The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coor-dinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability andmembrane rup-ture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomi-croscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual func-tion in maintaining the metastable state of the capsid by preventing premature disassembly andmediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electronmicroscopy, we suggest a re-fined disassembly mechanism upon entry. IMPORTANCE In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the mem-brane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal pro-tein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protei