Multifunctional Roles of the N-Terminal Region of HIV-1SF2Nef Are Mediated by Three Independent Protein Interaction Sites
Multifunctional Roles of the N-Terminal Region of HIV-1SF2Nef Are Mediated by Three Independent Protein Interaction Sites
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DOI：
10.1128/jvi.01398-19
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发表时间：
2020-01-01
影响因子：
5.4
通讯作者：
Facklera, Oliver T.
中科院分区：
文献类型：
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作者：
Ananth, Swetha;Morath, Katharina;Facklera, Oliver T.
HIV-1 Nef通过与多种宿主细胞蛋白相互作用改变宿主细胞转运和信号传导过程，促进病毒传播和疾病进展。HIV-1 Nef中包含残基12至39的N-末端区域涉及许多Nef活性，包括破坏CD 4 T淋巴细胞极化和归巢至淋巴结，拮抗SERINC 5限制病毒粒子感染性，下调细胞表面CD 4和主要组织相容性复合物I类（MHC-I），释放含Nef的细胞外囊泡，以及通过Nef相关激酶复合物（NAKC）的募集来磷酸化Nef。该区域如何介导这些多效性功能尚不清楚。一组丙氨酸突变体跨越N-末端区域，以确定特定的功能决定簇的表征显示，该区域是从HIV-1株SF 2（HIV-1（SF 2）Nef）对T细胞肌动蛋白组织和趋化性，重定向宿主细胞激酶Lck的反式高尔基体网络，并纳入Nef到细胞外囊泡的影响。MHC-I下调是特定于残基M20，Nef对T细胞极化的抑制需要整个区域的完整性。相反，下调细胞表面CD 4和SERINC 5拮抗作用是由一个特定的基序介导的，该基序包括残基32至39，这也是HIV在原代CD 4 T淋巴细胞中有效复制所必需的。最后，通过与NAKC的关联，Nef磷酸化由残基24至29内的两个EP重复介导，但不具有其他功能。这些结果确定了N-末端区域作为至少三种不同的宿主细胞配体的多功能相互作用模块，介导HIV-1（SF 2）Nef的独立功能，以促进免疫逃避和病毒spreads.Importance HIV-1 Nef关键决定病毒传播和疾病的进展，在感染的个体作为一个蛋白质相互作用适配器通过不完全定义的机制和配体。Nef的N末端附近的残基12至39已被描述为Nef相关激酶复合物（NAKC）的相互作用平台，并且最近被鉴定为广泛的Nef活性的必要决定因素。在这里，我们报告了一个系统的映射，揭示了存在三个独立的相互作用的图案与特定的配体和活动的氨基酸伸展。虽然细胞表面MHC-I的下调依赖于M20，但两个EP重复序列是NAKC的最小结合位点，残基32至39介导宿主细胞限制因子SERINC 5的拮抗作用以及细胞表面CD 4的下调。这些结果表明，HIV-1（SF 2）Nef的N-末端区域是一个通用的和多功能的蛋白质相互作用模块，通过独立的机制发挥致病因子的基本功能。
HIV-1 Nef promotes virus spread and disease progression by altering host cell transport and signaling processes through interaction with multiple host cell proteins. The N-terminal region in HIV-1 Nef encompassing residues 12 to 39 has been implicated in many Nef activities, including disruption of CD4 T lymphocyte polarization and homing to lymph nodes, antagonism of SERINC5 restriction to virion infectivity, downregulation of cell surface CD4 and major histocompatibility complex class I (MHC-I), release of Nef-containing extracellular vesicles, and phosphorylation of Nef by recruitment of the Nef-associated kinase complex (NAKC). How this region mediates these pleiotropic functions is unclear. Characterization of a panel of alanine mutants spanning the N-terminal region to identify specific functional determinants revealed this region to be dispensable for effects of Nef from HIV-1 strain SF2 (HIV-1(SF2)Nef) on T cell actin organization and chemotaxis, retargeting of the host cell kinase Lck to the trans-Golgi network, and incorporation of Nef into extracellular vesicles. MHC-I downmodulation was specific to residue M20, and inhibition of T cell polarization by Nef required the integrity of the entire region. In contrast, downmodulation of cell surface CD4 and SERINC5 antagonism were mediated by a specific motif encompassing residues 32 to 39 that was also essential for efficient HIV replication in primary CD4 T lymphocytes. Finally, Nef phosphorylation via association with the NAKC was mediated by two EP repeats within residues 24 to 29 but was dispensable for other functions. These results identify the N-terminal region as a multifunctional interaction module for at least three different host cell ligands that mediate independent functions of HIV-1(SF2)Nef to facilitate immune evasion and virus spread.IMPORTANCE HIV-1 Nef critically determines virus spread and disease progression in infected individuals by acting as a protein interaction adaptor via incompletely defined mechanisms and ligands. Residues 12 to 39 near the N terminus of Nef have been described as an interaction platform for the Nef-associated kinase complex (NAKC) and were recently identified as essential determinants for a broad range of Nef activities. Here, we report a systematic mapping of this amino acid stretch that revealed the presence of three independent interaction motifs with specific ligands and activities. While downmodulation of cell surface MHC-I depends on M20, two EP repeats are the minimal binding site for the NAKC, and residues 32 to 39 mediate antagonism of the host cell restriction factor SERINC5 as well as downmodulation of cell surface CD4. These results reveal that the N-terminal region of HIV-1(SF2)Nef is a versatile and multifunctional protein interaction module that exerts essential functions of the pathogenicity factor via independent mechanisms.