Structural characterization of the anti-HIV protein MX2 and its interactions with viral and cellular factors.

干扰素通过诱导多种干扰素刺激基因(ISGs)的表达，使细胞处于抗病毒状态。HIV-1的复制自然地被干扰素抑制，我们和其他人之前的工作已经建立了粘病毒抗性2(MX2/MXB)作为一种具有强大的抗病毒活性的ISG，抑制病毒DNA的核输入。MX2由一个具有GTPase活性的球状(G)结构域、一个促进其寡聚的茎结构域和一个氨基末端结构域(NTD)组成。NTD是MX2抗病毒活性的主要决定因素。它将MX2定位于核膜，调控与几种细胞成分的相互作用，并与其HIV-1蛋白靶标衣壳(CA)结合。病毒衣壳由约1,500个CA拷贝组成，组装成五聚体和六聚体，形成独特的圆锥形结构。重要的是，已报道了几种CA突变病毒，它们的氨基酸变化位于不同的CA表面，从而逃脱了MX2的限制1，2。虽然基于分子动力学模拟的MX2‘S与病毒衣壳相互作用的模型3，目前还没有直接的实验证据，NTD的结构也还没有确定。马利姆实验室最近获得的数据为MX2-CA相互作用提供了新的线索(S)。具体地说，我们已经证明：a)虽然MX2的NTD-CA结合是病毒抑制所必需的，但MX2也能够通过其G结构域与CA相互作用；b)抗MX2抑制的CA突变病毒(即具有P90A或T210K替换的)仍然与MX2的G结构域结合，但只有P90A与NTD相互作用；C)NTD磷酸化通过阻止CA结合(Betancor等人，在审查中)而丧失MX2‘S通过阻止CA结合来抑制艾滋病毒-1感染的能力；e)几个MX2残基的磷酸化(或突变为Asp，称为磷酸模拟突变)增强了抗病毒活性(称为多形态突变体)，以至于能够抑制上述CA突变病毒。通过了解所有这些残基如何平衡MX2-CA相互作用的结果，从而限制病毒，我们将为开发新的抗HIV-1疗法定义一个新的靶点。这一点很重要，因为目前的抗逆转录病毒治疗导致了耐药病毒株的出现，这使得发现新的治疗方法是必要的。因此，详细了解MX2-CA相互作用中涉及的特定残基/表面是至关重要的。到目前为止，唯一可用的MX2结构缺少关键的NTD6，7，因此，没有提供对MX2 NTD和CA之间的关键相互作用的洞察。通过这个项目，我们的目标是填补这一空白。参考文献：Goujon，C.等人。《自然》502，(2013)。Busnadiego，I.等人。J Virol 88，(2014)。Smaga，S.S.等人。结构27，(2019年)。Betancor，G.等人。细胞代表29，(2019)。Garbeli A.et al.《生物化学杂志》，474，(2014)。Friborgh，J.L.等人。细胞宿主微生物16，(2014)。阿尔瓦雷斯·F·J·D等人。SCI Adv 3，(2017)。Link J.O.等人，2020年。《自然》584，(2020)。目的：陈述主要的研究问题，并在适当的情况下测试主要的假设，该项目的目的是确定MX2和CA之间相互作用的分子细节。

Interferon (IFN) mobilizes a cellular anti-viral state by inducing the expression of numerous IFN-stimulated genes (ISGs). The replication of HIV-1 is naturally inhibited by interferon and previous work from us and others has established myxovirus resistance 2 (MX2/MxB) as an ISG with a robust anti-viral activity, suppressing the nuclear import of viral DNA. MX2 consists of a globular (G) domain with GTPase activity, a stalk domain that promotes its oligomerization, and an amino-terminal domain (NTD). The NTD is the main determinant for the anti-viral activity of MX2. It localizes MX2 to the nuclear membrane, governs the interaction with several cellular components and binds to its HIV-1 protein target, Capsid (CA). The viral capsid is composed of ~1,500 copies of CA, assembled into pentamers and hexamers, and forming a distinctive conical structure. Importantly, several CA mutant viruses, with amino acid changes located in different CA surfaces, have been reported to escape MX2 restriction1,2. While a molecular dynamic simulation-based model for MX2's interaction with the viral capsid has been proposed3, there is currently no direct experimental evidence for this, and the structure of the NTD has not been determined. Recent data obtained by the Malim lab has shed fresh light on the MX2-CA interaction(s). Specifically, we have demonstrated that: a) while the MX2 NTD-CA binding is essential for viral inhibition, MX2 is also able to interact with CA through its G domain4; b) CA mutant viruses resistant to MX2 inhibition (i.e., with the P90A or T210K substitutions) are still bound by the MX2 G domain, but only P90A interacts with the NTD; c) NTD phosphorylation abrogates MX2's ability to inhibit HIV-1 infection by impeding CA binding (Betancor et al., under review) and e) phosphorylation (or mutation to Asp, known as a phospho-mimetic mutation) of several MX2 residues enhances anti-viral activity (called hypermorphic mutants), to the extent of being able to inhibit the aforementioned CA mutant viruses. By understanding how all these residues balance the outcome of the MX2-CA interaction, and consequently viral restriction, we will be defining a novel target for the development of new anti-HIV-1 therapies. This is important, since current anti-retroviral treatments lead to the emergence of resistant viral strains5, making the discovery of new treatments a necessity. Therefore, it is critical to understand in detail the specific residues/surfaces involved in MX2-CA interaction. To date, the only MX2 structures available are missing the critical NTD6, 7, and consequently, do not provide insight into the pivotal interaction between the MX2 NTD and CA. Through this project, we aim to fill this gap. References: Goujon, C. et al. Nature 502, (2013). Busnadiego, I. et al. J Virol 88, (2014). Smaga, S. S. et al. Structure 27, (2019). Betancor, G. et al. Cell Rep 29, (2019). Garbelli A. et al. Biochem J. 474, (2014). Fribourgh, J. L. et al. Cell Host Microbe 16, (2014). Alvarez F. J. D, et al. Sci Adv 3, (2017). Link J. O. et al., 2020. Nature 584, (2020). Aim:State primary research question and where appropriate the primary hypotheses being tested The aim of this project is to determine the molecular details of the interaction between MX2 and CA.