Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, United States of America.
Department of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, United States of America.
PLoS One. 2019 Mar 19;14(3):e0212888. doi: 10.1371/journal.pone.0212888. eCollection 2019.
TRIM5α is an interferon inducible restriction factor which contributes to intrinsic defense against HIV infection by targeting the HIV capsid protein CA. Although human TRIM5α (huTRIM5α) does not potently inhibit HIV-1 infection, the ability of huTRIM5α to exhibit some control of HIV-1 infection is evidenced by a single nucleotide polymorphism in huTRIM5α which substitutes aspartic acid to glycine at position 249 (G249D) in the L2 region and is associated with higher susceptibility to HIV-1 infection. To understand the mechanistic basis for the reduced antiviral activity, we employed biophysical and cell biological methods coupled with molecular dynamics simulations to compare WT and the G249D polymorphism of huTRIM5α. We investigated the differences in conformational dynamics of rhesus and huTRIM5α Coiled Coil-Linker 2 (CC-L2) dimers utilizing circular dichroism and single molecule-Fluorescence Energy Transfer (sm-FRET). These methods revealed that the G249D dimer exhibits secondary structure and conformational dynamics similar to WT huTRIM5α. Homology modelling revealed that G249 was present on the hairpin of the antiparallel dimer, in a position which may act to stabilize the adjacent BBox2 domain which mediates the inter-dimeric contacts required for the formation of TRIM5 assemblies. We therefore asked if the G249D mutant forms assemblies in cells with the same efficiency as WT protein by expressing these proteins as YFP fusions and quantifying the number of assemblies in cells. In cells expressing comparable amounts of protein, the G249D mutant formed fewer assemblies than WT protein, in agreement with our homology modeling predictions and molecular dynamics simulations of dimers and higher oligomers of TRIM5α, providing a mechanistic explanation of the reduced antiviral activity of the G249D polymorphism.
TRIM5α 是一种干扰素诱导的限制因子，通过靶向 HIV 衣壳蛋白 CA 来帮助固有防御 HIV 感染。尽管人源 TRIM5α（huTRIM5α）不能有效地抑制 HIV-1 感染，但 huTRIM5α 能够对 HIV-1 感染进行一定程度的控制，这一事实证据是 huTRIM5α 中的一个单核苷酸多态性，该多态性在 L2 区将天冬氨酸替换为甘氨酸（G249D），并且与对 HIV-1 感染的更高易感性相关。为了理解抗病毒活性降低的机制基础，我们采用生物物理和细胞生物学方法，并结合分子动力学模拟，比较了 WT 和 huTRIM5α 的 G249D 多态性。我们利用圆二色性和单分子荧光能量转移（sm-FRET）研究了恒河猴和 huTRIM5α 卷曲螺旋连接子 2（CC-L2）二聚体构象动力学的差异。这些方法表明，G249D 二聚体表现出与 WT huTRIM5α 相似的二级结构和构象动力学。同源建模显示，G249 存在于反平行二聚体的发夹中，其位置可能有助于稳定相邻的 BBox2 结构域，该结构域介导形成 TRIM5 组装所需的二聚体间接触。因此，我们通过表达这些蛋白作为 YFP 融合蛋白并定量细胞中组装体的数量，询问 G249D 突变体是否以与 WT 蛋白相同的效率在细胞中形成组装体。在表达相当量蛋白的细胞中，G249D 突变体形成的组装体少于 WT 蛋白，这与我们的同源建模预测以及 TRIM5α 二聚体和更高寡聚体的分子动力学模拟一致，为 G249D 多态性降低抗病毒活性提供了机制解释。
