation with CDKL5 (B). Bars are mean ± SEM from 3 independent experiments. (E and F) CDKL5-KO HeLa cells reconstituted as in A were infected with WT SINV for 8 hours (MOI = 10) and lysates from uninfected (baseline) or infected cells subjected to endogenous p62 immunoprecipitation. Ubiquitin (Ub) was detected via a pan-ubiquitin antibody. (E) Representative blot and (F) quantification of ubiquitin normalized to the level of EV. Bars are mean ± SEM from 3 independent experiments. All P values were determined by 1-way ANOVA with Šidák’s multiple-comparison test. *P < 0.05.