第一部分全基因组分析鉴定2010~2019年中国大陆丙型肝炎病毒重组事件和正向选择位点   

摘要

背景：

丙型病毒性肝炎是由丙型肝炎病毒（Hepatitis Virus C，HCV）引起的一种病毒性肝炎，目前已成为全球性的公共卫生问题。根据2020年世界卫生组织官方数据显示，截至当年全世界约有七千一百万人（约占世界总人口的1%）患有慢性丙型肝炎，从而导致每年近四十万人死于肝硬化和肝癌。中国的丙型肝炎病毒感染率估计低于东地中海地区（2.3％）和欧洲地区（1.5％），与全球流行率（1％）相持平，但是，由于中国人口基础大，所以丙型肝炎病毒携带者的数量却是世界上最大的。尽管先前已在中国进行了一些小规模的人口调查和狭窄的地理区域关于HCV基因型的研究，但关于中国大陆流行的丙型肝炎病毒株的重组变异信息和正向选择位点知之甚少。

方法：

首先，基于NCBI数据库（http://www.ncbi.nlm.nih.gov），病毒病原体数据库（http://www.viprbrc.org）和HCV数据库（https://hcv.lanl.gov）收集了140条丙型肝炎病毒的序列，将所有的病毒株序列与参考株H77株序列对齐后，利用MEGA 10软件使用最大似然法构建了进化树进行系统发育分析。然后用RDP5软件和 SimPlot软件进行重组分析。在排除具有重组信号的病毒株序列，其余序列与参考序列进行对齐，最后使用Datamonkey服务器进行选择压力分析筛选氨基酸正向选择位点。

结果：

系统发育分析发现，基因型6型是中国大陆近十年最流行的丙肝病毒基因型之一。用RDP5和Simplot 软件进行重组分析确定了五个潜在重组事件，主要包括一个基因型间病毒重组株（HH075株）和四个基因型内病毒重组株（WYHCV286株、GB28株、GZ2983株和 HCV156株)。用Datamonkey服务器进行选择压力分析，发现大多数氨基酸位点处于负向选择，而通过SLAC、FEL和FUBAR法鉴定出的正向选择位点数目分别为5、12和7，其中SLAC法鉴定出了5个位点在另外两种分析方法的结果中也重复出现了。具体而言，五个正向选择位点分别为核心抗原基因中的两个氨基酸位点（氨基酸位点72和75），E2基因中的氨基酸位点395和NS5B基因中的两个氨基酸位点（氨基酸位点2537和2540）。

结论：

HCV在中国大陆具有遗传多样性，基因型和亚型内的重组可能是造成HCV遗传多样性的原因之一。选择压力分析结果表明大多数氨基酸位点处于负向选择状态，说明HCV变异主要是由于随机遗传漂移而积累的，而NS5B基因的氨基酸位点2537和2540处于正向选择状态，这可能对HCV复制和直接抗病毒药物（direct-acting antiviral agents, DAAs）的抗性有一定的影响。HCV的核心蛋白在免疫细胞中发挥重要抑制作用，核心抗原基因中两个氨基酸位点发生突变，可能是导致HCV慢性感染的部分原因。充分考虑变异、重组和选择压力对HCV遗传多样性和进化的影响，将有助于促进广谱抗病毒药物和疫苗的开发，也有助于建立一个适当的治疗性DAAs方案。

第二部分 SV40病毒T抗原基因产生环状RNA的机制研究

摘要

背景：

环状RNA（circular RNA, circRNA）于上世纪九十年代被鉴定出，是一类重要的非编码RNA，可作为竞争性内源RNA吸附miRNA分子（miRNA sponge），也可以与RNA结合蛋白（RNA binding protein，RBP）相结合，发挥转录或转录后调控作用。但以上只是部分环状RNA的功能，大量环状RNA的功能仍未可知，需要进一步探索。在本课题组前期工作中，我们系统分析了SV40病毒感染人成纤维细胞的circRNA表达谱，发现了1个病毒来源的circRNA，将其命名为circ-17kT，但其具体形成机制和功能仍然未知。

