Role of the Viral Capsid in HIV-1 Integration

摘要 正在进行的艾滋病毒大流行已导致美国约120万人感染，约3700万人感染 全世界。尽管艾滋病毒/艾滋病研究取得了相当大的进展，但抗逆转录病毒疗法(ART)仍然是 HIV-1感染的唯一治疗选择。虽然抗逆转录病毒疗法在控制病毒和 使艾滋病毒感染成为一种可控的疾病，ART方案中使用的药物昂贵，原因方面 影响，并面临病毒耐药性。因此，迫切需要继续开发针对 新的细胞和病毒靶点。HIV-1衣壳蛋白(CA)是一种重要的病毒治疗靶点 目前在临床上未被开发。最近，吉利德开发了一种高效的CA靶向抑制剂，这是下一步 一代又一代抗病毒药物。然而，这些CA特异性抑制剂的成功过渡到临床需要明确的 了解抗病毒机制及CA在HIV-1感染中的作用。我们的研究是 旨在了解HIV-1 CA控制病毒整合的机制。 HIV通过病毒膜与细胞膜的融合进入靶细胞，释放 病毒衣壳核心进入靶细胞的细胞质。HIV-1变异株的功能研究表明 衣壳核心的正确组装、形态和稳定性对HIV-1的传染性都是必不可少的。虽然它是 众所周知，HIV-1 CA有助于逆转录，最近的数据表明CA也是一个关键 HIV-1进入靶细胞细胞核的能力的决定因素。特别是，CA在基因上是 功能上涉及逆转录前病毒的核进入，通过介导与 细胞因素。一个关键的知识缺口是CA在病毒整合中的作用-这是后核进入的关键步骤 HIV-1感染。我们假设HIV-1 CA蛋白直接影响病毒DNA整合。为了测试 在这一假设中，我们提出了三个具体目标：目标1.确定衣壳稳定性对HIV-1的影响 整合前复合体(PIC)活性和病毒DNA完整性。目标2.确定融合活动是否 取决于图片中的CA水平。目的3.确定已知的衣壳结合宿主蛋白在 定义与PIC相关的CA级别和PIC活动。测试HIV-1 CA和病毒DNA之间的直接联系 集成，我们开发了一种新的模型，通过耦合定量的生化方法 使用CA特异性抑制剂作为探针，测量PIC相关的整合活性。此外，我们 组建了一支拥有PIC生物化学(Dash)、衣壳生物学(Aiken)和 逆转录病毒整合(恩格尔曼)。因此，拟议的研究将产生关于 CA调节HIV-1整合的机制及CA抑制剂的抗病毒作用 开发以CA为基础的新型抗病毒疗法(美国国立卫生研究院的艾滋病毒/艾滋病重点研究领域)。

ABSTRACT The ongoing HIV pandemic has resulted in ~1.2 million infections in the US and ~37 million infections worldwide. In spite of considerable progress in HIV/AIDS research, anti-retroviral therapy (ART) remains the only treatment option for HIV-1 infection. While ART has been highly effective in controlling the virus and making HIV infection a manageable disease, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. Thus, there is an urgent need for continued development of drugs against novel cellular and viral targets. HIV-1 capsid protein (CA) is an important viral therapeutic target that is currently clinically unexploited. Recently, Gilead developing a highly potent CA-targeting inhibitor as next generation of antivirals. However, successful transition of these CA-specific inhibitors to the clinic require clear understanding of the anti-viral mechanisms and the delineating CA’s role in HIV-1 infection. Our studies are designed to understand the mechanism by which HIV-1 CA controls viral integration. HIV enters the target cell by fusion of the viral membrane with the cellular membrane, releasing the viral capsid core into the cytoplasm of the target cell. Functional studies of HIV-1 variants indicate that the proper assembly, morphology, and stability of the capsid core are all essential for HIV-1 infectivity. While it is well established that HIV-1 CA facilitates reverse transcription, recent data shows that CA is also a key determinant of the ability of HIV-1 to enter the nucleus of the target cell. In particular, CA is genetically and functionally implicated in nuclear entry of the reverse transcribed provirus, by mediating interactions with cellular factors. A key knowledge gap is the role of CA in viral integration-a critical post-nuclear entry step of HIV-1 infection. We hypothesize that HIV-1 CA protein directly influences viral DNA integration. To test this hypothesis we propose three specific aims: Aim 1. To define the effects of capsid stability on HIV-1 preintegration complex (PIC) activity and viral DNA integrity. Aim 2. To determine whether integration activity is dependent on CA levels in the PICs. Aim 3. To determine the role of known capsid-binding host proteins in defining PIC-associated CA levels and PIC activity. To test a direct link between HIV-1 CA and viral DNA integration, we have developed a novel model by coupling a biochemical approach that quantitatively measures PIC-associated integration activity to the use of the CA-specific inhibitor as probes. In addition, we have assembled a multidisciplinary team with expertise in PIC biochemistry (Dash), capsid biology (Aiken) and retroviral integration (Engelman). Therefore, the proposed studies will generate new knowledge on the mechanism by which CA regulates HIV-1 integration and define the antiviral effects of CA-inhibitors to facilitate the development of novel CA-based anti-viral therapies (a High-Priority HIV/AIDS research area of the NIH).