The role of the Nup358-CRM1-axis in nuclear import of adenoviral genomes

• Applicant: Professor Dr. Ralph Kehlenbach
• Subject Area: Virology ; Cell Biology
• Term: since 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 286487595

Adenoviruses (Ad) are important respiratory viruses but also play a major role as vaccination vectors, e.g. during the recent COVID-19 pandemic. They are large, non-enveloped DNA viruses with a capsid diameter of 90 nm that replicate in the nucleus of infected cells. During interphase, the nuclear envelope (NE) is a major barrier on the way into the nucleus. Due to their size, import of intact capsids is not supported. Instead, they disassemble at the nuclear pore complex (NPC), which is then used for import of the released genome into the nucleus. Since nuclear import of DNA is not a physiological process, Ads overcome this problem by exploiting the cellular nucleo-cytoplasmic transport machinery. Here we aim to investigate the mechanisms underlying the nuclear delivery of the Ad genome by focusing on the nucleoporin Nup358, which localizes to the cytoplasmic side of the NPC, and the export factor CRM1. Having worked on the nuclear protein export functions of CRM1 for almost 25 years, we recently identified CRM1 as a factor that promotes Ad capsid disassembly and trafficking from the microtubule organizing centre to the NPC, where Nup358 coordinates genome import. Importantly, the protein export function of CRM1 is not required for the observed effects, since LMB, a CRM1 inhibitor, affects capsid disassembly also in mitotic cells, i.e. in the absence of a functional NE-barrier. Instead, a unique domain in a flexible hinge region of CRM1 that we identified in a small mutagenesis screen is involved in viral genome release. Point mutations in that region affect genome release, but not nuclear protein export. Furthermore, we found soluble Nup358 fragment with and without known binding sites for CRM1 that block genome import and show that Nup358 and CRM1 form an export-independent complex in situ. Over the last couple of years, our labs have developed several assay systems to analyze the molecular details of AdV-genome release and nuclear import. In addition, we will apply novel tools, e.g. cell lines that allow degron- and auxin-dependent degradation of nucleoporins. The goal of this proposal is to investigate the role of Nup358 and CRM1 in viral genome release and in nuclear import and to address a possible interplay of the two transport factors - in interphase cells as well as in mitosis. Furthermore, we will address the question of which viral and/or cellular protein(s) can be linked to the Nup358/CRM1-effects. We will address these questions using biochemical, cell biological and virological approaches, taking advantage of the combined expertise in the two laboratories. Since in the context of viral genome import, CRM1 clearly has functions that are distinct from its canonical role as a nuclear protein export factor, we expect to identify novel cellular functions of CRM1 as well.

• DFG Programme: Research Grants
• International Connection: France
• Cooperation Partner: Professor Dr. Harald Wodrich