Research Accomplishment Reports 2007

Defining RNA and Protein Factors Affecting Tombusvirus Replication

P.D. Nagy
Department of Plant Pathology

 

Project Description

Significance: Viruses cause major losses in agriculture. Replication of viruses in infected hosts is central to virus pathogenesis. Tombusviruses are exceptionally suitable for replication studies because of recent major advances made in the PI’s and others’ labs. These include the development of tractable in vitro RNA replication assays by the PI based on purified tombusvirus replicase obtained from yeast and plants. In addition, the PI has developed a powerful in vivo RNA replication system in yeast, an excellent model host, in addition to the standard whole plant and single cell (protoplast) hosts. Therefore, it is now possible to identify the roles of viral and host protein factors and the viral RNA in replication. Interactions of viral proteins and the viral RNA with host factors are likely critical determinants of host range, susceptibility and resistance of plants against particular viruses. The knowledge obtained from this research may be useful for designing novel antiviral strategies. Given that tombusviruses are important and emerging pathogens in vegetable-growing areas and that they are also similar to many other important plants, animal and human virus pathogens, advances in this area of research are anticipated to have broad and significant consequences for control of these pathogens.

Progress made: the PI's lab has shown that the tombusviral p33 replication protein has RNA chaperone activity, which facilitates initiation of RNA synthesis by the viral replicase. In vitro experiments demonstrated that the purified recombinant p33 facilitated RNA synthesis on plus-stranded and double-stranded RNA templates up to 5-fold by the viral replicase. Altogether, the RNA chaperone activity of the auxiliary replication protein of a tombusvirus is essential for replication.

The PI's lab has also demonstrated that cdc34p ubiquitin conjugating protein is present within the tombusvirus replicase complex. This was achieved by testing the yeast proteome microarray carrying 4088 purified host proteins. Fifty eight host proteins were identified that interacted with the p33 replication protein. Down-regulation of cdc34p expression in yeast, which supports robust replication of a tomato bushy stunt virus (TBSV) RNA, reduced repRNA accumulation by 3-fold. Also, cdc34p was able to ubiquitinate p33 in vitro in the absence of e3 ligase. Ubiquitination of p33 did not affect its stability, but likely affects TBSV replication.

The PI's lab has shown that a subverted host metabolic enzyme participated in the retention of the viral RNA template in the replicase complex. Viruses transform host cells into viral factories by subverting host proteins. A key, co-opted host protein in the tbsv replicase is glyceraldehyde-3-phosphate dehydrogenase (GAPDH). During TBSV replication, gapdh is demonstrated to re-localize from the cytosol to the peroxisomal membrane surface, the site of viral RNA synthesis. Down-regulation of GAPDH inhibited TBSV replication, resulting in an ~1:1 ratio of (+)/(-)RNA, instead of the hallmark asymmetric synthesis leading to excess (+)RNA. Also, down-regulation of the plant cytosolic GAPDH decreased TBSV accumulation in a natural host.

Impact

Identification of host factors involved in viral RNA in replication is a major frontier in virology. Interactions of viral proteins and the viral RNA with host factors are likely critical determinants of host range, susceptibility and resistance of plants against particular viruses. The knowledge obtained from this research may be useful for designing novel antiviral strategies. Given that tombusviruses are important and emerging pathogens in vegetable-growing areas and that they are also similar to many other important plants, animal and human virus pathogens, advances in this area of research are anticipated to have broad and significant consequences for control of these pathogens.

The PI's group has screened over 95% of host genes affecting tombusvirus replication and recombination, based on yeast with 6,000 genes. Currently, this is the highest coverage for any host - pathogen interactions. This will allow the PI and others to develop new antiviral strategies.

Accomplishments:

The PI's group has

• performed a genome-wide screen that identified 100 host genes affecting replication of an RNA virus
• shown that the tombusviral p33 replication protein has RNA chaperone activity.
• demonstrated that Cdc34p ubiquitin-conjugating protein is present within the tombusvirus replicase complex.
• shown that a subverted host metabolic enzyme participated in the retention of the viral RNA template in the replicase complex.
• uncovered that an alteRNAtive subcellular location exists for replication by showing that Tombusvirus replication switches to the endoplasmic reticulum in the absence of peroxisomes.
• demonstrated that the host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins.
• shown that Expression of the Arabidopsis Xrn4p 5'-3' exoribonuclease facilitates degradation of tombusvirus RNA and promotes rapid emergence of viral variants in plants
• performed a proteomics analysis of the Tombusvirus replicase, which led to the discovery that Hsp70 molecular chaperone is associated with the replicase and enhances viral RNA replication
• dentified essential host factors affecting tombusvirus RNA replication based on the yeast yTHC collection.

Publications

Jonczyk, M., Pathak, K. B., Sharma, M., and Nagy, P. D. 2007. Exploiting alternative subcellular location for replication: Tombusvirus replication switches to the endoplasmic reticulum in the absence of peroxisomes. Virology 362(2), 320-30.

Jaag, H. M., Stork, J., and Nagy, P. D. 2007. Host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins. Virology 368, 388-404.

Cheng, C. P., Jaag, H. M., Jonczyk, M., Serviene, E., and Nagy, P. D. 2007. Expression of the Arabidopsis Xrn4p 5'-3' exoribonuclease facilitates degradation of tombusvirus RNA and promotes rapid emergence of viral variants in plants. Virology 368, 338-248.