Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA.
J Virol. 2012 Feb;86(3):1421-32. doi: 10.1128/JVI.05993-11. Epub 2011 Nov 16.
For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2-mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2-mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking.
对于 HIV-1 和鼠白血病病毒（MLV）等逆转录病毒，在病毒进入时会发生活跃的受体募集和运输。然而，该过程中涉及的潜在机制和细胞因子在很大程度上仍未得到阐明。嗜性 MLV（eMLV）的病毒受体是鼠阳离子氨基酸转运蛋白 1（mCAT-1），它是逆转录病毒感染机制和发病机制的经典模型。生长因子受体结合蛋白 2（GRB2）是一种衔接蛋白，已被证明可将细胞表面受体（如表皮生长因子受体（EGFR）和肝细胞生长因子受体）与细胞内信号事件偶联。在这里，我们研究了 GRB2 是否也可以通过影响受体功能在控制逆转录病毒感染中发挥作用。GRB2 的 RNA 干扰（RNAi）介导的内源性 GRB2 抑制导致病毒结合和膜融合的一致且显著减少。通过免疫沉淀检测到 eMLV 与细胞的结合促进了 GRB2-mCAT-1 相互作用的增加。一致地，通过共聚焦显微镜检测到 GRB2 和 mCAT-1 信号的增加共定位。这种关联是时间依赖性的，并与细胞-病毒膜融合的动力学平行。有趣的是，与 GRB2 和生长因子受体的典型结合模式不同，GRB2-mCAT-1 结合不依赖于 GRB2-SH2 结构域介导的对受体酪氨酸磷酸化的识别。内源性 GRB2 的抑制导致 mCAT-1 表面水平降低，这通过免疫沉淀和使用重组 MLV 包膜蛋白受体结合域（RBD）的直接结合测定来检测。与这一观察结果一致的是，显性负 GRB2 突变体（R86K）的表达导致 mCAT-1 从细胞表面隔离到细胞内囊泡中。总之，这些发现表明 GRB2 在促进 mCAT-1 运输方面在嗜性 MLV 进入和感染中发挥了新的作用。
