戊型肝炎病毒（HEV）是导致急性肝炎的重要病原体，但目前对于HEV基因组上调控病毒复制的顺式作用元件和参与病毒复制的宿主因子尚不清楚，研究HEV的复制机制对治疗HEV感染意义重大。新型冠状病毒（SARS-CoV-2）的研究需要在生物安全等级三级（BSL-3）实验室进行，但BSL-3实验室数量有限，阻碍了科研人员对SARS-CoV-2的研究和抗病毒药物的开发。因此，建立一个安全高效的可以在BSL-2实验室操作的SARS-CoV-2感染系统具有重要意义。在本研究中，利用HEV复制酶反式互补系统和HEV分泌型荧光素酶复制子，对HEV基因组上调控病毒复制的顺式作用元件进行系统筛选。结果显示，基因组编码区存在对病毒复制必需的功能性顺式作用元件，单碱基同义突变能够使HEV彻底丧失复制和感染能力。顺式作用元件在8种HEV基因型中保守，并且是不同基因型HEV复制所必需的结构。机制研究表明，位于ORF2的顺式作用元件可以跟3’端非翻译区碱基互补配对形成茎环结构，促进HEV的复制。顺式作用元件对于病毒复制酶识别病毒基因组至关重要。通过HEV复制酶互作组和复制复合体组成研究，我们鉴定到PRMT5和WDR77是HEV感染的限制性宿主因子。为了构建SARS-CoV-2反式互补系统，本研究中将SARS-CoV-2基因组上对病毒组装必需的核衣壳蛋白（N）替换成绿色荧光蛋白（GFP），通过在细胞里异位表达病毒N蛋白的方式建立细胞系依赖的病毒感染体系。该系统产生的具有转录和复制能力的SARS-CoV-2病毒样颗粒（trVLP）只能在表达N的细胞中持续感染，保证了系统的安全性。trVLP连续传代过程中，未检测到N基因重组到病毒基因组中。利用该系统，我们鉴定了对N蛋白发挥功能至关重要的磷酸化位点；通过同义突变验证了病毒基因组上RNA二级结构对病毒复制的重要意义。同时，本研究系统评估了中和抗体、干扰素、瑞德西韦等多种药物的抗病毒效果，建立了可以在BSL-2实验室操作的抗病毒药物高通量筛选系统。最后，我们从377个小分子化合物中鉴定出5个能够有效抑制SARS-CoV-2感染的药物。综上所述，本文系统鉴定了调控HEV复制的顺式作用元件和限制性宿主因子，阐明了HEV复制酶识别病毒基因组进行高效复制的机制，为开发以RNA为基础的治疗方案和高效的HEV感染系统提供了依据。同时，建立了SARS-CoV-2 N蛋白反式互补系统，为研究病毒与宿主相互作用和开发抗病毒药物提供了工具。
Hepatitis E virus （HEV） is an important pathogen causing acute hepatitis, but the cis-acting elements on viral genome and host factors regulating viral replication are still unclear. Studying the mechanism of HEV replication is significant to treat HEV infection. Research on severe acute respiratory syndrome coronavirus 2 （SARS-CoV-2） needs to be conducted in biosafety level three （BSL-3） laboratory. However, the limited number of BSL-3 laboratories prevents researchers from studying SARS-CoV-2 biology and developing antivirals. Therefore, it is important to establish a safe and efficient SARS-CoV-2 infection system that can be operated in the BSL-2 laboratory.In this thesis, we ultilized HEV ORF1 trans-complementation system and HEV secreted luciferase replicon to systematically screen the cis-acting elements on viral genome regulating HEV replication. We identified two functional cis-acting elements within coding region required for viral replication, and single-nucleotide synonymous mutation completely abolishes HEV replication and infection. Besides, the identified cis-acting elements are conserved among eight HEV genotypes and necessary for the replication of differenct HEV genotypes. Mechanistic study shows that cis-acting element located in ORF2 can form a stem-loop structure with the 3‘ untranslated region, which promotes viral replication. Cis-acting elements are essential for ORF1 replicase to recognize viral genome. From HEV ORF1 interactome and replication complex, we identified PRMT5 and WDR77 as restriction factors for HEV infection.In order to develop SARS-CoV-2 trans-complementation system, the nucleocapsid protein （N） necessary for virus assembly on the SARS-CoV-2 genome is replaced with green fluorescent protein （GFP）. We established a cell line-dependent infection system by ectopically expressing N protein in cells. The transcription and replication-competent SARS-CoV-2 virus-like-particles （trVLP） produced can only continuously infect N-expressing cells, ensuring the safety of the system. During the continuous passage of trVLP, no recombination of the N gene into the viral genome was detected. Using this system, we have identified phosphorylation sites that are essential for the function of N protein; synonymous mutations verify the importance of RNA secondary structure on SARS-CoV-2 genome for viral replication. At the same time, we systematically evaluated the antiviral effects of neutralizing antibodies, interferon, remdesivir and other drugs, and established a high-throughput screening system for antivirals that can be operated in the BSL-2 laboratory. Finally, 5 drugs that can effectively inhibit SARS-CoV-2 infection are identified from 377 small molecule compounds.In conclusion, we have systematically identified cis-acting elements and restriction factors that regulate HEV replication, and explained the mechanism by which HEV replicase recognizes the viral genome for efficient replication. This is important for developing RNA-based therapeutics and efficient HEV inection model. At the same time, we have established SARS-CoV-2 N trans-complementation system, which provides a powerful tool for studying virus-host interactions and developing antivirals.
