慢病毒核壳蛋白的热稳定性及在反转录中与RNA结合的研究

Nucleocapsid (NC) refers to both the NC domain of the retroviral Gag proteins and the NC proteins in the mature viral particles. The NC domain of the Gag protein is responsible for the recognition of packaging signal and the encapsidation of the viral genomic RNA into the virion. Mutations of several amino acids in the NC domain or in the packaging signal sequences lead to the loss of viral infectivity. The NC proteins in the mature virion, which are cleaved from the Gag proteins, can bind to the genomic RNA, coating and protecting the viral genome by forming the stable and compact nucleoprotein complexes. Upon entry into the cell, NC also plays a critical role in assuring the specificity and efficiency of reverse transcription. On one hand, NC is significantly important, since it plays an important role in essentially every step of the viral replication cycle, however, on the other hand, detailed mechanisms of how NC functions are still missing. We have found that NC is unusually thermostable, and the heat-treated NC protein remained its ability to bind with RNA and to inhibit the in vitro reverse transcription. Based on preliminary studies, we propose to compare the NC protein with the NC domain-containing fusion protein in their secondary conformations and their affinities and sequence selectivities to bind with RNA; to detect the temperature variation during CA polymerization and characterize the significance of temperature variation on the activities of the NC protein and the reverse transcriptase; to study whether the NC proteins modulate the activity of the reverse transcriptase, and whether dynamic dissociations of the NC proteins from the template RNA occur during the reverse transcription process. Detailed understanding of NC’s properties and functions in the viral replication cycle will facilitate the elucidation of the mechanism of viral replication, and facilitate the development of effective and rational therapeutic strategies against retroviruses.

核壳（NC）既指反转录病毒Gag蛋白的NC结构域，又指成熟毒粒中的NC蛋白。Gag蛋白的NC结构域负责识别包装信号，引导基因组RNA包装入病毒颗粒，它和包装信号中多个位置的突变都会使病毒丧失感染力；切割后的成熟NC蛋白则与病毒基因组RNA形成稳定紧凑的核蛋白复合物，起贴身保护作用，入侵细胞后在调控反转录的效率和特异性方面也发挥作用，但其中很多细节并不清楚。我们的前期研究证实，NC蛋白具有热稳定性，热处理后仍保持结合RNA和抑制体外反转录的能力。本研究拟比较慢病毒Gag蛋白的NC结构域和成熟NC蛋白在分子构象、结合RNA的特异性和亲和力方面的差异；分析毒粒成熟过程是否伴随温度变化及变温对NC和反转录酶活性的影响；研究NC蛋白及其热稳定性对反转录酶活性的影响，以及反转录酶通过时NC与RNA的结合如何改变。深入研究NC的热稳定性及功能，有助于阐明病毒的复制机制，为研发新的抗病毒治疗策略奠定基础。

慢病毒的核壳蛋白（nucleocapsid, NC）是Gag蛋白的NC结构域在病毒成熟时被蛋白酶切割出来的。Gag蛋白的NC结构域负责在病毒装配时特异性识别并结合病毒包装信号（packaging signal, Psi），将2拷贝的基因组RNA包装进病毒颗粒。而NC蛋白被认为是核酸分子伴侣，在成熟病毒颗粒中非特异性结合并保护病毒基因组。在本项研究中，我们证实了HIV-1、SIV、EIAV的NC蛋白具有热稳定性，100°C孵育60 min后仍有超40%保持可溶，且可溶NC保持了其RNA结合活性、Psi结合活性、以及高序列覆盖度下的反转录抑制活性。HIV-1的反转录酶在42°C短暂处理后可溶性和活性也未受到影响。为比较NC结构域和成熟NC蛋白的RNA结合特性，我们建立了基于RT-qPCR的定量分析在众多无关RNA存在的情况下蛋白质选择性富集病毒Psi的策略。发现HIV-1的Gag在大肠杆菌体内和在体外结合反应中分别将Psi富集了17.70和21.05倍，而NCp7只富集了1.97和2.31倍。利用SPR技术测定出Gag结合Psi中SL3的亲和力比NCp7高一个数量级。由于NCp7结合Psi不依赖其锌指基序和二级结构，我们测定了Gag和NCp7的锌离子含量，发现Gag结合2个锌，而NCp7只结合1个，推测是N-端锌指基序丧失了大部分的锌结合能力。结合2个锌的Gag截短体富集Psi的能力和结合SL3的亲和力与Gag相当，均高于NCp7和结合1个锌的Gag截短体和突变体。利用加热及其后的不同冷却方式，我们制备了含0、1、2个锌的NCp7，发现2NCp7选择Psi-RNA的能力及与SL3的亲和力与Gag大致相当；而0NCp7则与H23&44K双突变的NCp7相当。我们还纯化了HIV-1的蛋白酶，通过测定酶切反应前后游离的及蛋白结合的锌离子浓度，发现由NC结构域切割出成熟NC时可能伴随锌离子的释放。通过本项目的研究，我们不仅回答了申请时提出的科学问题，证实了NC的热稳定性及其在高序列覆盖度下对反转录的抑制，揭示了Gag和NC蛋白在选择病毒Psi及结合SL3亲和力方面的显著差异，还首次分析了NC结构域和成熟NC蛋白具有不同RNA结合特性的可能原因，提示锌离子在病毒生活周期调控方面可能发挥重要作用。这项工作将有助于对慢病毒复制调控过程的理解，为基于锌离子的慢病毒治疗策略研发提供依据。