Symons Jori, Chopra Abha, Malatinkova Eva, De Spiegelaere Ward, Leary Shay, Cooper Don, Abana Chike O, Rhodes Ajantha, Rezaei Simin D, Vandekerckhove Linos, Mallal Simon, Lewin Sharon R, Cameron Paul U
The Peter Doherty Institute for Infection and Immunity, University of Melbourne and Royal Melbourne Hospital, 792 Elizabeth St, Melbourne, VIC, 3000, Australia.
Institute for Immunology and Infectious Diseases (IIID), Murdoch University, Murdoch, WA, Australia.
Retrovirology. 2017 Jan 13;14(1):2. doi: 10.1186/s12977-016-0325-2.
Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position.
We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells.
Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.
评估潜伏感染细胞中HIV整合位点的位置和频率，可能有助于我们理解在联合抗逆转录病毒治疗期间HIV是如何持续存在的。我们开发了一种新型的高通量测序方法，用于评估潜伏感染细胞系中的HIV整合位点，以确定在这些细胞系中是否存在病毒复制或克隆扩增，这可表现为同一位置的多个整合事件。
我们修改了先前报道的使用随机DNA剪切和PCR的方法，以实现高通量机器人处理，从而确定潜伏感染细胞系中HIV整合的位点和频率。用完整病毒感染的潜伏感染细胞系显示出多个不同的HIV整合位点（每150,000个细胞中，U1细胞系有28个不同位点，ACH-2细胞系有110个，J1.1细胞系有117个）。相比之下，用无复制能力病毒感染的细胞系（J-Lat细胞）显示出单个整合位点。ACH-2细胞系在体外传代后，我们观察到独特HIV整合位点的频率显著增加，并且前病毒DNA中存在多个突变和大片段缺失。当ACH-2细胞系与整合酶抑制剂拉替拉韦一起培养时，独特HIV整合位点的数量显著减少，并且与这些细胞中的病毒复制一致的2-LTR环频率短暂增加。
用完整HIV潜伏感染的细胞系显示出多个独特的HIV整合位点，表明这些细胞系不是克隆性的，并且在ACH-2细胞系中有低水平病毒复制的证据。这些发现对于将潜伏感染细胞系用作HIV潜伏模型以及将这些细胞用作标准具有重要意义。
