Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
J Virol. 2014 May;88(9):4839-52. doi: 10.1128/JVI.03359-13. Epub 2014 Feb 12.
Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFβ was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFβ remains unclear. Several studies noted reduced Vif expression in CBFβ knockdown cells while others saw no significant impact of CBFβ on Vif stability. Here, we confirmed that CBFβ increases Vif steady-state levels. CBFβ affected expression of neither viral Gag nor Vpu protein, indicating that CBFβ regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBFβ on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBFβ to interact with Vif. Importantly, at comparable Vif levels, CBFβ further enhanced A3G degradation, suggesting that CBFβ facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFβ also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFβ had no detectable effect on RUNX1 metabolic stability. We propose that CBFβ acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G.
In this study, we show that CBFβ has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBFβ. Kinetic studies revealed that CBFβ increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBFβ were nonresponsive to CBFβ. Our data suggest that CBFβ exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature protein conformation to ensure proper function of Vif and RUNX1. Thus, we identified a novel mechanism of protein regulation that affects both viral and cellular factors and thus has broad implications beyond the immediate HIV field.
Vif 是一种慢病毒辅助蛋白，通过诱导 APOBEC3G（A3G）的蛋白酶体降解，部分调节病毒感染力。最近，发现 CBFβ 有助于 Vif 依赖的 A3G 降解。然而，CBFβ 的确切作用仍不清楚。一些研究注意到 CBFβ 敲低细胞中 Vif 的表达减少，而其他研究则没有看到 CBFβ 对 Vif 稳定性的显著影响。在这里，我们证实 CBFβ 增加了 Vif 的稳态水平。CBFβ 既不影响病毒 Gag 蛋白也不影响 Vpu 蛋白的表达，表明 CBFβ 在后转录水平上调节 Vif 的表达。动力学研究表明 CBFβ 对 Vif 的代谢稳定性和生物合成速率都有影响。这些影响依赖于 CBFβ 与 Vif 相互作用的能力。重要的是，在可比的 Vif 水平下，CBFβ 进一步增强了 A3G 的降解，表明 CBFβ 通过增加 Vif 的水平并独立增强 Vif 靶向 A3G 降解的能力来促进 A3G 的降解。CBFβ 还通过增强 RUNX1 的生物合成来增加 RUNX1 的表达。然而，与 Vif 不同的是，CBFβ 对 RUNX1 的代谢稳定性没有可检测的影响。我们提出 CBFβ 作为一种伴侣蛋白，在合成过程中和合成后稳定 Vif，并促进 Vif 与细胞辅助因子相互作用，从而有效地降解 A3G。
在这项研究中，我们表明 CBFβ 对 HIV-1 感染力因子 Vif 和细胞转录因子 RUNX1 的表达有深远的影响，这两种蛋白与 CBFβ 物理相互作用。动力学研究表明，CBFβ 在翻译水平上增加了 Vif 和 RUNX1 的生物合成速率。不能与 CBFβ 物理相互作用的 Vif 突变体对 CBFβ 没有反应。我们的数据表明，CBFβ 发挥伴侣样活性（i）通过结合新生蛋白来最小化缺陷核糖体产物（DRiPs）的产生，以防止过早终止，（ii）稳定成熟蛋白构象，以确保 Vif 和 RUNX1 的正常功能。因此，我们确定了一种新的蛋白质调节机制，它影响病毒和细胞因子，因此超出了 HIV 领域的直接影响。
