Rab9 depletion enhances human adenovirus type 26 transduction efficiency through increased internalization and reduced late endosomal/lysosomal retention

Abstract Understanding intracellular trafficking is critical for viral pathogenesis and for the rational design of viral vectors, as endocytic routing determines genome release, immune sensing, and overall transduction efficiency. Human adenovirus type 26 (HAdV-D26) presents a promising platform for vector design due to its low preexisting immunity, potent immune stimulation, scalable production, and versatile genetic engineering capacity. Although increasingly significant, the fundamental mechanisms governing HAdV-D26 intracellular trafficking are still not fully understood. Our study demonstrates that compared to well-described human adenovirus type 5 (HAdV-C5), HAdV-D26 undergoes prolonged intracellular trafficking, transiently localizing to early endosomes before residing in late endosomes/lysosomes for up to four hours post-infection. Inhibition of lysosomal acidification modestly enhances HAdV-D26 transduction efficiency, whereas blocking transport from early to late endosomes/lysosomes does not. Strikingly, Rab9 knockdown reduces HAdV-D26 late endosomal/lysosomal localization while increasing both virus internalization and genome delivery of HAdV-D26. These findings indicate that late endosomal sorting pathways actively influence HAdV-D26 infection outcomes. Together, our results provide new mechanistic insight into HAdV-D26 intracellular trafficking, highlight serotype-specific differences in adenovirus entry pathways, and identify endosomal trafficking steps that may be targeted to improve adenoviral vector performance. Author summary Human adenovirus type 26 is an important platform for vaccines and gene therapy due to its low preexisting immunity and versatility as a vector. Despite its growing importance, how HAdV-D26 enters and moves within host cells is still not well understood. In this study, we show that compared to HAdV-C5, HAdV-D26 exhibits prolonged intracellular trafficking, transiently passing through early endosomes and accumulating in late endosomes and lysosomes for several hours after entry. We further identify the host trafficking protein Rab9 as an important regulator of HAdV-D26 infection, as altering Rab9 levels changes viral internalization, late endosomal localization, and delivery of the viral genome. These findings reveal previously unrecognized mechanisms that control adenovirus intracellular trafficking and demonstrate that different adenovirus types use distinct cellular entry routes. Understanding these pathways provides insights that may guide the development of more effective and better-controlled adenovirus-based vaccines and gene delivery vectors.