Endosomal trafficking factor RAB5c acts as a host restriction factor against FAdV-4 propagation through interaction with ORF1B.

Rab small GTPases (RABs), a family of key regulators governing intracellular membrane trafficking and endosomal dynamics, are extensively implicated in diverse physiological and pathological processes. RAB5c, a critical isoform of the RAB5 subfamily that controls early endosome maturation and vesicle transport, also exerts indispensable functions during viral infection. Previous mass spectrometry analysis preliminarily identified RAB5c as a potential interacting partner of the ORF1B protein encoded by fowl adenovirus serotype 4 (FAdV-4). In this study, we further verified the interaction between RAB5c and ORF1B using co-immunoprecipitation (Co-IP) assays, and confocal microscopy revealed that these two proteins were co-localized in the cytoplasm of leghorn male hepatoma (LMH) cells. Furthermore, the expression pattern of RAB5c and its regulatory role during FAdV-4 infection was evaluated. Results demonstrated that both mRNA and protein levels of RAB5c were significantly upregulated upon FAdV-4 infection. Notably, RAB5c acted as a negative regulator of FAdV-4 replication in LMH cells. Viral titers and viral protein synthesis were remarkably reduced in RAB5c-overexpressing cells, but significantly increased in RAB5c-knockdown cells compared to the control ones. Collectively, these results identified RAB5c as a crucial factor that antagonized FAdV-4 replication.