Author ORCID Identifier
https://orcid.org/0000-0001-5094-6492
Date Available
7-20-2026
Year of Publication
2025
Document Type
Doctoral Dissertation
Degree Name
Doctor of Philosophy (PhD)
College
Agriculture, Food and Environment
Department/School/Program
Plant Pathology
Faculty
Peter Nagy
Abstract
Positive-strand RNA viruses, such as coronaviruses and flaviviruses, are obligate parasites whose genomic RNAs function as mRNAs. These viruses infect a wide range of hosts-including plants, animals, and humans-resulting in significant economic losses and posing serious threats to public health. Due to the relatively simple structure of the genome of positive-strand RNA viruses, many host factors and physiological processes are co-opted to support virus infection and replication. In our previous studies, we identified numerous host factors and associated physiological processes involved in the replication of tombusviruses, a cytosolic-replicating model that includes tomato bushy stunt virus (TBSV) and carnation Italian ringspot virus (CIRV). Among these, several nuclear proteins were identified, including exportin 1 (XPO1), a nuclear shuttle protein. However, it remains unclear how these cytosolic tombusviruses gain access to and co-opt nuclear proteins.
XPO1 is a nuclear transport receptor that mediates the export of nuclear proteins by binding leucine-rich nuclear export signal of the cargos. Previous work in the lab shows that XPO1 affects tombusviruses replication in yeast. In this dissertation, I elucidated the antiviral function of XPO1 in planta by assessing tombusviruses replication levels under conditions of transient XPO1 overexpression and VIGS (Virus Induced Gene Silencing)-mediated knockdown in Nicotiana benthamiana. XPO1 interacts with the CIRV replication protein p36 and the TBSV replication protein p33, being recruited to cytosolic virus replication organelles (VROs) where it colocalizes with these proteins during tombusvirus replication. Argonaute 2 (AGO2), Centromere-specific Histone H3 (CenH3), and Double-stranded RNA Binding protein 4 (DRB4) were identified as cargos of XPO1 contributing to the inhibitory effect during tombusviruses replication. These cargos were also recruited into VROs, and colocalized with replication proteins together with XPO1. The relocation of XPO1 as well as its cargos were actin dependent. It was identified that XPO1, cargos, and the regulatory proteins were localized in virus induced condensates (vir-condensates) reflecting the complex role of vir-condensates as the battle ground during virus replication.
In addition, by utilizing an optimized neutral reporter system, I found that the nuclear export process was significantly attenuated but nuclear import was not during tombusviruses replication. Further studies employing the FLIP (Fluorescence Loss In Photobleaching) method revealed that viral condensates induced by viral replication proteins sequester XPO1, thereby inhibiting its nucleocytoplasmic shuttling. Leveraging AlphaFold2 Multimer (AFM), the interaction model of p33-XPO1 predicted that residues 13E and 15F of p33 interact with residue K537 of XPO1, thereby facilitating M7/ I8/ W9 of p33 to block XPO1’s cargo‐loading groove by binding to the α‐helix residues that comprise this groove. Substitution mutations in XPO1a K537A, K537D and K537R showed enhanced inhibitory effect to TBSV replication. Notably, these amino acid substitution mutants did not affect the cargo export efficiency of the nuclear export receptors, even during TBSV replication or p33 expression in planta. This result presents a novel strategy for redesigning an antiviral factor to enhance its antiviral performance.
In this research I also identify a protein, Upstream framshit 1 (Upf1), as the nuclear cargo of XPO1. Upf1 is usually reported to be localized in cytosol. In Chapter 4, treatment with Leptomycin B-an inhibitor of XPO1-mediated cargo loading, knockdown of NbXPO1, and fusion of Upf1 with a nuclear retention signal (NRS) resulted in the nuclear accumulation of Upf1 in the cells of N. benthamiana, as observed via confocal laser microscopy. Surprisingly, Upf1 was rapidly exported to the cytosol by XPO1, indicating its predominantly cytosolic localization. Upf1 exhibited significant antiviral activity against tombusvirus replication. Similarly, as the cargo of XPO1, Upf1 was also recruited into the vir-condensates and colocalized with viral replication proteins and XPO1 in VROs.
Overall, this dissertation elucidates XPO1-mediated antiviral mechanisms by identifying several nuclear antiviral factors that serve as its cargos. It demonstrates a novel mechanism by which cytosolic RNA viruses access nuclear resources by hijacking XPO1 and recruiting it into viral replication organelles (VROs) along actin filament networks. Furthermore, it reveals how tombusviruses attenuate nuclear export to counteract XPO1-mediated antiviral functions and leads to a novel strategy to redesign and enhance the antiviral effect of XPO1.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2025.277
Recommended Citation
Sun, Biao Dr., "EXPORTIN 1 IS THE CENTRAL HUB IN ANTIVIRAL DEFENSIVE MOBILIZATION OF NUCLEAR FACTORS AND THE TARGET OF COUNTER-DEFENCE BY TOMBUSVIRUSES" (2025). Theses and Dissertations--Plant Pathology. 47.
https://uknowledge.uky.edu/plantpath_etds/47
