流感（Influenza）是由流感病毒引起的一种严重威胁人类健康和公共卫生安全的急性呼吸道传染病。其中以A型流感病毒的危害最大，其宿主范围广，抗原变异率高，容易造成大范围流行，全球的几次流感大爆发都是由A型流感病毒引起的。流感病毒属于正粘病毒科（Orthomyxoviridae），是一种有包膜的基因组分节段的RNA病毒。其中A型流感病毒的基因组由8个单股负链RNA片段组成，每一条单股负链RNA又可分为具有调控功能的位于两端的非编码区（non-coding region, NCRs），以及位于中间的编码区（coding regions, CDS)。目前，国际主流学术界关注点主要集中在流感病毒致病的分子机制和禽流感病毒跨种属传播机制的研究，而研究对象也主要是侧重于基因编码区的功能及编码区突变对病毒的影响。然而，研究显示流感病毒基因组的非编码区在病毒进化中起着不可或缺的作用。与此同时，流感病毒基因组的非编码区还可以影响基因组的腺苷酸化、合成、转录及病毒选择性包装等多个方面，但其中的具体机制需要进一步研究。因此，我们将对病毒非编码区功能进行系统性研究，为全面理解流感病毒复制、致病及传播的分子生物学过程提供理论基础。

通过对A型流感病毒基因非编码区进行生物信息学分析我们发现：流感病毒各片段的片段特异性非编码区序列（segment specific NCR, ssNCR）具有较高保守性。其中，所有亚型PB2 vRNA 3＇端的13-14位都是高度保守的“AG”核苷酸碱基；而5＇端的14＇-16＇位是保守的“UCG”碱基。由于病毒启动子Corkscrew结构的存在，上述3＇端13-14位和5＇端的14＇-15＇位可以形成核苷酸碱基配对，从而在一定程度上使得病毒启动子更稳定。流感病毒的非编码区是病毒包装信号的一部分，科学研究发现流感病毒的某一片段的包装信号缺失，不仅能降低该片段本身的包装效率，也能降低其余片段的包装效率。而这种影响在PB2片段中表现的最明显。这提示在病毒包装过程中是存在着一种等级制度，而PB2片段在包装所有8个基因片段过程中起着最为重要的作用。所以本研究在A型流感病毒PB2片段上，探讨毗邻启动子保守碱基的功能。

首先，为了阐明这些位点的碱基在病毒复制时的作用，我们以A/WSN/1933(H1N1)作为病毒骨架，在PB2片段上引入系列突变，运用反向遗传学操作技术来拯救WSN（wild type）以及WSN（mutations）病毒株。我们通过比较各组病毒生长曲线发现，PB2基因3＇端13-15位与5＇端14＇-16＇位的碱基突变对病毒复制效率产生差异影响，并且这种影响作用会随突变位点离启动子距离增大而减小。

为了确定这些位点的碱基是否通过调节聚合酶活性来影响病毒的生长繁殖，我们在小型复制子中引入上述突变，来说明RNA聚合酶活性的与病毒的增殖能力之间的关系。结果显示，PB2基因3＇端13-15位与5＇端14＇-16＇位的碱基突变对报告基因HA蛋白及RNA表达水平有不同程度的影响，并且PB2基因3＇端13位与5＇端14＇位的碱基的聚合酶活性和病毒的增殖能力是严格的对应关系。这提示了这些位点突变很可能是通过影响聚合酶活性来影响病毒复制效率。

我们之前研究报道A型流感病毒HA的亚型特异性非编码区会影响流感病毒HA片段包装，而且病毒的包装能力也是影响病毒增殖能力的因素之一。此外，我们发现PB2基因3＇端14-15位与5＇端15＇-16＇位的碱基的聚合酶活性和病毒的增殖能力并不是对应关系，为了确定这些位点的病毒包装效率与病毒增殖能力的关系以及更深入了解上述位点的功能，我们利用病毒包装实验对这些位点对病毒包装的影响进行了检测。结果显示，PB2基因3＇端14-15位与5＇端15＇-16＇位的碱基突变对病毒的包装效率有差异影响，并且这影响和病毒复制能力有严格的一致性。这提示这些位点的病毒包装能力是决定病毒复制能力的主要因素。

