Figueras Novoa, Carmen; (2024) Manipulation of host autophagy machinery and cell death pathways by viral ion channels. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Characterizing exploitation of host cellular mechanisms by viruses is key to understanding pathogenesis. This work explores the hijacking of the autophagy and cell death pathways by two viral ion channel proteins, the Influenza A virus (IAV) Matrix 2 (M2) and SARS-CoV-2 Envelope (E) viroporins. In this thesis, a predicted caspase cleavage motif in the M2 C-terminal domain was characterized, which led to the identification of amino acid D85 as the aspartic acid recognized and cleaved by caspases. Using caspase deficient cell lines, caspase-6 and, to a lesser extent, caspase-3 were identified as responsible for mediating M2 cleavage. The 82SAVD85 motif described in this work is upstream of an LC3 interacting region (LIR) reported in the literature (91FVSI94). Removal of the last 12 amino acids in the C-terminal region, mimicking cleavage, abolished the M2-LC3B interaction and led to reduced levels of LC3B lipidation. While an IAV mutant containing an uncleavable M2 (M2D85A) localized similarly to wild-type M2, IAV expressing truncated M2 (M2∆86-97) was deficient in M2 plasma membrane localization. The M2∆86-97 mutant was also substantially attenuated when compared to wild-type virus and M2D85A titers. Purification of M2D85A and M2∆86-97 virions showed that the M2 LIR motif mediates LC3B incorporation into virions. Due to the similarities between E and M2, LC3B lipidation during SARS-CoV-2 infection and E expression was studied. Here, the E ion channel activity was discovered to activate V-ATPase/ATG16L1-induced LC3 lipidation (VAIL). Furthermore, both VAIL inhibitor SopF and infection with a SARS-CoV-2 mutant virus deficient in E ion channel activity resulted in a decreased viral titer, pointing to a role for VAIL in viral replication. Together, this work presents different mechanisms employed by viral ion channels to hijack cellular pathways and directly interact with host proteins.

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