Institute for Human Infections and Immunity, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Glenn Biggs Institute for Alzheimer's & Neurodegenerative Diseases, University of Texas Health San Antonio, San Antonio, TX 78229, USA.
Viruses. 2020 Jun 19;12(6):660. doi: 10.3390/v12060660.
Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.
RNA 病毒的报告基因由于可能发生的 RNA 重组事件而不稳定，这些重组事件会切除插入的核酸。在甲病毒中，RNA 重组已被证明与复制保真度共同调节，但尚不清楚少数变异体水平的重组事件如何作用，这对于疫苗和转基因传递设计很重要。因此，我们试图描述在多个复制周期中，低保真度甲病毒突变体对报告基因的去除。为此，将 GFP 插入到 TC-83 中，TC-83 是一种用于甲型虫委内瑞拉马脑炎病毒的活减毒疫苗，以及 TC-83 的低保真度变体，并传代至荧光不再观察到为止。使用 ClickSeq 进行短读 RNA 测序，以确定病毒基因组的哪些区域发生了重组，以及随着多个复制周期的变化情况。观察到 GFP 基因的快速去除，病毒群体中的少数变体积累了小的缺失，这些缺失在传代过程中逐渐增大。最终，这些小的缺失融合完全去除了 GFP 基因。在低保真度 TC-83 的传代过程中，这种去除显著增强，表明增加的重组水平是该突变体的一个特征。
