Author ORCID Identifier

https://orcid.org/0009-0005-9631-2432

Date Available

10-16-2024

Year of Publication

2024

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Agriculture, Food and Environment

Department/School/Program

Plant Pathology

Advisor

Dr. Peter D. Nagy

Abstract

Tombusviruses are positive-strand RNA viruses, which utilize numerous co-opted host factors and massive de-novo synthesized membrane resources to modify host membranes and form large viral replication organelles (VROs). Among plenty of host cellular processes, autophagy emerges as a potential membrane resource for VROs formation due to its ability to de-novo generate the double membrane structures, known as autophagosomes. Besides, as an important host defense pathway, autophagy is also a target for tombusviruses to prevent its antiviral activities. In this study, I identify the role of key autophagy related genes, Atg11, Atg8, and Atg2, which are involved in autophagosome formation, and the selective autophagy receptor NBR1, in tombusviruses replication. Based on genomics studies, I found that deletion of Autophagy-related 11 (ATG11/FIP200), a component of the Atg1/13 complex involved in selective autophagy, inhibited TBSV and the closely related carnation Italian ringspot virus (CIRV) replication in yeast and similarly in plants. I demonstrated that Atg11 directly interacts with the TBSV p33 replication protein in vivo and in vitro, facilitating its recruitment into VROs. Interestingly, I observed that the subverted Atg11 affects the recruitment of additional membrane contact site (MCS) proteins, such as Sac1, Scs2 VAP, and Osh6 OSBP1-like proteins, to support the formation of the virus-induced MCS (vMCS), which is important to sterol transfer to VROs. These results indicate a novel function of Atg11 in TBSV replication. Additionally, I show that the highly conserved Atg8 autophagy regulator proteins are co-opted by both TBSV and CIRV via direct interactions with their viral replication proteins. Knockdown of Atg8f in Nicotiana benthamiana plants resulted in reduced tombusvirus replication, thus indicating a pro-viral function for Atg8f. Using RavZ, a Legionella effector that inhibits host autophagy through irreversible Atg8 deconjugation, I demonstrated that Atg8-PE is essential for TBSV replication. Monitoring autophagy flux activity via the ratio of GFP/GFP-Atg8 and Atg8-PE/Atg8 revealed that TBSV replication inhibits host autophagy progress. One possible mechanism is non-PE conjugated Atg8 forming bio-condensates with NBR1 selective autophagy receptor, resulting in their sequestration in condensates near and in VROs. Furthermore, NBR1 also appears to facilitate the accumulation of phospholipids in VROs, indicating a complex and finely regulated mechanism of exploitation of host autophagy by tombusviruses. Also, Atg2 was identified as a pro-viral host factor, with its function and protein structure closely related to its role as a lipid transfer protein (LTP), where its N-terminal hydrophobic channel can nonspecifically bind and transfer various phospholipids crucial for VRO formation. Subsequent experiments confirmed that silencing Atg2 in plants or its deletion in yeast significantly reduces the enrichment of key phospholipids in VROs as well as inhibits tombusviruses replication. Overall, through the study of three specific autophagy-related genes, I have identified three independent lipid transport pathways potentially exploited by the tombusviruses. These pathways provide the virus with lipid sources, enabling the rapid establishment of VROs and facilitating viral replication. Ultimately, these advancements open up a new chapter in tombusvirus-host interactions.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.60

Funding Information

  • National Science Foundation MCB1517751 in 2015;
  • National Science Foundation IOS-1922895 in 2019;
  • National Institute of Food and Agriculture NIFA, 2020-70410-32901 in 2020;
  • National Institute of Food and Agriculture hatch grant (KY012042) in 2016.

Download

Share

COinS