Student Theses and Dissertations

Date of Award

2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Rout Laboratory

Abstract

Human-infecting viruses have evolved diverse strategies to enter cells and hijack the host machinery to promote their self-replication. Viruses deploy their proteins to subvert a number of host functions, such as the cell cycle, cellular metabolism, protein synthesis, nuclear and RNA transport across the nuclear pore complex, apoptosis, and innate immune responses. Picornaviruses are the most dominant human disease-causing viruses and present an excellent clinical target for research studies into their molecular mechanisms. Picornaviruses have an RNA genome that is translated as a single polyprotein, which is processed into individual components by two proteases, termed 2A and 3C. In addition to polyprotein processing, these proteases also subvert host cell function through cleavage of specific protein targets. The 2A protease is especially critical during the initial stages of picornaviral infections. We developed protein expression platforms to further characterize the 2A protease’s function, interacting partners, and targeting mechanisms, showing that it makes surgical strikes against eIF4G and Nup98, key players in host protein synthesis and nucleocytoplasmic trafficking of proteins and RNA, respectively. We subsequently utilized those protease expression platforms in combination with transport reporter assays to interrogate nuclear import and export through the NPC. By studying the interactome of 2A protease, we discovered that it seems to employ two different cleavage mechanisms for its primary targets, Nup98 and eIF4G. It directly binds and degrades Nup98, and may alternatively bind eIF3L and utilize the eIF3 complex as a targeting platform to cleave eIF4G. Cellular fractionation revealed Nup98 cleavage by 2A protease results in observable dissociation of Rae1 from the NPC as well as cytoplasmic accumulation of proteins normally transported by Karyopherins that interact with Nup98 FG motifs. We developed fluorescent transport reporters with various nuclear import and export signals to elucidate their transport mechanism and dependence on Nup98. Nuclear localization signals promiscuously recognized by a variety of Importins were marginally, if at all, affected by Nup98 depletion, while export and import signals depending on Crm1 or Rae1 mediated RNA export were severely affected by the absence of full-length Nup98. We propose a novel cleavage mechanism for the 2A protease, as well as report on our development of a suite of molecular tools to further characterize 2A protease function and nucleocytoplasmic transport mediated by Nup98. These tools can be adapted to include a diverse variety of viral proteins to further characterize host subversion mechanisms as well as other aspects of nuclear pore complex function.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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