Regulation of HPV-16 infection by ubiquitination of the L1 major capsid protein

The L1 major capsid protein of the papillomavirus is critical for both viral assembly and entry into host cells, but the impact of its post-translational modifications on the HPV life cycle remains poorly understood. In this study, we show, for the first time, that HPV16 L1 is ubiquitinated at several conserved lysine residues (K20, K64, K152, K217, K437, K452, and K454) identified by mass spectrometry. Mutations at K64 and K152 prevent the formation of pseudovirions, while other lysine mutants permit the formation of pseudovirions, but these display lower infectivity in several epithelial cell lines. Pre-incubation of HPV-16 pseudovirions with an anti-ubiquitin antibody also significantly reduces their infectivity. Ubiquitination does not appear to be essential for virus attachment, but K452 and K454 are critical for efficient infectious entry, and they impact intracellular trafficking and processing. Taken together, these results demonstrate a critical link between the ubiquitin-conjugating system and HPV capsid proteins and highlight the potential and novel role of L1 protein ubiquitination in regulating different stages of the HPV infection. IMPORTANCE: Despite vaccination efforts, HPV infection remains a major global health threat. Understanding how the virus interacts with human cells is therefore crucial for the development of new therapeutic strategies. This study reveals a previously unknown role of ubiquitination of the HPV16 L1 capsid protein, which is critical for both virus assembly and infectious entry. The Ub-acceptor sites identified by mass spectrometry are highly conserved between different papillomavirus types (HPV-16 and BPV-1), indicating a conserved function within the L1 protein and the viral capsid. We show that L1 ubiquitination plays important but diverse roles during HPV infection, potentially influencing both correct intracellular virus trafficking and HPV virion assembly. Overall, these studies demonstrate a critical link between the ubiquitin-conjugating system and HPV capsid proteins and highlight the novel role of ubiquitination during HPV infection.