Activity, Template Preference, and Compatibility of Components of RNA Replicase of Eastern Equine Encephalitis Virus.
Activity, Template Preference, and Compatibility of Components of RNA Replicase of Eastern Equine Encephalitis Virus.
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DOI：
10.1128/jvi.01368-22
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发表时间：
2023-01-31
影响因子：
5.4
通讯作者：
中科院分区：
文献类型：
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东部马脑炎病毒（EEEV）通常在黑尾库利塞特蚊子和鸟类之间循环;然而，它也可以感染人类。EEEV有一个正义RNA基因组，在感染的细胞中，作为P1234多蛋白的mRNA。P1234经历一系列精确的切割事件，产生四种非结构蛋白（nsP 1 -4），代表RNA复制酶的亚基。在这里，我们报告了EEEV的反式复制酶的构建和性质。EEEV的模板RNA被证明可以通过不同甲病毒的复制酶复制。另一方面，EEEV复制酶在复制源自塞姆利基森林病毒复合体的甲病毒的模板RNA方面表现出有限的能力。利用P123和nsP 4两个组分成功地构建了EEEV的复制酶。EEEV P123与异源nsP 4形成功能性RNA复制酶的能力使用EEEV模板RNA比使用异源甲病毒模板RNA更有效。这一发现表明，与先前研究的塞姆利基森林复合体甲病毒不同，P123和/或其加工产物在EEEV模板RNA识别中起主导作用。用EEEV或西方马脑炎病毒感染携带EEEV模板RNA的HEK 293 T细胞显著激活了模板RNA中编码的报告基因的表达;对于其他甲病毒感染的影响要小得多，并且在黄病毒感染后检测不到。同时，EEEV感染仅导致基孔肯雅病毒模板RNA的有限激活。因此，含有携带病毒的模板RNA的细胞可以用作不同甲病毒的敏感和选择性生物传感器。重要性人类EEEV感染可导致严重的神经系统疾病，致死率约为30%。虽然人类感染很罕见，但2019年记录了破纪录的数字。EEEV的复制对宿主因素有独特的要求，但研究很少，部分原因是病毒需要生物安全3级设施，这可能限制实验范围;同时，这些研究对于开发抗病毒方法至关重要。本研究成功的EEEV复制酶为EEEV的研究提供了一种安全、通用的研究工具。使用该系统，分析EEEV复制酶组分与来自其他甲病毒的对应物的相容性。所获得的数据可用于开发独特的生物传感器，其提供用于检测、鉴定、定量和中和与高通量、半自动化方法兼容的活甲病毒的替代方法。
Eastern equine encephalitis virus (EEEV) usually cycles between Culiseta melanura mosquitoes and birds; however, it can also infect humans. EEEV has a positive-sense RNA genome that, in infected cells, serves as an mRNA for the P1234 polyprotein. P1234 undergoes a series of precise cleavage events producing four nonstructural proteins (nsP1–4) representing subunits of the RNA replicase. Here, we report the construction and properties of a trans-replicase for EEEV. The template RNA of EEEV was shown to be replicated by replicases of diverse alphaviruses. The EEEV replicase, on the other hand, demonstrated limited ability in replicating template RNAs originating from alphaviruses of the Semliki Forest virus complex. The replicase of EEEV was also successfully reconstructed from P123 and nsP4 components. The ability of EEEV P123 to form functional RNA replicases with heterologous nsP4s was more efficient using EEEV template RNA than heterologous alphavirus template RNA. This finding indicates that unlike with previously studied Semliki Forest complex alphaviruses, P123 and/or its processing products have a leading role in EEEV template RNA recognition. Infection of HEK293T cells harboring the EEEV template RNA with EEEV or Western equine encephalitis virus prominently activated expression of a reporter encoded in the template RNA; the effect was much smaller for infection with other alphaviruses and not detectable upon flavivirus infection. At the same time, EEEV infection resulted only in a limited activation of the template RNA of chikungunya virus. Thus, cells harboring reporter-carrying template RNAs can be used as sensitive and selective biosensors for different alphaviruses. IMPORTANCE Infection of EEEV in humans can cause serious neurologic disease with an approximately 30% fatality rate. Although human infections are rare, a record-breaking number was documented in 2019. The replication of EEEV has a unique requirement for host factors but is poorly studied, partly because the virus requires biosafety level 3 facilities which can limit the scope of experiments; at the same time, these studies are crucial for developing antiviral approaches. The EEEV trans-replicase developed here contributes significantly to research on EEEV, providing a safe and versatile tool for studying the virus RNA replication. Using this system, the compatibility of EEEV replicase components with counterparts from other alphaviruses was analyzed. The obtained data can be used to develop unique biosensors that provide alternative methods for detection, identification, quantitation, and neutralization of viable alphaviruses that are compatible with high throughput, semiautomated approaches.
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DOI：
10.3390/v14061327
发表时间：
2022-06-17
期刊：
Viruses
影响因子：
--
作者：
通讯作者：
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DOI：
10.1073/pnas.1900656116
发表时间：
2019-05-07
影响因子：
11.1
作者：
Law, Yee-Song;Utt, Age;Luo, Dahai
通讯作者：
Luo, Dahai
影响因子：
5.4
作者：
Hellstrom, Kirsi;Kallio, Katri;Ahola, Tero
通讯作者：
Ahola, Tero
影响因子：
5.4
作者：
Kallio, Katri;Hellstrom, Kirsi;Ahola, Tero
通讯作者：
Ahola, Tero
影响因子：
4.7
作者：
Phelps, Amanda L.;O'Brien, Lyn M.;Ulaeto, David O.
通讯作者：
Ulaeto, David O.