Jaag Hannah M, Stork Jozsef, Nagy Peter D
Department of Plant Pathology, University of Kentucky, Plant Science Building, Lexington, KY 40546, USA.
Virology. 2007 Nov 25;368(2):388-404. doi: 10.1016/j.virol.2007.07.003. Epub 2007 Aug 8.
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 p92(pol) 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 p92(pol) 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 p92(pol) 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 p92(pol) 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 p92(pol) 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.
先前的全基因组筛选鉴定出100多个宿主基因，其缺失/下调会影响番茄丛矮病毒的复制，还鉴定出32个宿主基因，它们在酵母（番茄丛矮病毒复制的模式宿主）中影响番茄丛矮病毒的RNA重组。几个已鉴定宿主基因的下调影响了p33和p92（pol）复制蛋白的积累水平，这增加了这些宿主因子可能参与病毒复制蛋白数量调控的可能性，因此，它们间接参与了番茄丛矮病毒的复制和重组。为了验证这一模型，我们开发了一种用于在酵母中严格调控重组p33和p92（pol）复制蛋白表达的系统。我们证明，p33的高积累水平促进了有效的病毒RNA复制，而p33水平对RNA重组的影响则不太明显。另一方面，高水平的p92（pol）积累比RNA复制更有效地促进了番茄丛矮病毒的RNA重组。正如预期的那样，作为聚合酶II复合体一部分的Rpb11p影响了p33和p92（pol）的积累水平，同时也改变了RNA复制和重组。用番茄丛矮病毒复制酶进行的体外试验进一步支持，Rpb11p仅通过调节p33和p92（pol）水平间接影响番茄丛矮病毒的复制和重组。相比之下，Rpt4p内肽酶/ATP酶和Mps1p苏氨酸/酪氨酸激酶影响番茄丛矮病毒重组的机制与Rpb11p的不同。我们提出了一个模型，即病毒复制酶内复制蛋白的浓度（分子拥挤）是影响病毒复制和重组的一个因素。
