Maturation of adenovirus via a new type of biochemistry

描述（由申请人提供）：腺病毒蛋白酶（AVP）在新生病毒颗粒内被激活;然后它切割用于病毒体组装的六种不同病毒体前体蛋白的多个拷贝，以使病毒颗粒具有感染性。我们观察到一个难题，所有这些蛋白质都是序列独立的DNA结合蛋白。鉴于病毒体中DNA的高浓度，这些蛋白质基本上不可逆地与病毒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，通过显示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，我们要问的是，这些蛋白质在DNA上滑动的生物物理机制是什么？在具体目标1中，我们提出了初步的证据表明，11个氨基酸的肽（pVIc）从C-末端的pVI，腺病毒蛋白VI的前体，可以滑动本身的DNA。我们建议的参数，使滑动的特点。这些信息将用于具体目标2，我们提出了初步证据，证明pVIc是一种“分子雪橇”，不仅能够自行滑动，而且还能够 我们继续提出，我们将通过直接可视化，使用双色全内反射荧光显微镜，显示AVP如何被底物滑动激活，以及活性AVP-pVIc复合物如何通过酶滑动病毒体前体蛋白来处理。 然后，我们将扩展我们的结果，看看类似的蛋白质：蛋白质的相互作用发生在哺乳动物细胞核中通过一维扩散。具体目标1中描述的初步证据表明，pVIc中可以滑动的最小序列是KRRR，其是功能性核定位信号（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，其必须在蛋白质的表面上才能被核输入机制识别。因此，预期这些蛋白质将结合到在染色体DNA的一维轮廓上相互作用的DNA上并沿着DNA滑动。这种新形式的生物化学，一维生物化学，有一些独特的性质。例如，如果酶及其底物通过经由NLS结合到DNA而最佳地共取向，则相对于不存在DNA，由滑动介导的生产性酶-底物碰撞的分数可以增加许多数量级，可能高达1.0。 这项工作提出了一个新的范例，蛋白酶和它们的底物如何可以相互作用，一个新的范例，病毒体成熟，一个新的车辆在分子生物学中，“分子雪橇”，甚至是一种新形式的生物化学，可能适用于所有的双分子相互作用，发生在哺乳动物细胞的细胞核。

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.