Fiber-2 induces autolysosome dysfunction and impairs autophagic flux to promote FAdV-4 replication.

Fowl adenovirus serotype 4 (FAdV-4) causes hepatitis-hydropericardium syndrome in poultry, yet viral subversion of host autophagy remains unclear. Here, we investigated the role of the viral structural protein Fiber-2 in modulating the host autophagy-lysosome pathway. Transcriptomic profiling of LMH cells expressing Fiber-2 revealed coordinated upregulation of autophagy-related genes, validated by quantitative PCR. Functional assays demonstrated that Fiber-2 expression leads to autophagosome accumulation and impaired autophagic flux, as shown by LC3-II/p62 analysis, tandem fluorescent LC3 reporter imaging, Bafilomycin A1 treatment, and transmission electron microscopy. Fiber-2 predominantly localizes to late endosomes and lysosomes, and GFP-Galectin-3 reporter assays indicate that it disrupts lysosomal membrane integrity rather than blocking autophagosome-lysosome fusion. Co-immunoprecipitation (Co-IP) and enrichment analyses identified BECN1, a key autophagy regulator, as a Fiber-2-interacting protein, and associated host proteins were enriched in autophagy-related pathways. Finally, CRISPR/Cas9-mediated deletion of Fiber-2 significantly reduced FAdV-4 replication at later stages of infection, consistent with a proviral role for Fiber-2. These findings indicate that Fiber-2 disrupts autophagic flux by inducing lysosomal dysfunction and membrane damage, thereby promoting FAdV-4 replication and uncovering a previously unrecognized mechanism of viral subversion of host degradative pathways.