Data_Sheet_6_The Signature Amino Acid Residue Serine 31 of HIV-1C Tat Potentiates an Activated Phenotype in Endothelial Cells.PDF

The natural cysteine to serine variation at position 31 of Tat in HIV-1C disrupts the dicysteine motif attenuating the chemokine function of Tat. We ask if there exists a trade-off in terms of a gain of function for HIV-1C Tat due to this natural variation. We constructed two Tat-expression vectors encoding Tat proteins discordant for the serine 31 residue (CS-Tat vs. CC-Tat), expressed the proteins in Jurkat cells under doxycycline control, and performed the whole transcriptome analysis to compare the early events of Tat-induced host gene expression. Our analysis delineated a significant enrichment of pathways and gene ontologies associated with the angiogenic signaling events in CS-Tat stable cells. Subsequently, we validated and compared angiogenic signaling events induced by CS- vs. CC-Tat using human umbilical vein endothelial cells (HUVEC) and the human cerebral microvascular endothelial cell line (hCMEC/D3). CS-Tat significantly enhanced the production of CCL2 from HUVEC and induced an activated phenotype in endothelial cells conferring on them enhanced migration, invasion, and in vitro morphogenesis potential. The ability of CS-Tat to induce the activated phenotype in endothelial cells could be of significance, especially in the context of HIV-associated cardiovascular and neuronal disorders. The findings from the present study are likely to help appreciate the functional significance of the SAR (signature amino acid residues) influencing the unique biological properties.

HIV-1C型Tat蛋白第31位天然发生的半胱氨酸到丝氨酸变异，会破坏其双半胱氨酸基序，削弱Tat的趋化因子功能。本研究旨在探究该天然变异是否会使HIV-1C型Tat出现功能增益层面的权衡效应。我们构建了两种编码丝氨酸31位点存在差异的Tat蛋白的Tat表达载体（CS-Tat与CC-Tat），在强力霉素诱导调控下于Jurkat细胞中表达上述蛋白，并开展全转录组分析，以比较Tat诱导宿主基因表达的早期事件。分析结果显示，CS-Tat稳定细胞株中与血管生成信号通路相关的通路及基因本体显著富集。随后，我们分别利用人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）与人脑微血管内皮细胞系（human cerebral microvascular endothelial cell line, hCMEC/D3），验证并比较了CS-Tat与CC-Tat诱导的血管生成信号事件。实验结果表明，CS-Tat可显著增强HUVEC中CCL2的产生，并诱导内皮细胞形成激活表型，使其迁移、侵袭及体外形态发生能力得到提升。CS-Tat诱导内皮细胞激活表型的能力具有重要研究价值，尤其在HIV相关心血管及神经系统疾病的研究背景下。本研究结果将有助于阐明特征氨基酸残基（signature amino acid residues, SAR）对蛋白质独特生物学特性的功能重要性。