Date of Award
Doctor of Philosophy (PhD)
Human papillomaviruses (HPV) are important pathogens that cause 5% of all human cancers worldwide, including essentially all cases of cervical cancer. Prophylactic vaccines against HPV exist, however these are not widely utilized in all communities, cannot cure existing infection, and do not protect against all subtypes. Therefore, study of HPV infection may lead to important new therapeutics to reduce the spread of the virus and disease burden. In addition, thorough investigation of HPV infection is likely to provide new insights into many aspects of cell biology and biochemistry, as the study of viruses has in the past. HPV requires multiple cellular proteins for proper viral trafficking during virus entry, including both γ-secretase and retromer. γ-secretase is a complex of four cellular transmembrane (TM) proteins that typically binds to and cleaves TM proteins within their TM domain. During HPV infection, γ-secretase binds to the viral capsid protein L2 and facilitates its insertion into the endosomal membrane. As a result of L2 insertion, L2 protrudes into the cytoplasm, a vital step in infection that allows HPV to bind other cellular factors such as retromer. Retromer is a cytoplasmic complex of three proteins that binds to and sorts cellular cargo and HPV into the retrograde trafficking pathway. In this thesis, we further characterize the interaction between γ-secretase and HPV L2. We show that γ-secretase is required for membrane protrusion of L2 and that L2 associates strongly with the PS1 catalytic subunit of γ-secretase. HPV infection also stabilizes the γ-secretase complex. Mutational studies of a putative TM domain in HPV16 L2 revealed that it cannot be replaced with a foreign TM domain, that infectivity of HPV TM mutants is tightly correlated with γ-secretase binding and stabilization, and that the L2 TM domain is required for protrusion of the L2 protein into the cytoplasm. Additionally, we show that retromer and γ-secretase interact in infected and uninfected cells, and that both proteins are required for L2 protrusion into the cytoplasm. Retromer binding to L2 is required for the interaction between L2 and γ-secretase and for γ-secretase stabilization. Constitutively active Rab7, which causes retromer to remain associated with L2 on the endosomal membrane, affects complex formation between γ-secretase and L2. Finally, γ-secretase activity is required for the trafficking of a cellular retromer cargo, DMT1-II. These results provide new insight into the interaction between γ-secretase and L2 and highlight the importance of the native L2 TM domain for proper HPV trafficking during virus entry. They also show that γ-secretase and retromer cooperate to mediate L2 membrane insertion, and suggest that binding to a cytoplasmic protein other than retromer can stabilize L2 in the endosomal membrane. Furthermore, interactions between γ-secretase and retromer may be important for the normal sorting activity of retromer. Overall, my work in the DiMaio lab highlights the importance of γ-secretase, the HPV L2 transmembrane domain, and the interaction between γ-secretase and retromer in HPV infection. Our results highlight that detailed mechanistic analysis of individual steps in viral entry lead to discoveries that can identify novel functions of proteins that are important for cellular biology.
Crite, Mac, "The Role of γ-Secretase in Human Papillomavirus Infection" (2022). Yale Graduate School of Arts and Sciences Dissertations. 579.