Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N, Wolfe Street, Baltimore, MD 21205, USA.
Retrovirology. 2014 Jan 14;11:4. doi: 10.1186/1742-4690-11-4.
HIV-1 Vif promotes the degradation of host anti-retroviral factor family, APOBEC3 proteins via the recruitment of a multi-subunit E3 ubiquitin ligase complex. The complex is composed of a scaffold protein, Cullin 5 (Cul5), RING-box protein (Rbx), a SOCS box binding protein complex, Elongins B/C (Elo B/C), as well as newly identified host co-factor, core binding factor beta (CBF-β). Cul5 has previously been shown to bind amino acids within an HCCH domain as well as a PPLP motif at the C-terminus of Vif; however, it is unclear whether Cul5 binding requires additional regions of the Vif polypeptide.
Here, we provide evidence that an amino terminal region of full length Vif is necessary for the Vif-Cul5 interaction. Single alanine replacement of select amino acids spanning residues 25-30 (25VXHXMY30) reduced the ability for Vif to bind Cul5, but not CBF-β or Elo B/C in pull-down experiments. In addition, recombinant Vif mutants had a reduced binding affinity for Cul5 compared to wild-type as measured by isothermal titration calorimetry. N-terminal mutants that demonstrated reduced Cul5 binding were also unable to degrade APOBEC3G as well as APOBEC3F and were unable to restore HIV infectivity, in the presence of APOBEC3G. Although the Vif N-terminal amino acids were necessary for Cul5 interaction, the mutation of each residue to alanine induced a change in the secondary structure of the Vif-CBF-β-Elo B/C complex as suggested by results from circular dichroism spectroscopy and size-exclusion chromatography experiments. Surprisingly, the replacement of His108 to alanine also contributed to the Vif structure. Thus, it is unclear whether the amino acids contribute to a direct interaction with Cul5 or whether the amino acids are responsible for the structural organization of the Vif protein that promotes Cul5 binding.
Taken together, we propose a novel Vif N-terminal motif that is responsible for Vif recruitment of Cul5. Motifs in Vif that are absent from cellular proteins represent attractive targets for future HIV pharmaceutical design.
HIV-1 Vif 通过招募一个多亚基 E3 泛素连接酶复合物来促进宿主抗病毒因子家族 APOBEC3 蛋白的降解。该复合物由支架蛋白 Cullin 5（Cul5）、RING 盒蛋白（Rbx）、SOCS 盒结合蛋白复合物、Elongins B/C（Elo B/C）以及新鉴定的宿主共因子核心结合因子β（CBF-β）组成。先前已经表明，Cul5 结合 Vif 的 HCCH 结构域内的氨基酸以及 C 末端的 PPLP 基序；然而，Cul5 结合是否需要 Vif 多肽的其他区域尚不清楚。
在这里，我们提供的证据表明全长 Vif 的氨基末端区域是 Vif-Cul5 相互作用所必需的。跨越残基 25-30 的选定氨基酸的单个丙氨酸取代（25VXHXMY30）降低了 Vif 结合 Cul5 的能力，但在下拉实验中不降低与 CBF-β或 Elo B/C 的结合能力。此外，与野生型相比，通过等温滴定量热法测量，重组 Vif 突变体对 Cul5 的结合亲和力降低。与 Cul5 结合能力降低的 N 端突变体也不能降解 APOBEC3G 以及 APOBEC3F，并且在存在 APOBEC3G 的情况下不能恢复 HIV 感染性。尽管 Vif 的氨基末端氨基酸对于 Cul5 相互作用是必需的，但是每个残基突变为丙氨酸会导致 Vif-CBF-β-Elo B/C 复合物的二级结构发生变化，这一点从圆二色性光谱和大小排阻色谱实验的结果中可以看出。令人惊讶的是，His108 突变为丙氨酸也有助于 Vif 结构。因此，尚不清楚这些氨基酸是否有助于与 Cul5 的直接相互作用，或者这些氨基酸是否负责促进 Cul5 结合的 Vif 蛋白的结构组织。
总之，我们提出了一个新的 Vif N 末端基序，该基序负责 Vif 招募 Cul5。Vif 中不存在于细胞蛋白中的基序是未来 HIV 药物设计的有吸引力的靶标。
