Year of Publication

2011

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Agriculture

Department

Plant Pathology

First Advisor

Dr. Peter Nagy

Abstract

Positive-strand RNA virus group are the most abundant among viruses affecting plants and animals. To successfully achieve replication, these viruses usurp or co-opt host proteins. To facilitate the discovery of host factors involved in Tomato bushy stunt virus (TBSV), yeast has been developed as a surrogate model host. Genome-wide approaches covering 95% of yeast genes, has revealed approximately hundred factors that could affect virus replication. Among the identified host factors, there are fourteen yeast genes, which affect/regulate lipid metabolism of the host.

One of the identified host gene is ERG25, which is an important factor for sterol biosynthesis pathway, affecting viral replication. Sterols present in eukaryotes affect the lipid composition of membranes, where tombusviruses, similar to other plus-strand viruses of tobacco, replicate. Since potent inhibitors of sterol synthesis are known, I have tested their effects on tombusvirus replication. We demonstrated that these sterolsynthesis inhibitors reduced virus replication in tobacco protoplasts. Virus replication is resumed to the wild type level by providing phytosterols in tobacco protoplasts confirming the role of sterols in RNA virus replication in tobacco.

We have also identified INO2, a transcription factor for many phospholipid biosynthetic genes, reduces virus replication in its deletion background. When we provided this gene product in the mutant background, viral replication was back to normal, confirming the role of Ino2p in tombusvirus replication. Further biochemical assays showed that the viral inhibition is because of alteration in the formation of the viral replicase complex. Using confocal microscopy, we showed that the viral replication protein, termed p33, is forming large and few punctate structures rather than the small and many by overexpressing Ino2p in the wild type yeast cells. Over-expression of Opi1, an inhibitor of Ino2p led to greatly reduced viral replication, further supporting the roles of the phospholipid pathway in tombusvirus replication.

One of the phospholipid, which is regulated by this pathway, is cardiolipin an important component of the mitochondrial as well as peroxisomal membranes. We further characterized how cardiolipin is playing an important role for tombusvirus replication by using different biochemical approaches.

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