SARS-CoV-2 Orf6 requires the Nup98/Rae1 complex to inhibit host nuclear transport
Creators: Love, Nicole D
Publisher: name: University of Alberta Library
Year: 2023

<p>The nucleus is a hallmark organelle of eukaryotes. It separates the genetic material from the rest of the cytoplasm using a selectively permeable double phospholipid bilayer called the nuclear envelope (NE). The NE contains numerous pores with embedded macromolecular structures called nuclear pore complexes (NPCs), which are formed from ~30 different proteins called nucleoporins (Nups). In conjunction with soluble nuclear transport factors, cellular proteins and RNAs are able to move through the NPC. Although humans and the budding yeast Saccharomyces cerevisiae have been separated by approximately a billion years of evolution, both organisms contain a striking conservation in overall NPC structure and function, with many Nups, transport factors, and transport pathways functionally conserved. Of interest, the human Nup98/Rae1 complex is related to the S. cerevisiae Nup116/Gle2 complex, and both have been shown to have important functions in transport across the NPC.<br>
Many viruses, including the SARS-CoV-2 virus, have been shown to target host NPCs in order to create an environment conducive for viral replication. Numerous viral proteins, one of which is the SARS-CoV-2 Orf6 protein, achieve this by binding to the Nup98/Rae1 complex to inhibit the bidirectional nuclear transport of host proteins and RNAs. The viral proteins that interact with the Nup98/Rae1 complex contain a common motif, termed a Nup98/Rae1 interaction motif.<br>
Interestingly, residues within the human Nup98/Rae1 complex that form major interactions with the Nup98/Rae1 interaction motif of Orf6 are conserved in the S. cerevisiae Nup116/Gle2 complex. On the basis of this similarity, we hypothesized that the SARS-CoV-2 Orf6 protein interacts with the Nup116/Gle2 complex and would inhibit the bidirectional nuclear transport of conserved nuclear import and export pathways in yeast. We show here that Orf6 can be expressed and localizes to the NE of S. cerevisiae, potentially to a subset of NPCs. However, we observed no detectable inhibition of two protein import pathways or bulk mRNA export defects associated with Orf6 expression. Thus, the consequences of Orf6 association at the yeast NE remain to be determined.<br>
Although Orf6 has been shown to target the mammalian Nup98/Rae1 complex, how Orf6 inhibits the nuclear transport of host proteins and RNAs remains unclear. To further investigate the functional relationship between Orf6 and the Nup98/Rae1 complex, the Orf6-mediated block of nuclear transport was examined in Vero cells depleted of either Nup98 or Rae1. We show that depletion of Rae1 abolishes the Orf6-mediated block of both STAT1 nuclear import and poly-A RNA nuclear export. Similarly, depletion of Nup98 also suppresses Orf6-mediated inhibition of STAT1 nuclear import. Collectively, these studies suggest Orf6 requires the Nup98/Rae1 complex to function, and further points to an important role for Rae1 in facilitating the block of nuclear transport caused by Orf6 expression. Since the Nup98/Rae1 complex is commonly targeted by viral proteins to cause a dysregulated immune response, understanding how Orf6 inhibits nuclear transport by targeting Nup98 or Rae1 could provide important insights into therapeutic treatments for viral infection, and may also further elucidate the roles of Nup98 and Rae1 in nuclear transport. </p>