Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

DiMaio, Daniel

Abstract

Approximately 5% of human cancer worldwide is caused by Human Papillomavirus (HPV). Although there are highly effective prophylactic vaccines, HPV remains a serious public health challenge due to low vaccine uptake and the inability of the vaccine to treat anyone already infected. Understanding how HPV enters and infects cells is important, as this work may lead to the development of new antivirals or therapeutics and may also reveal new aspects of cell and protein biology. HPVs are non-enveloped viruses, containing an ~8000 base pair double-stranded DNA genome encapsidated by 360 copies of L1 major capsid protein and up to 72 copies of L2 minor capsid protein. Previous work showed that a C-terminal cell-penetrating peptide allows most of the L2 protein to protrude through the endosomal membrane into the cytoplasm. This unique activity enables L2 to interact with cellular factors required for trafficking of the virus to the nucleus by exposing binding sites throughout the central and C-terminal region of L2 to the cytoplasmic compartment. Despite the unique function and importance of L2 during infection, the structure of L2 remains unknown. We predicted the structure of the L2 protein using various structural prediction software, including AlphaFold2. Modeling predicts that most of L2 is unstructured, but that it contains a central three-stranded antiparallel β-sheet conserved in highly divergent HPV types. We designed and tested HPV16 pseudoviruses containing mutations in the predicted β-sheet region. Mutations predicted to disrupt the β-sheet led to a dramatic reduction in infectivity, but nearby mutations predicted to preserve the β-sheet structure were tolerated. Mutational analysis of a predicted pair of interacting residues far apart on the linear sequence is consistent with these residues participating in a strong salt bridge interaction. We then replaced the entire β-sheet, or each individual β-strand, with an artificial computer-generated amino acid sequence predicted to recapitulate the β-sheet. These artificial β-sheet mutations were not tolerated, indicating that the amino acid sequence of the β-sheet is important. We also show that several HPV mutants with these defective artificial sequences are able to reach the Golgi apparatus during infection and accumulate in this compartment. In vitro binding experiments similarly suggest that binding of L2 to COPI, a cellular trafficking complex, through a binding site in the L2 central region is a dynamic process that is tightly regulated in the Golgi. Finally, we provide modeling and mutational evidence that the HPV16 β-sheet is pH sensitive, and propose that unfolding at low pH allows protrusion of this segment of L2 into the cytoplasm. Collectively, these results suggest the presence of a previously unknown structured region in L2 that is conserved across a diverse range of papillomaviruses and required for infection. This central structure in L2 appears to be important for trafficking in the Golgi and the results shown here are consistent with sequential protrusion of L2 from the C-terminus to N-terminus that gradually exposes different portions of L2 that act at different times during infection. Evidence that the central structure is remarkably stable at neutral pH but unfolds at low pH supports a model where the folded central structure prevents protrusion of this segment of L2 until the proper time during infection, where it then unfolds at low pH and enters the cytoplasm, acting to regulate Golgi trafficking and potentially downstream steps.

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