Capsid-dependent innate sensing and restriction or retroviral replication complexes

这个项目的主要目的是阐明完整的逆转录病毒衣壳在功能复制复合体的形成和核进入过程中的不同作用，并伴随着对病毒核酸的先天感知的逃避。我们的研究主要集中在HIV-1(部分是HIV-2)和MLV上，因为这两种病毒在相关功能上表现出根本的差异。在第一个资助期，我们已经：(I)建立和验证了检测和表征两种病毒的功能复制复合体的方法，(Ii)通过结构分析观察和表征了HIV和MLV成熟衣壳结构的意外差异，(Iii)确定了衣壳结合宿主因子CPSF6在HIV-1复制复合体的核进入和核质定位中的功能，以及(Vi)获得了衣壳靶向药物PF74在阻止HIV复制方面的不同机制的证据。加上最近报告的IP6是天然衣壳稳定分子，以及NONO是潜在的桥梁因子，目的是通过衣壳结合将cGAS感应到核反转录病毒复合体，这些结果为第二个资助期的拟议工作方案提供了基础。主要的假设仍然是，逆转录病毒衣壳保护病毒核酸免受先天感知，而它本身是抗病毒识别的目标，这些方面因细胞类型和逆转录病毒而异。具体地说，我们为第二个资助期制定了以下目标：(I)对野生型和突变型HIV-1和MLV衣壳的结构和稳定性进行比较表征，(Ii)分析逆转录病毒复制复合体与已知和推定的宿主因子和核酸传感分子的关联，以及(Iii)真正复制复合体的分离和表征，以及相关宿主因子的鉴定。

The main objective of this project is to elucidate the differential role of intact retroviral capsids regarding formation and nuclear entry of functional replication complexes with concomitant evasion from innate sensing of the viral nucleic acids. Our studies focus on HIV-1 (and partially HIV-2) and MLV since these two viruses exhibit fundamental differences in the relevant functions. In the first funding period, we have: (i) established and validated assays to detect and characterize functional replication complexes for both viruses, (ii) observed and characterized unexpected differences in the architecture of HIV and MLV mature capsids by structural analysis, (iii) identified a function of the capsid-binding host factor CPSF6 in nuclear entry and nucleoplasmic localization of HIV-1 replication complexes, and (vi) obtained evidence for a different mechanism of the capsid-targeting drug PF74 in blocking HIV replication. Together with the recently reported identification of IP6 as natural capsid-stabilizing molecule and of NONO as a potential bridging factor targeting cGAS sensing to the nuclear retroviral complex via capsid binding, these results provide the basis for the proposed work programme in the second funding period. The main hypothesis remains that the retroviral capsid shields the viral nucleic acids from innate sensing, while itself being the target of antiviral recognition, and these aspects differ depending on cell type and retrovirus. Specifically, we have the following aims for the second funding period: (i) comparative characterization of structure and stability of wild-type and mutant HIV-1 and MLV capsids, (ii) analysis of the association of retroviral replication complexes with known and presumed host factors and nucleic acid sensing molecules, and (iii) isolation and characterization of bona fide replication complexes and identification of associated host factors.