Author ORCID Identifier

https://orcid.org/0000-0002-9021-4920

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Plant Pathology

First Advisor

Dr. Peter D. Nagy

Abstract

Positive-stranded (+) RNA viruses are the largest family of viruses that infect plants, causing important economic losses in different crops. Tomato bushy stunt virus (TBSV), a small positive-stranded RNA virus, has emerged as a model virus to study virus-host interactions. TBSV encodes for only five proteins, therefore, to infect the host cell TBSV co-opts selected host components and subverts specific molecular pathways.

Firstly, I performed a proteomic screening using Arabidopsis proteins. I found that TBSV viral replication proteins interact with 88 host proteins, including the ubiquitin-conjugating enzyme 2 (Ubc2), fructose 1,6 biphosphate aldolase (Fba1), and several members of the Hps70 family. Ubc2 and its yeast ortholog Rad6 act as pro-viral factors promoting TBSV replication in plants and yeasts. Ubc2 and Rad6 ubiquitinate TBSV replication proteins to subvert ESCRT (endosomal sorting complexes required for transport) proteins for the viral replication complex (VRC) assembly.

Similar to the yeast cytosolic Hsp70 (SSA1 and SSA2) I found that tombusviruses co-opt the constitutively expressed plant Hsp70-2 and the plant-specific stress-inducible Erd2 (early responsive to dehydration 2) to assemble the VRC and activate the TBSV RdRp, named p92pol. More, tombusviruses increase the accumulation levels of Hsp70 and Erd2 proteins in the cell during infection. These discoveries demonstrate that TBSV is able to co-opt more than one member of the Hsp70 family to promote viral replication.

In addition, I found that TBSV viral replication proteins interact with Fructose 1,6 biphosphate aldolase (FBA1), a key enzyme in plants involved in glycolysis and glucogenesis. I discovered that FBA1 is a pro-viral factor necessary to locally produce ATP within the viral replication compartments and support tombusvirus replication.

I used Legionella pneumophila effectors to disrupt actin dynamics and I found that the expression of the RavK effector, which cleaves the actin filaments, reduces tombusvirus replication in yeast and plants whereas the VipA effector, which polymerizes and stabilizes the actin filaments, enhances tombusvirus replication. Using RavK and VipA effectors as tools to study virus-hosts interactions, I found that actin dynamics is important for the efficient recruitment of glycolytic enzymes into the VRC to productively generate ATP at the replication sites. More, tombusviruses use Rpn11 deubiquitinase pro-viral host factor to recruit the glycolytic and fermentation enzymes to the site of replication via the actin network, thus the virus co-opts entire pathways facilitating viral replication and infection.

Finally, I discovered that actin dynamics also affect the recruitment of cell-intrinsic restriction factors (CIRFs), like cyclophilins, co-chaperons, and helicases, to the replication sites. The stabilized actin network enhances the recruitment of pro-viral factors and reduces the recruitment of CIRFs into the VRC. Manipulation of the host actin network by tombusviruses during the early stage of infection is key for the tug-of-war between the virus and the host.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.031

Funding Information

National Science Foundation

- (Molecular and Cellular Biosciences) MCB-1517751 (09/01/2015 - 08/31/2019)

- (Integrative Organismal Systems) IOS-1922895 (08/01/2019 - 07/31/2022)

Available for download on Sunday, October 03, 2021

Share

COinS