Using prototype foamy virus integrase as a model for detailed structural studies of retroviral DNA integration

为了实现有效感染，艾滋病毒必须将其基因组的DNA拷贝插入人类细胞的染色体中。这一过程由整合酶协调，整合酶是病毒携带的一种酶。一旦整合完成，病毒基因组就成为细胞中的永久居民。从那里，它将启动新的传染性颗粒的产生，或者它可能会在很长一段时间内处于休眠状态，不被发现。融合是艾滋病毒感染持续存在的部分原因。然而，艾滋病毒对融合的依赖也是一个可以利用的弱点。一类新的抗逆转录病毒药物破坏整合酶的酶活性，利用这一弱点来对抗艾滋病毒。这些药物也被称为整合酶抑制剂，开发这些药物的主要障碍是我们对艾滋病毒整合的结构方面的了解有限。2010年，我们确定了与病毒DNA末端结合的逆转录病毒整合酶长期寻找的三维结构。在这些研究中，我们选择使用源自原型泡沫病毒(PFV)的IN，与大多数其他逆转录病毒IN相比，当从其自然环境中移除时，IN更具溶解性和活性。另一方面，所有的逆转录病毒整合酶在结构组成上都非常相似。因此，尽管PFV不是人类病原体，但其整合酶是作为HIV整合酶替代品的理想实验模型。至关重要的是，它已经让我们能够解释艾滋病毒整合酶抑制剂是如何发挥作用的。目前的项目旨在利用PFV整合酶的独特生化特性来了解逆转录病毒整合过程的细节。以X射线结晶学为主要工具，我们将在整合过程的不同阶段确定PFV整合酶的三维结构。此外，利用它与HIV整合酶的相似性，我们将利用它来理解HIV有时是如何逃避整合酶抑制药物的作用的。我们还将产生更多的结构信息，药物开发人员将使用这些信息来提高整合酶抑制剂的效力。我们的数据将对学术和私人团体的药物发现和开发具有重大价值，并将有助于降低成本和改善最终治疗的可获得性。

To achieve productive infection, HIV must insert a DNA copy of its genome into a chromosome of a human cell. This process is orchestrated by integrase, an enzyme carried by the virus. Once integration is complete, the viral genome becomes a permanent resident in the cell. From there it will initiate production of new infectious particles or it might stay dormant and undetected for a long period of time. Integration is partly responsible for the persistence of HIV infection. Yet, the dependence of HIV on integration is also an exploitable weakness. A new class of antiretroviral drugs disrupts enzymatic activity of integrase taking advantage of this weakness to fight HIV. The main impediment to the development of these drugs, also known as integrase inhibitors, is our limited understanding of the structural aspects of HIV integration. In 2010 we determined the long-sought after three-dimensional structure of retroviral integrase bound to viral DNA ends. For these studies we chose to use IN derived from prototype foamy virus (PFV), which is much more soluble and active when removed from its natural environment compared to most of other retroviral INs. On the other hand, all retroviral integrase enzymes are very similar in their structural makeup. Therefore, although PFV is not a human pathogen, its integrase is an ideal model for experimentation as a proxy for HIV integrase. Crucially, it already allowed us to explain how HIV integrase inhibitors work. The current project aims use the unique biochemical properties of PFV integrase to understand the retroviral integration process down to a fine detail. Using X-ray crystallography as our main tool, we will determine three-dimensional structures of PFV integrase at various stages of the integration process. Furthermore, taking advantage of its similarity to HIV integrase, we will use it to understand how HIV can sometimes evade the action of integrase inhibiting drugs. We will also generate more structural information that drug developers will use to improve potency of integrase inhibitors. Our data will be of substantial value for drug discovery and development by both academic and private groups and will serve to reduce the costs and improve availability of the eventual treatments.