cryo-ET redefines HPV disassembly and transport paradigms.

Human papillomavirus (HPV) causes 4.5% of all human cancers, although a complete cellular basis of infection remains unclear. The mechanisms of virus disassembly and transport are particularly enigmatic. Here, we use cryo-electron tomography (cryo-ET) to provide direct visualization of high-risk HPV16 during its infection cycle. We demonstrate that, when HPV reaches the lysosome, it remains intact and infection competent, contrary to the prevailing view of viral inactivation within lysosomes. We challenge the current model of HPV trafficking, which predicts progressive disassembly of the L1 capsid during retrograde transport, by showing that HPV remains intact when it reaches the Golgi to the nucleus for infection. Finally, we provide snapshots of the HPV containing transport vesicles budding from the Golgi lumen in a COPI coat-dependent fashion and show that HPV is connected to the overlying membrane by proteinaceous structural bridges, possibly formed by the L2 capsid, to guide virus trafficking. Our results redefine the dominant models of HPV entry, revealing the virus capsid's resilience to disassembly during retrograde trafficking, a coat-dependent budding mechanism for transport, and demonstrate that the lysosome serves as a reservoir for infectious HPV.