Previous genome-wide screens identified over 100 host genes whose deletion/down-regulation affected tombusvirus replication and 32 host genes that affected tombusvirus RNA recombination in yeast, a model host for replication of Tomato bushy stunt virus (TBSV). Down-regulation of several of the identified host genes affected the accumulation levels of p33 and p92pol replication proteins, raising the possibility that these host factors could be involved in the regulation of the amount of viral replication proteins and, thus, they are indirectly involved in TBSV replication and recombination. To test this model, we developed a tightly regulated expression system for recombinant p33 and p92pol replication proteins in yeast. We demonstrate that high accumulation level of p33 facilitated efficient viral RNA replication, while the effect of p33 level on RNA recombination was less pronounced. On the other hand, high level of p92pol accumulation promoted TBSV RNA recombination more efficiently than RNA replication. As predicted, Rpb11p, which is part of the polII complex, affected the accumulation levels of p33 and p92pol as well as altered RNA replication and recombination. An in vitro assay with the tombusvirus replicase further supported that Rpb11p affects TBSV replication and recombination only indirectly, via regulating p33 and p92pol levels. In contrast, the mechanism by which Rpt4p endopeptidase/ATPase and Mps1p threonine/tyrosine kinase affect TBSV recombination is different from that proposed for Rpb11p. We propose a model that the concentration (molecular crowding) of replication proteins within the viral replicase is a factor affecting viral replication and recombination.
|Number of pages||17|
|State||Published - Nov 25 2007|
Bibliographical noteFunding Information:
We thank Dr. Piia Leskinen (University of Turku, Finland) for providing pSAL1 and Dr. Elena Serviene for sharing pYC/DI-AU and pGBK-His33/CUP1. The authors also thank Drs. Judit Pogany and Daniel Barajas for their comments on the manuscript. This work was supported by NSF (IOB 0517218) and NIH-NIAID.
Copyright 2009 Elsevier B.V., All rights reserved.
- Cucumber necrosis virus
- Host factor
- RNA recombination
- RNA replication
- Tomato bushy stunt virus
ASJC Scopus subject areas