Wertz Gail W, Moudy Robin, Ball L Andrew
Department of Microbiology, University of Alabama School of Medicine, Birmingham, Alabama 35294, USA.
J Virol. 2002 Aug;76(15):7642-50. doi: 10.1128/jvi.76.15.7642-7650.2002.
Gene expression of the nonsegmented negative strand (NNS) RNA viruses is controlled primarily at the level of transcription by the position of the genes relative to the single transcriptional promoter. We tested this principle by generating engineered variants of vesicular stomatitis virus in which an additional, identical, transcriptional unit was added to the genome at each of the viral gene junctions. Analysis of transcripts confirmed that the level of transcription was determined by the position of the gene relative to the promoter. However, the position at which a gene was inserted affected the replication potential of the viruses. Adding a gene between the first two genes, N and P, reduced replication by over an order of magnitude, whereas addition of a gene at the other gene junctions had no effect on replication levels. All genes downstream of the inserted gene had decreased levels of expression, since transcription of the extra gene introduced an additional transcriptional attenuation event. The added gene was stably maintained in the genome upon repeated passage in all cases. However, expression of the added gene was stable at only three of the four positions. In the case of insertion between the N and P genes, a virus population arose within two passages that had restored replication to wild-type levels. In this population, expression of the additional gene as a monocistronic mRNA was suppressed by mutations at the end of the upstream (N) gene that abolished transcriptional termination. Because transcription is obligatorily sequential, this prevented transcription of the inserted downstream gene as a monocistronic mRNA and resulted instead in polymerase reading through the gene junction to produce a bicistronic mRNA. This eliminated the additional attenuation step and restored expression of all downstream genes and viral replication to wild-type levels. These data show that transcriptional termination is a key element in control of gene expression of the negative strand RNA viruses and a means by which expression of individual genes may be regulated within the framework of a single transcriptional promoter. Further, these results are directly relevant to the use of NNS viruses as vectors and vaccine delivery agents, as they show that the level of expression of an added gene can be controlled by its insertion position but that not all positions of insertion yield stable expression of the added gene.
非节段性负链（NNS）RNA病毒的基因表达主要在转录水平上受基因相对于单一转录启动子的位置控制。我们通过构建水疱性口炎病毒的工程变体来验证这一原理，即在每个病毒基因连接处向基因组中添加一个额外的、相同的转录单元。转录本分析证实，转录水平由基因相对于启动子的位置决定。然而，基因插入的位置影响病毒的复制潜力。在第一个基因N和P之间添加一个基因，复制能力降低了一个数量级以上，而在其他基因连接处添加基因对复制水平没有影响。插入基因下游的所有基因表达水平均下降，因为额外基因的转录引入了额外的转录衰减事件。在所有情况下，经多次传代后，添加的基因在基因组中稳定维持。然而，添加基因的表达仅在四个位置中的三个位置稳定。在N和P基因之间插入的情况下，在两代内出现了一个病毒群体，其复制能力恢复到野生型水平。在这个群体中，上游（N）基因末端的突变抑制了额外基因作为单顺反子mRNA的表达，这些突变消除了转录终止。由于转录是强制性连续的，这阻止了插入的下游基因作为单顺反子mRNA的转录，而是导致聚合酶通读基因连接处产生双顺反子mRNA。这消除了额外的衰减步骤，并使所有下游基因的表达和病毒复制恢复到野生型水平。这些数据表明，转录终止是负链RNA病毒基因表达调控的关键因素，也是在单一转录启动子框架内调节单个基因表达的一种方式。此外，这些结果与将NNS病毒用作载体和疫苗递送剂直接相关，因为它们表明添加基因的表达水平可通过其插入位置控制，但并非所有插入位置都能使添加基因稳定表达。
