PDF(Pubmed)
Abstract：
Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced GTPase that inhibits human immunodeficiency virus-1 (HIV-1) infection by preventing nuclear import of the viral preintegration complex. The HIV-1 capsid (CA) is the major viral determinant for sensitivity to MX2, and complex interactions between MX2, CA, nucleoporins (Nups), cyclophilin A (CypA), and other cellular proteins influence the outcome of viral infection. To explore the interactions between MX2, the viral CA, and CypA, we utilized a CRISPR-Cas9/AAV approach to generate CypA knock-out cell lines as well as cells that express CypA from its endogenous locus, but with specific point mutations that would abrogate CA binding but should not affect enzymatic activity or cellular function. We found that infection of CypA knock-out and point mutant cell lines with wild-type HIV-1 and CA mutants recapitulated the phenotypes observed upon cyclosporine A (CsA) addition, indicating that effects of CsA treatment are the direct result of blocking CA-CypA interactions and are therefore independent from potential interactions between CypA and MX2 or other cellular proteins. Notably, abrogation of GTP hydrolysis by MX2 conferred enhanced antiviral activity when CA-CypA interactions were abolished, and this effect was not mediated by the CA-binding residues in the GTPase domain, or by phosphorylation of MX2 at position T151. We additionally found that elimination of GTPase activity also altered the Nup requirements for MX2 activity. Our data demonstrate that the antiviral activity of MX2 is affected by CypA-CA interactions in a virus-specific and GTPase activity-dependent manner. These findings further highlight the importance of the GTPase domain of MX2 in regulation of substrate specificity and interaction with nucleocytoplasmic trafficking pathways.
摘要：
人类粘病毒抗性2（MX2/MXB）是干扰素诱导的GTP酶，通过防止病毒整合前复合物的核输入来抑制人类免疫缺陷病毒1（HIV-1）感染。HIV-1衣壳（CA）是对MX2敏感性的主要病毒决定因素，以及MX2，CA，核孔蛋白(Nups)，亲环蛋白A（CypA），和其他细胞蛋白影响病毒感染的结果。探索MX2，病毒CA，还有CypA,我们利用CRISPR-Cas9/AAV方法来产生CypA敲除细胞系以及从其内源性基因座表达CypA的细胞。但具有特定的点突变，可以消除CA结合，但不应该影响酶活性或细胞功能。我们发现，用野生型HIV-1和CA突变体感染CypA敲除和点突变细胞系可以概括添加环孢菌素A（CsA）后观察到的表型，表明CsA处理的效果是阻断CA-CypA相互作用的直接结果,因此独立于CypA与MX2或其他细胞蛋白之间的潜在相互作用。值得注意的是,当CA-CypA相互作用被废除时，MX2对GTP水解的废除赋予了增强的抗病毒活性，这种效应不是由GTP酶结构域中的CA结合残基介导的，或通过MX2在T151位的磷酸化。我们还发现,GTP酶活性的消除也改变了对MX2活性的Nup需求。我们的数据表明，MX2的抗病毒活性受CypA-CA相互作用以病毒特异性和GTP酶活性依赖性方式影响。这些发现进一步突出了MX2的GTP酶结构域在调节底物特异性和与核质运输途径相互作用中的重要性。
