New form of HPV18 L1 found in native virions contributes to virion stability and infectivity

Current dogma states that papillomavirus virions consist of only one form of the L1 major capsid protein. Human papillomaviruses (HPVs) have been studied using recombinant particles, thereby bypassing the need to grow the virus in a model of differentiating epithelium. Expression vectors engineered to produce L1 utilize a consensus methionine found in many HPV types. However, HPV18 has two additional in-frame methionine residues located 61 and 26 amino acids upstream of the consensus methionine. These methionine residues are excluded from the L1 expression vector used to create the recombinant virus. On the other hand, HPV produced from organotypic raft culture allows virions to be assembled in a differentiating epithelium in the presence of its native promoters and complete genome. Here, we have utilized this system to show that the wild-type HPV18, produced in a more natural setting, translates a larger form of L1 from the upstream methionine, which is 61 amino acid residues upstream from the consensus methionine, and a smaller form from the consensus methionine, with both sizes assembled in the newly formed virion. Ablation of the upstream methionine residue at position 61 altered the virion capsid conformation, and also decreased virion stability and infectivity. IMPORTANCE: The present study investigates whether the papillomavirus virions contain more than one isoform of the L1 major capsid protein. By using organotypic raft cultures that mimic the naturally differentiating epithelium, we revealed that wild-type HPV18 expresses and incorporates two distinct L1 isoforms into the virion, including a previously unrecognized longer form translated from an upstream methionine. Our study has further shown that disruption of the upstream methionine impairs virion stability and infectivity, demonstrating that the longer L1 isoform contributes to proper capsid architecture and viral function. These findings offer a new understanding of HPV capsid biology and have significant implications for the future development of HPV vaccines, diagnostics, and antiviral therapies.