东部马脑炎病毒(EEEV)通常在库利塞塔蚊子和鸟类之间循环;但是,它也可以感染人类。EEEV有一个正向RNA基因组,在受感染的细胞中,作为P1234多蛋白的mRNA。P1234经历一系列精确的切割事件,产生代表RNA复制酶亚基的四种非结构蛋白(nsP1-4)。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显示可通过多种甲病毒的复制酶复制。EEEV复制酶,另一方面,证明了复制源自SemlikiForest病毒复合体的甲病毒的模板RNA的能力有限。EEEV的复制酶也成功地从P123和nsP4组件重建。EEEVP123与异源nsP4形成功能性RNA复制酶的能力使用EEEV模板RNA比异源甲病毒模板RNA更有效。这一发现表明,与先前研究的SemlikiForest复杂甲病毒不同,P123和/或其加工产物在EEEV模板RNA识别中具有主导作用。用EEEV或西方马脑炎病毒感染带有EEEV模板RNA的HEK293T细胞,显着激活了模板RNA中编码的报道分子的表达;对于其他甲病毒感染,效果要小得多,而在黄病毒感染时无法检测到。同时,EEEV感染仅导致基孔肯雅病毒模板RNA的有限激活。因此,携带报道分子的模板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EEEV在人类中的感染可引起严重的神经系统疾病,死亡率约为30%。虽然人类感染很少见,2019年记录了一个破纪录的数字。EEEV的复制对宿主因素有独特的要求,但研究甚少,部分原因是该病毒需要生物安全3级设施,这可能会限制实验范围;同时,这些研究对于开发抗病毒方法至关重要。这里开发的EEEV反式复制酶为EEEV的研究做出了重要贡献,为研究病毒RNA复制提供安全和通用的工具。使用这个系统,对EEEV复制酶组分与其他甲病毒的对应物的相容性进行了分析.获得的数据可用于开发独特的生物传感器,为检测提供替代方法,identification,定量,和中和与高通量相容的活的甲病毒,半自动方法。
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.