CDKL5 regulates p62-mediated selective autophagy and confers protection against neurotropic viruses
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
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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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 1

CDKL5 is necessary for SINV–induced autophagy and host antiviral response to diverse viruses.

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CDKL5 is necessary for SINV–induced autophagy and host antiviral respons...
WT CDKL5/GFP-LC3 and 2 CDKL5-KO/GFP-LC3 HeLa clones (KO1 and KO2) were reconstituted with CDKL5 or empty vector (EV). (A) Representative fluorescence micrographs of GFP-LC3 puncta (autophagosomes) in mock-infected cells or cells infected with WT SINV at a multiplicity of infection (MOI) of 10 for 6 hours. Scale bar: 20 μm. Data presented here and in Supplemental Figure 1C are from an experiment performed contemporaneously. (B) Western blot of CDKL5 expression in WT and CDKL5-KO cells reconstituted with CDKL5 or EV. (C) Quantification of GFP-LC3 puncta, with bars representing mean ± SEM of triplicate samples and with at least 100 cells per sample. Statistical analysis was by 1-way ANOVA with Šidák’s correction for multiple comparisons, including the data in Supplemental Figure 1D. Results are representative of 3 independent experiments. (D) Western blot of LC3 conversion and (E) quantification by densitometry comparing WT and CDKL5-KO cells reconstituted with EV, WT CDKL5, or a kinase dead (KD) CDKL5, infected with SINV (MOI = 10, 6 hours) and treated with either DMSO (vehicle) or BafA1 for 2 hours prior to harvesting cells. Statistical analysis performed by 1-way ANOVA with Dunnett’s test for multiple comparisons. (F–H) Survival of 7-day-old CDKL5-WT and CDKL5-KO mice infected (F) intracerebrally (i.c.) with SINV (1 × 103 PFU) or (G) subcutaneously with CHIKV (1 × 105 PFU) and (H) 8- to 10-week-old CDKL5-WT and CDKL5-KO mice infected i.c. with HSV-1ΔBBD (5 × 104 PFU). Number of mice per genotype and P value (log-rank test) are indicated in F–H. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.