方法：

SV40病毒感染Vero细胞后，利用核糖核酸酶R（Ribonuclease R，RNase R）抗性分析实验证明circ17-kT是环状RNA分子。首先我们利用剪接小体能识别RNA前体的剪接位点并催化剪接反应，以及5'剪接位点（5'splice site, 5'SS）和3'剪接位点（3'splice site, 3'SS）两个经典的剪接位点是可被剪接小体识别的RNA前体中内含子和外显子连接的接头位点这两个重要特征，利用用剪接小体抑制、剪接位点突变两个实验对RNA剪接过程相关原件在该环状RNA形成过程中的作用进行研究。其次，我们通过探究外显子及其两侧翼内含子的功能来研究circ-17kT形成机制。内含子在环状RNA形成过程中起到辅助作用，有的环状RNA形成过程中需要内含子的帮助。因此在预先确定第二个外显子（Exon 2）两侧内含子无重复和反向互补序列特征后，我们设计了第二个外显子（Exon 2）两侧翼内含子被截短的内含子截短实验。外显子跳跃是形成环状RNA的一种重要方式，因此我们设计了第一个外显子（Exon 1）到第三个外显子（Exon 3）的外显子跳跃实验。最后，采用质粒过表达和反义寡核苷酸（antisense oligonucleotide, ASO）干扰抑制circ-17kT表达的策略研究circ-17kT对其母本基因大T抗原（large T antigen，LT）基因的作用关系及其与病毒增殖能力的关系。

结果：

经RNase R消化证明circ-17kT是SV40病毒感染Vero细胞产生的环状RNA；当剪接小体被异银杏双黄酮抑制时，SV40病毒感染Vero细胞产生的circ-17kT表达量无明显变化；将5'剪接位点（5'splice site, 5'SS）和3'剪接位点（3'splice site, 3'SS）剪接位点进行突变后构建质粒转染细胞，转染后Vero细胞仍然能正常表达circ-17kT；内含子截短实验发现当形成circ-17kT的T基因Exon 2两侧翼内含子发生截短时，circ-17kT形成没有影响；第一个外显子（Exon 1）到第三个外显子（Exon 3）的外显子跳跃实验表明circ-17kT可能不是由包含Exon 2的套索前体加工产生。过表达circ-17kT能正反馈促进SV40 大T抗原基因mRNA的表达，但与对照组相比，SV40病毒滴度没有明显变化；而抑制circ-17kT表达时，LT表达显著下降且SV40病毒滴度显著降低。

结论：

circ-17kT形成过程中不需要由由剪接小体提供剪接功能，且circ-17kT形成过程中不需要形成套索前体，也不依赖于两侧翼内含子的帮助。初步实验表明circ-17kT不是由外显子跳跃驱动形成的。当剪接位点发生突变和Exon 2两侧翼内含子被截短不影响circ-17kT形成,提示circ-17kT形成存在其他由RBP或反式因子驱动的环化机制。初步功能分析显示，circ-17kT能正反馈促进其母本基因LT的表达，初步结果提示circ-17kT抑制表达时可抑制SV40病毒增殖。

Part I Complete genome analysis identifies recombinant events and positive  selection sites of hepatitis C virus from mainland China during 2010–2019

Abstract

Background: Viral hepatitis C is a type of viral hepatitis caused by the hepatitis C virus, it has become a global public health problem. According to statistics from the World Health Organization, more than 71 million people (around 1% of the world’s population) worldwide suffering from chronic hepatitis C infection as of 2020, and resulting in approximately 400,000 deaths each year mainly due to cirrhosis and hepatocellular carcinoma. Lower than the Eastern Mediterranean Region (2.3 %) and European Region (1.5 %), the estimated infection rate of hepatitis C virus in China is nearly equal to global prevalence (1%). However, regarding the population base, the number of hepatitis C virus carriers in China is the largest in the world. Although some previous studies on HCV genotypes, conducted on small populations and narrow geographical areas, have been performed in China, little is known about the recombinant variant information and positive selection sites of circulating HCV strains in mainland China. After all sequences are aligned with the reference sequence, a phylogenetic tree is constructed by MEGA10 software using the maximum likelihood method for phylogenetic analysis.

Methods: Based on NCBI nucleotide sequence database

(http://www.ncbi.nlm.nih.gov), Virus Pathogen Database (http://www.viprbrc.org) and HCV database (https://hcv.lanl.gov), 140 sequences of HCV were included in this study. After aligning all the virus strain sequences with the reference strain H77 strain sequence, phylogenetic analysis was carried out by MEGA10 software using the maximum likelihood method, and recombination analysis was carried out by RDP5 software and SimPlot software. After the sequences with recombination signals are excluded, and the rest of the sequences are compared with the reference sequences, at last, the Datamonkey network server is used to analyze the selection pressure.

Results: By constructing the maximum likelihood tree, it was found that genotype 6 was the most popular hepatitis C genotype in Chinese mainland in recent ten years. Five groups of potential recombination events were identified by RDP5 and Simplot software,

including one inter-genotype recombinant virus strain (strain HH075) and four intra-genotype recombinants virus strain (strains WYHCV286, GB28, GZ2983, and HCV156) . Using Datamonkey network server to analyze the selection pressure, it was found that most of the codon sites were negative selection, while the number of positive selection sites identified by SLAC, FEL and FUBAR methods were 5, 12 and 7, respectively. Five positive selection sites identified by SLAC method also appeared in the results of the other two analysis methods. Specifically, the two sites (codon 72 and 75) in the core gene, the codon site 395 in the E2 gene, and two codon sites (2537 and 2540) in the NS5B gene were under positive selection are being selected.

