High-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing.
High-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing.
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DOI：
10.1371/journal.ppat.1001146
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发表时间：
2010-10-14
期刊：
影响因子：
6.7
通讯作者：
Schmaljohn CS
中科院分区：
文献类型：
--
作者：
Beitzel BF;Bakken RR;Smith JM;Schmaljohn CS
我们开发了一种高分辨率基因组作图技术，将转座子介导的插入突变与毛细管电泳或大规模并行测序相结合，以识别委内瑞拉马脑炎病毒（VEEV）基因组的功能重要区域。我们最初使用毛细管电泳方法来深入了解 VEEV 非结构蛋白 3 (nsP3) 在病毒复制中的作用。我们在 nsP3 中发现了几个不能容忍小（15 bp）插入的区域，因此可能具有重要的功能。我们还确定了 nsP3 中的 9 个独立区域，这些区域可以在低温（30°C）下耐受小插入，但不能在较高温度（37°C 和 40°C）下耐受。由于我们发现这种方法在识别温度敏感（ts）突变方面非常有效，但受到毛细管电泳能力的限制，因此我们用大规模并行测序取代了毛细管电泳，并使用改进的方法生成了整个 VEEV 基因组的功能图谱。我们在整个基因组中发现了数百个潜在的 ts 突变，并使用反向遗传学生成的病毒进行单周期生长曲线实验，验证了 nsP2、nsP3、E3、E2、E1 和衣壳中的几个突变。我们进一步证明，两种 nsP3 ts 突变体在小鼠中的毒力减弱，但可以引发针对野生型 VEEV 攻击的保护性免疫。重组ts突变体将成为进一步研究VEEV复制和毒力的有价值的工具。此外，我们开发的方法适用于为任何具有强大反向遗传学系统的病毒生成此类工具。委内瑞拉马脑炎病毒 (VEEV) 是一种新世界甲病毒，于 1938 年在委内瑞拉首次发现。VEEV 通常在啮齿动物群体中传播，但在疫情爆发期间，它可以传播到马和人类身上，导致衰弱甚至可能致命的疾病。目前尚无针对 VEEV 的疫苗或抗病毒药物获准用于人类。在这项研究中，我们描述了一种我们开发的技术，可以快速识别病毒突变体，这对于研究病毒复制的基础生物学很有用。这些突变体还可用于生产预防野生型病毒感染的疫苗。我们通过识别遍布整个 VEEV 基因组的 200 多个突变来证明该技术的实用性，这些突变使病毒无法在较高温度（37°C 或 40°C）下有效复制。此外，我们表明其中两种突变病毒可作为疫苗，并保护小鼠免受 VEEV 的致命感染。该技术可应用于研究其他病毒，并可快速识别多种候选疫苗。
We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system. Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that was first identified in Venezuela in 1938. VEEV normally circulates in rodent populations, but during outbreaks it can jump to horses and humans where it can cause debilitating and potentially fatal disease. There are currently no vaccines or antiviral agents against VEEV licensed for use in humans. In this study, we describe a technique that we have developed that allows for the rapid identification of viral mutants that can be useful for studying the basic biology of viral replication. These mutants can also be used to generate vaccines that protect against infection with wild-type virus. We demonstrate the utility of this technique by identifying over 200 mutations spread throughout VEEV genome that make the virus unable to replicate efficiently at higher temperatures (37°C or 40°C.) Furthermore, we show that two of the mutant viruses work as vaccines, and protect mice against lethal infection with VEEV. This technique can be applied to studying other viruses, and may allow for the rapid identification of numerous vaccine candidates.
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影响因子：
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作者：
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通讯作者：
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影响因子：
3.5
作者：
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通讯作者：
Kääriäinen, L
DOI：
10.1073/pnas.82.14.4648
发表时间：
1985-01-01
影响因子：
11.1
作者：
HAHN, CS;STRAUSS, EG;STRAUSS, JH
通讯作者：
STRAUSS, JH