Virus promoters determine interference by defective RNAs:: Selective amplification of mini-RNA vectors and rescue from cDNA by a 3′ copy-back ambisense rabies virus
Virus promoters determine interference by defective RNAs:: Selective amplification of mini-RNA vectors and rescue from cDNA by a 3′ copy-back ambisense rabies virus
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DOI：
10.1128/jvi.73.5.3818-3825.1999
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发表时间：
1999-05-01
影响因子：
5.4
通讯作者：
Conzelmann, KK
中科院分区：
文献类型：
--
作者：
Finke, S;Conzelmann, KK
典型的缺陷干扰（DI） rna在病毒聚合酶的竞争中比亲本（辅助）病毒更成功，这主要是由于DI启动子组成的改变。狂犬病毒（RV）内部缺失rna具有真正的RV末端启动子，因此具有转录活性，可以作为外源基因表达的载体，在RV感染的细胞中繁殖不良，不干扰RV的复制。为了允许从这种mini-RNA载体中进行di样扩增和高水平的基因表达，我们使用了一种工程化的3'复制回（双义）辅助RV，其中抗基因组的强复制启动子被弱50倍的基因组启动子取代。在双感辅助病毒和编码氯霉素乙酰转移酶（CAT）和荧光素酶的mini-RNAs共同感染的细胞中，mini-RNAs被高水平扩增。这与干扰辅助病毒复制有关，最终导致mini- rna明显优于辅助病毒。然而，在不添加外源辅助病毒的情况下，可以实现mini- rna的高效连续传代和高水平的报告基因活性，显示出相当中等程度的干扰，但不排除大量的HV繁殖。与表达CAT的重组RV载体感染相比，丰富的mini-RNA模板的可用性导致CAT mRNA水平增加，从而使CAT活性增加高达250倍，而病毒基因转录保持在最低水平。我们还利用了内部缺失模型RNA的行为与DI RNA相似的发现，并在ambisense辅助病毒存在下选择性扩增，首次证明了在表达T7 RNA聚合酶的ambisense rv感染细胞中转染mini-RNA cDNA后，rv支持cDNA的拯救。
Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.