Conclusion: HCV has genetic diversity in Chinese mainland, and the recombination within genotypes and subtypes may be one of the reasons for HCV genetic diversity.

The results of selection pressure analysis showed that most codon sites were in negative selection state, indicating that HCV variation was mainly accumulated due to random genetic drift, while codon 2537 and 2540 of NS5B gene were under positive selection, which may have some influences on HCV replication and the resistance of DAAs. Core protein of HCV exerts inhibitory functions in many vitally important immune cells, two positive selection sites identified in this study within the core gene may be partially responsible for the HCV chronic infection. Fully considering the impact of mutations, recombination, and selective pressure in shaping the genetic diversity and evolution of HCV will facilitate the development of broad-spectrum antiviral drugs and effective vaccines, and also helps to establish an appropriate therapeutic DAAs program.

Part II  Study on the mechanism of SV40 virus T antigen gene producing circular RNA

Abstract

Background: Circular RNA（circRNA） was identified in the 1990s as an important type of non-coding RNA. It can be used as a competitive endogenous RNA to adsorb miRNA molecules (miRNA sponge), or it can be combined with RNA binding protein (RBP), both of which can play a role in transcription The role of post-regulation; there is also a part of circRNA that plays a role of transcriptional regulation. However, the above are the functions of some special circular RNAs, and the functions of a large number of circular RNAs are still unknown and require further exploration by researchers. In the previous study, we systematically analyzed the circRNA expression profile of SV40, and discovered a virus-derived circRNA—circ-17kT for the first time, but its formation mechanism and function are still unknown.

Methods: After SV40 virus infects Vero cells, the RNase R resistance analysis experiment proved that circ17-kT is a circular RNA molecule. First, we use splice bodies to recognize the splice site of the RNA precursor and catalyze the splicing reaction, as well as the 5'splice site (5'SS) and 3'splice site (3'SS). The classic splice site is the two important features of the joint site where the intron and exon in the RNA precursor can be recognized by the splicing body. The two experiments of splicing body suppression and splicing site mutation are used. The role of the elements related to the RNA splicing process in the formation of the circular RNA is studied. Secondly, we investigate the formation mechanism of circ-17kT by exploring the functions of exons and their flanking introns. Introns play an auxiliary role in the formation of circular RNAs, and some circular RNAs need the help of introns during the formation of circular RNAs. Therefore, after pre-determining the non-repetitive and reverse complementary sequence characteristics of the introns on both sides of the second exon (Exon 2), we designed the second exon (Exon 2) to be truncated. The intron truncation experiment. Exon skipping is an important way to form circular RNA, so we designed an exon skipping experiment from the first exon (Exon 1) to the third exon (Exon 3). Finally, the strategy of plasmid overexpression and antisense oligonucleotide (ASO) interference with circ-17kT expression was used to study the effect of circ-17kT on the large T antigen (LT) gene of its maternal gene. Its relationship with the ability of the virus to multiply.

Results: RNase R digestion proved that circ-17kT is a circular RNA produced by SV40 virus infecting Vero cells; when the splicing body is inhibited by Isoginkgetin, the expression of circ-17kT produced by SV40 virus infecting Vero cells has no significant change; The 5'splice site ( 5'SS) and 3'splice site (3'SS) are mutated to construct plasmid transfected cells, and Vero cells can still be normal after transfection Expression of circ-17kT. Intron truncation experiments found that when the flanking introns of Exon 2 of the T gene forming circ-17kT were truncated, the formation of circ-17kT had no effect, the first exon (Exon 1) to the third The exon skipping experiment of two exons (Exon 3) indicated that circ-17kT may not be produced by lasso precursor processing containing Exon 2. This part of the results requires further experimental verification. Overexpression of circ-17kT can positively promote the expression of SV40 large T antigen gene mRNA, but compared with the control group, the SV40 virus titer does not change significantly; while inhibiting the expression of circ-17kT, the LT expression significantly decreases and the SV40 virus titer Significantly reduced.

Conclusion: During the formation of circ-17kT, splicing bodies do not need to provide the splicing function, and the formation of circ-17kT does not require the formation of lasso precursors, nor does it rely on the help of flanking introns. Preliminary experiments show that circ-17kT is not formed by exon skipping. When the splicing site is mutated and the flanking introns of Exon 2 are truncated, it does not affect the formation of circ-17kT, suggesting that there are other cyclization mechanisms driven by RBP or trans factors in the formation of circ-17kT. Preliminary functional analysis showed that circ-17kT can positively promote the expression of its maternal gene LT. The preliminary results suggest that circ-17kT can inhibit the proliferation of SV40 virus when the expression is inhibited.