van Dinten L C, den Boon J A, Wassenaar A L, Spaan W J, Snijder E J
Department of Virology, Institute of Medical Microbiology, Leiden University, The Netherlands.
Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):991-6. doi: 10.1073/pnas.94.3.991.
Equine arteritis virus (EAV) is a positive-strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of six subgenomic mRNAs from which its structural genes are expressed. A stable bacterial plasmid (pEAV030) containing a full-length cDNA copy of the 12.7-kb EAV genome was constructed. After removal of a single point mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BHK-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus. The potential of pEAV030 as a tool to express foreign genes was demonstrated by the efficient expression of the chloramphenicol acetyltransferase (CAT) reporter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to result in a particularly intriguing phenotype: RNA carrying this mutation can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mutant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least partially, different and that these processes may be separated experimentally.
马动脉炎病毒（EAV）是一种正链RNA病毒，它利用不连续转录机制产生一组嵌套的六个亚基因组mRNA，其结构基因由此表达。构建了一个稳定的细菌质粒（pEAV030），其中包含12.7 kb EAV基因组的全长cDNA拷贝。在去除复制酶基因中的一个单点突变后，从pEAV030体外产生的RNA转录本在电穿孔导入BHK - 21细胞后显示具有感染性。在cDNA水平引入遗传标记突变，并从子代病毒基因组中恢复。氯霉素乙酰转移酶（CAT）报告基因从两个不同的亚基因组mRNA高效表达，证明了pEAV030作为表达外源基因工具的潜力。最初使全长克隆无感染性的单点突变被发现导致了一种特别有趣的表型：携带这种突变的RNA可以高效复制，但不产生结构蛋白表达所需的亚基因组mRNA。据我们所知，这种突变体首次证明动脉炎病毒基因组复制和不连续mRNA合成的要求至少部分不同，并且这些过程可以通过实验分离。
