An emerging and variant viral promoter of HIV-1 subtype C exhibits low-level gene expression noise.
An emerging and variant viral promoter of HIV-1 subtype C exhibits low-level gene expression noise.
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DOI：
10.1186/s12977-021-00572-2
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发表时间：
2021-09-19
期刊：
影响因子：
3.3
通讯作者：
Ranga U
中科院分区：
文献类型：
--
作者：
Ali H;Bhange D;Mehta K;Gohil Y;Prajapati HK;Byrareddy SN;Buch S;Ranga U
近年来，我们观察到印度出现了几种启动子变异病毒株。变体病毒启动子含有存在于病毒调节区或增强子中的转录因子结合位点的额外拷贝，包括RBEIII、LEF-1、Ap-1和/或NF-κB。这些位点对于控制病毒基因表达和潜伏期至关重要。在此，我们推断，与典型的RN 3-LTR或不同的变体R2 N4-LTR（RBEIII位点和NF-κ B基序的重复）相比，含有两个拷贝的RBF-2结合位点（RBEIII位点重复）和三个拷贝的NF-κ B基序的变体病毒启动子R2 N3-LTR可能显示出低水平的基因表达噪音。为了证明这一点，我们构建了一组启动子变体LTR的亚基因组病毒载体，在达特和Rev的双重控制下共表达两种报告蛋白（mScarlet和Gaussia荧光素酶）。我们建立了CEM. NKR-CCR 5细胞（CEM-CCR 5 RL报告细胞）的稳定池，并在不同的细胞活化条件下评估了报告基因的表达。R2 N3-LTR建立了严格的潜伏期，与典型的RN 3-或变体R2 N4-LTR相比，该潜伏期对有效的细胞激活剂如TNF-α或PMA的逆转，甚至对激活剂的混合物具有高度抗性。R2 N3-LTR在没有细胞活化的情况下表现出低水平的基础基因表达，其在活化时略微但显著地增强。在达特和Rev存在下，以感染性病毒的形式反式互补，R2 N3-LTR表现出与野生型病毒启动子相当的基因表达水平。R2 N3-LTR对源自代表不同遗传亚型的病毒株的达特和Rev因子有应答。由于转录噪音极低，R2 N3-LTR可以作为一个很好的模型来研究HIV-1潜伏期的建立、维持和逆转。R2 N3-LTR也将是开发高通量筛选测定以鉴定有效的潜伏期逆转剂的理想病毒启动子，因为LTR不受细胞的通常背景噪声的影响。
We observe the emergence of several promoter-variant viral strains in India during recent years. The variant viral promoters contain additional copies of transcription factor binding sites present in the viral modulatory region or enhancer, including RBEIII, LEF-1, Ap-1 and/or NF-κB. These sites are crucial for governing viral gene expression and latency. Here, we infer that one variant viral promoter R2N3-LTR containing two copies of RBF-2 binding sites (an RBEIII site duplication) and three copies of NF-κB motifs may demonstrate low levels of gene expression noise as compared to the canonical RN3-LTR or a different variant R2N4-LTR (a duplication of an RBEIII site and an NF-κB motif). To demonstrate this, we constructed a panel of sub-genomic viral vectors of promoter-variant LTRs co-expressing two reporter proteins (mScarlet and Gaussia luciferase) under the dual-control of Tat and Rev. We established stable pools of CEM.NKR-CCR5 cells (CEM-CCR5RL reporter cells) and evaluated reporter gene expression under different conditions of cell activation. The R2N3-LTR established stringent latency that was highly resistant to reversal by potent cell activators such as TNF-α or PMA, or even to a cocktail of activators, compared to the canonical RN3- or the variant R2N4-LTR. The R2N3-LTR exhibited low-level basal gene expression in the absence of cell activation that enhanced marginally but significantly when activated. In the presence of Tat and Rev, trans-complemented in the form of an infectious virus, the R2N3-LTR demonstrated gene expression at levels comparable to the wild-type viral promoter. The R2N3-LTR is responsive to Tat and Rev factors derived from viral strains representing diverse genetic subtypes. With extremely low-level transcriptional noise, the R2N3-LTR can serve as an excellent model to examine the establishment, maintenance, and reversal of HIV-1 latency. The R2N3-LTR would also be an ideal viral promoter to develop high-throughput screening assays to identify potent latency-reversing agents since the LTR is not affected by the usual background noise of the cell.
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