Mechanism of Viral Genome Delivery into Cells

项目摘要 在发现第一种病毒近120年后，我们对病毒如何将基因组运送到 克服生物膜复杂性的细胞仍然有限。虽然一部浩瀚的科学文献 存在于病毒表面蛋白及其与宿主受体和免疫系统的相互作用中，很少 重点一直致力于研究将整个病毒基因组送入细胞。例如，如何 噬菌体通过革兰氏阴性细菌的细胞膜排出DNA？或者，在人类中，如何 疱疹病毒通过核孔复合体(NPC)将~200kb的基因组送入细胞核？ 在25年的时间里，首先是作为实习生(1995-2003)，从2004年开始作为首席调查员，我一直 研究了核质转运和病毒基因组包装的机制。我的工作已经 结果发表了近85篇论文，有助于阐明关键的原子结构和调节 与核进口和病毒基因组包装有关的因素。在这个R35中，我建议将研究结合起来 这两个看似截然不同的生物过程，通过重点研究病毒基因组传递的机制 变成活细胞。具体地说，我将问两个生物学问题，试图比较和对比有多简单 细菌病毒(或噬菌体)通过疱疹病毒的传递方式将它们的DNA喷射到细菌中。 将复杂的基因组送入真核细胞的核中。第一个问题是探索噬菌体是如何 通过革兰氏阴性菌的细胞膜排出~45kb的基因组。长期以来一直被认为是一个简单的 压力驱动注射，这个过程使用了病毒编码的纳米机器，我们已经开始在 我的实验室。第二个问题探索了疱疹病毒如何通过 人类细胞的核孔复合体(NPC)进入细胞核。这是一种由信号和能量介导的 使用宿主输入蛋白和GTP酶RAN的过程，利用细胞运输机制促进 外源基因组进入细胞核。总体而言，了解病毒如何转移基因 通过生物膜进入细胞和细胞器的信息对破译分子至关重要 毒力机制以及新的治疗方法的发展。平凡的 这个R35的分母在于我们对生物的结构和运输机制的兴趣 大分子。我们的研究方法结合了成熟的科学，如蛋白质生物化学和X射线 用低温电子显微镜(Cryo-EM)观察生物大分子的结晶学 近乎原生的条件。我们相信，这一R35 Mira筹资机制将推动创新和勤奋 寻求对我们提出的问题的答案，使我们的研究计划能够实现重要的 结构生物学的进展。

Project Summary Nearly 120 years since the discovery of the first virus, our understanding of how viruses deliver genomes into cells overcoming the complexity of biological membranes remains limited. While a vast scientific literature exists on viral surface proteins and their interaction with host receptors, and the immune system, little emphasis has been devoted to studying the delivery of entire viral genomes into cells. For instance, how do bacteriophages eject DNA through the cell envelope of Gram-negative bacteria? Or, in humans, how do herpesviruses deliver ~200 kb genome through the Nuclear Pore Complex (NPC) into the cell nucleus? For a quarter of a century, first as a trainee (1995-2003), and since 2004 as a principal investigator, I have investigated the mechanisms of nucleocytoplasmic transport and viral genome packaging. My work has resulted in close to 85 publications that contributed to elucidating the atomic structure and regulation of crucial factors implicated in nuclear import, and viral genome packaging. In this R35, I propose to combine the study of these two seemingly distinct biological processes by focusing on the mechanisms of viral genome delivery into living cells. Specifically, I will ask two biological questions that seek to compare and contrast how simple bacterial viruses (or bacteriophages) eject their DNA into bacteria with how Herpesviruses deliver their complex genomes into the nucleus of eukaryotic cells. The first question explores how bacteriophages eject ~45 kb genomes through the cell envelope of gram-negative bacteria. Long-thought to be a simple pressure-driven injection, this process uses a virus-encoded nanomachine, which we have begun to study in my laboratory. The second question explores how Herpesviruses deliver their large genome through the Nuclear Pore Complex (NPC) of human cells into the cell nucleus. This is a signal- and energy-mediated process that uses host importins and the GTPase Ran, exploiting the cellular transport machinery to promote entry of an exogenous genome into the nucleus. Overall, understanding how viruses transfer genetic information through biological membranes into cells and organelles is vital for deciphering the molecular mechanisms of virulence as well as the development of novel therapeutic approaches. The common denominator of this R35 lies in our interest in the structure and transport mechanisms of biological macromolecules. Our research approach marries established sciences like protein biochemistry and X-ray crystallography with the power of cryo-electron microscopy (cryo-EM) to visualize biological macromolecules in near-native conditions. We believe that this R35 MIRA funding mechanism will fuel the creative and diligent pursuit of answers to the questions we pose, permitting our research program to achieve significant advancements in structural biology.