综上，我们在A型流感病毒PB2基因3＇端13-15位与5＇端14＇-16＇位上的碱基构建系列突变，运用反向遗传学操作技术、小型复制子系统和病毒包装实验探究这些保守核苷酸碱基的生物学功能，进一步丰富了人们对于A型流感病毒非编码区的认识，也为我们理解A型流感病毒的基因表达调控提供了一个新视角。同时，为流感病毒疫情的防治提供理论基础。

Influenza is an acute, highly contagious respiratory disease caused by Influenza virus. In the past few decades most influenza pandemics, which were caused by influenza A viruses (IAV), are the major threat to human health and results in significant morbidity and mortality worldwide. Influenza viruses belong to the Orthomyxoviridae, a family of enveloped negative sense, single-stranded RNA viruses with segmented genomes. The influenza A virus genome is composed of eight negative sense RNA segments, each RNA segment contains coding regions (CDS) and non-coding regions (NCRs). At present, most of international scientists paid close attention to the pathogenic molecular mechanism of influenza virus and cross species transmission mechanism of avian influenza virus. Most of research are mainly focused on the coding region of viral genome. The NCR of influenza A virus, as an important component of viral genome, contains the signals required for transcription, replication and selectively package of the viral genes. But the mechanism of these sides are unclear. Therefore, there is a demand a systematic study of the role of the NCR in both transcription and translation to be undertaken.

In our previous study, we analyzed all available sequences of the non-coding regions (NCR) of influenza A viruses in the NCBI Influenza Virus Resource. We found the segment-specific NCR (ssNCR) of viral genome comprise highly conversed sequences in all eight RNA segments. The conservative nucleotide adjoining to viral promoter sequence of the PB2 vRNA are partially complementary and can form duplex structures. In addition, the PB2 vRNA is more critical for efficient infectious virion generation. Herein, we conducted a mutational study to elucidate the function of the conservative nucleotide next to viral promoter sequence of IAV PB2.

To assess the role of the PB2 conservative nucleotide adjoining to viral promoter sequence in influenza A virus, we generated viruses with groups of mutations using H1N1 WSN reverse genetics. To determine the relationship of these nucleotides with viral reproduction, we examined the virus growth efficiencies of rescued mutant viruses in MDCK cells. These results suggested that both base pairing and alternative base pairs are essential for influenza A virus replication and base pairing is more important than alternative base pairs in influenza A virus replication. Moreover, this influence of base pairing and alternative base pairs will attenuate in?uenza A virus when the location from the conserved promoter is far.

The activity of the virus RNA polymerase is closely related to the influence of virus reproduction. To clarify further the effect of all these mutants on influenza virus RNA and protein production, we used Minireplicon assay derived from the WSN virus. These results suggested that the reductions in viral replication ef?ciencies were mainly due to the effects of PB2 conservative nucleotides at 3＇end 13 and 5＇end 14＇ position adjoining to viral promoter sequence on viral RNA synthesis and viral protein translation.

Previously, we reported that the HA ssNCR of influenza A viruses can significantly modulate virus replication efficiency by affecting HA segment virion incorporation. In this study, we observed that the effects of some mutations at 3＇end 14-15 and 5＇end 15＇-16＇ position on the RNA synthesis in RNP system do not correlate with their effects on virus replication efficiencies. Therefore, the effects of the mutations on virus genome packaging efficiency were analyzed by RNA-staining and RT-qPCR. These results suggested that above conservative nucleotides at 3＇end 14-15 and 5＇end 15＇-16＇ position played a critical role in mediating incorporation of virus genome RNA into virus particle.

In summary, this study focused on the mutational analyses of IAV PB2 conservative nucleotide adjoining to viral promoter sequence, indicating the biological significance of these sites under pressure of evolution. These sites, in NCR of PB2 segment of H1N1 influenza virus, regulated RNA expression level and genome packaging efficiency differentially, contributed on viral proliferation. These findings also can provide a new insight into the regulating effect of NCR on the life cycle of influenza A virus.