HSV-2 Triggers Upregulation of MALAT1 in CD4+ T cells and Promotes HIV Latency Reversal (scRNA-Seq). HSV-2 Triggers Upregulation of MALAT1 in CD4+ T cells and Promotes HIV Latency Reversal (scRNA-Seq)

HSV-2 coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well-defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2 infected and bystander 2D10 cells. Bulk and single-cell RNA sequencing studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2-infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms including upregulation of MALAT1 to release epigenetic silencing. Overall design: CD4+ T cells were isolated by immunomagnetic negative selection, activated by anti-CD3/CD28 beads for 72 hours, infected with HSV-2(SD90), and isolated at 0 (mock), 6 and 24 hours for scRNAseq analysis.

单纯疱疹病毒2型（HSV-2）合并感染与人类免疫缺陷病毒1型（HIV-1）病毒载量升高及组织病毒库扩增相关，但其具体分子机制尚未完全阐明。HSV-2复发会导致活化CD4+ T细胞向病毒复制部位浸润，并使外周血中活化CD4+ T细胞数量增加。本研究推测HSV-2可通过调控此类细胞的相关变化，促进HIV-1的激活与复制，并在人CD4+ T细胞以及HIV-1潜伏模型2D10细胞中验证了这一假说。实验结果显示，HSV-2可促进受感染及旁观者2D10细胞的潜伏病毒激活。对活化原代人CD4+ T细胞的批量及单细胞RNA测序分析显示，在HSV-2感染细胞与旁观者细胞中，HIV-1限制因子的表达均出现下调，而可促进HIV复制的转录本（包括转移相关肺腺癌转录本1（MALAT1））的表达则显著上调。将调控转录的HSV-2编码蛋白VP16转染至2D10细胞后，可显著上调MALAT1的表达，降低组蛋白H3第27位赖氨酸三甲基化水平，并触发HIV潜伏病毒的激活。敲除2D10细胞中的MALAT1可消除其对VP16的应答，并减弱其对HSV-2感染的应答反应。上述结果表明，HSV-2可通过多种机制促进HIV-1的激活，其中包括通过上调MALAT1的表达解除表观遗传沉默。实验整体设计：采用免疫磁珠阴性分选法分离CD4+ T细胞，经抗CD3/CD28磁珠活化72小时后，以HSV-2(SD90)毒株感染细胞，并分别于0小时（mock对照）、6小时及24小时收集细胞进行单细胞RNA测序（scRNA-seq）分析。