Maturation of adenovirus via a new type of biochemistry

描述(申请人提供)：腺病毒蛋白水解酶(AVP)在新生病毒颗粒内被激活；然后它切割六个不同的病毒颗粒前体蛋白的多个副本，用于病毒颗粒的组装，使病毒颗粒具有感染性。我们观察到一个难题，因为所有这些蛋白质都是序列无关的DNA结合蛋白。鉴于病毒粒子中的DNA浓度很高，这些蛋白质基本上不可逆转地与病毒DNA结合。但是，在没有三维扩散的情况下，这些蛋白质如何相互作用以形成有效的碰撞，即导致结合的碰撞？我们解决了这个难题，证明了这些生化反应发生在病毒DNA的一维轮廓上，这些蛋白质通过一维扩散沿着DNA滑动，这是对蛋白质：蛋白质相互作用的前所未有的观察。在这里，我们问这些蛋白质是通过什么生物物理机制在DNA上滑动的？在特定的目标1中，我们提出了来自腺病毒蛋白VI的前体PVI C-末端的11个氨基酸的多肽(PVIc)可以在DNA上自行滑动的初步证据。我们建议对允许滑动的参数进行表征。然后这些信息将被用于特定的目标2，在那里我们提出了初步证据，表明pVIc是一种不仅能够自行滑动而且能够 滑动的异种货物附在它上面。我们进一步提出，我们将用双色全内反射荧光显微镜直观地展示AVP是如何被底物滑动激活的，以及AVP-pVIc活性复合体是如何通过酶通过滑动来滑动病毒粒子前体蛋白的过程。然后，我们将扩展我们的结果，看看类似的蛋白质：蛋白质相互作用是否通过哺乳动物细胞细胞核中的一维扩散发生。在特定目的1中描述的初步证据表明，pVIc中可以滑动的最小序列是KRRR，它是一个功能性核定位信号(NLS)。因此，推测pVIc通过其NLS与DNA结合并在DNA上滑动。许多核蛋白具有NLS，它必须位于蛋白质的表面才能被核进口机构识别。因此，这些蛋白质有望与DNA结合并沿着DNA滑动，在染色体DNA的一维轮廓上相互作用。这种生物化学的新形式，一维生物化学，具有一些独特的性质。例如，如果一种酶和它的底物通过NLS与DNA结合以最佳共定向，那么与没有DNA的情况相比，滑动介导的生产性酶-底物碰撞的比例可能增加许多个数量级，可能高达1.0个数量级。这项工作提出了一种新的蛋白酶及其底物如何相互作用的范式，一种病毒粒子成熟的新范式，一种分子生物学的新工具--“分子雪橇”，甚至一种可能适用于发生在哺乳动物细胞核中的所有双分子相互作用的新形式的生物化学。

DESCRIPTION (provided by applicant): The adenovirus proteinase (AVP) is activated inside nascent virus particles; it then cleaves multiple copies of six, different virion precursor protein used in the assembly of the virion to render the virus particle infectious. We observed a conundrum in that all these proteins are sequence-independent DNA binding proteins. Given the high concentration of DNA in the virion, these proteins are essentially irreversibly bound to the viral DNA. But then, how do these proteins interact to form productive collisions, i.e. collisions that lead to binding, in the absence of three-dimensional diffusion? We solved this conundrum by showing that these biochemical reactions take place on the one-dimensional contour of the viral DNA by these proteins sliding along the DNA via one-dimensional diffusion, an unprecedented observation for protein:protein interactions. Here we ask by what biophysical mechanisms do these proteins slide on DNA? In Specific Aim 1, we present preliminary evidence that an 11-amino acid peptide (pVIc) from the C-terminus of pVI, the precursor to adenovirus protein VI, can slide by itself on DNA. We propose to characterize the parameters that enable sliding. That information will then be used in Specific Aim 2 where we present preliminary evidence that pVIc is a "molecular sled" capable not only of sliding by itself but also sliding heterologous cargoes attached to it. We go on to propose that we shall show by direct visualization, using two color total internal reflection fluorescence microscopy, how AVP is activated by substrate sliding and how active AVP-pVIc complexes process by enzyme sliding the virion precursor proteins by sliding. We shall then extend our results to see if similar protein:protein interactions occur via one-dimensional diffusion in the nucleus of mammalian cells. Preliminary evidence described in Specific Aim 1 indicated that the minimum sequence in pVIc that can slide is KRRR which is a functional nuclear location signal (NLS). Thus, presumably pVIc binds to and slides on DNA via its NLS. Many nuclear proteins have an NLS that must be on the surface of the proteins to be recognized by the nuclear import machinery. Therefore, those proteins would be expected to bind to and slide along DNA interacting on the one-dimensional contour of chromosomal DNA. This new form of biochemistry, one-dimensional biochemistry, has some unique properties. For example, if an enzyme and its substrate are optimally co-oriented by being bound to DNA via an NLS, the fraction of productive enzyme-substrate collisions mediated by sliding may be increased many orders of magnitude, possibly up to 1.0, relative to in the absence of DNA. This work presents a new paradigm for how proteinases and their substrates can interact, a new paradigm for virion maturation, a new vehicle in molecular biology, the "molecular sled," and even a new form of biochemistry that may be applicable to all bimolecular interactions that take place in the nucleus of mammalian cells.