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

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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.