Gurgo Corrado, Fenizia Claudio, McKinnon Katherine, Hsia Ru-Ching, Franchini Genoveffa
Animal Models and Retroviral Vaccines Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy.
Retrovirology. 2025 Mar 17;22(1):2. doi: 10.1186/s12977-025-00658-1.
Like all retroviruses, two kinds of viral DNA are present in the nucleus of HIV-infected cells: integrated DNA and a pool of unintegrated DNA containing linear and circular forms. For the most part, it has been difficult to examine the role of the unintegrated DNA forms in the viral life cycle in the presence of the integrated form, or to distinguish the respective contributions of the two circular DNA forms in the context of the unintegrated DNA.
In our approach, we constructed a 1-LTR circular form of HIV in order to study its expression in isolation from the other forms; we derived a linear genomic HIV DNA lacking the 5'-LTR (1-LTR) from a molecular clone of HIV. This linear form is transcriptionally incompetent, but via circularization becomes a transcriptionally competent 1-LTR circle. When transfected into cells lacking CD4 where neither the spread of virus nor reinfection can occur, the linear or in vitro circularized form produces a fully infectious HIV. Virus expression is stable throughout cell division as measured on a per cell basis by flow cytometry. A progressive accumulation of copies of the circular form is observed in the presence of the cell growth inhibitor aphidicolin, suggestive of episomal amplification, for which we propose a model.
We demonstrate in this study that production of infectious virus is initiated and completed by the 1-LTR episomal form of HIV DNA in the absence of reinfection and integration. In addition, we show that the 1-LTR episomal form replicates in the absence of an origin of replication, and we propose a model for its amplification. In line with the work of others but following a different approach, we provide support for a potential role of episomal forms in HIV persistence. Our data highlight the biological complexity of HIV replication and the potential of the episomal form to contribute to the persistence of HIV.
与所有逆转录病毒一样，在感染HIV的细胞细胞核中存在两种病毒DNA：整合DNA和一组包含线性和环状形式的未整合DNA。在很大程度上，在整合形式存在的情况下，很难研究未整合DNA形式在病毒生命周期中的作用，或者在未整合DNA的背景下区分两种环状DNA形式各自的作用。
在我们的方法中，我们构建了一种HIV的1-LTR环状形式，以便独立于其他形式研究其表达；我们从HIV分子克隆中获得了一种缺乏5'-LTR（1-LTR）的线性基因组HIV DNA。这种线性形式在转录上无活性，但通过环化成为转录活性的1-LTR环。当转染到缺乏CD4的细胞中（在这些细胞中病毒既不能传播也不能再次感染）时，线性或体外环化形式产生完全有感染性的HIV。通过流式细胞术在每个细胞基础上测量，病毒表达在整个细胞分裂过程中是稳定的。在存在细胞生长抑制剂阿非迪霉素的情况下，观察到环状形式的拷贝逐渐积累，提示游离型扩增，我们为此提出了一个模型。
我们在本研究中证明，在没有再次感染和整合的情况下，感染性病毒的产生由HIV DNA的1-LTR游离型启动并完成。此外，我们表明1-LTR游离型在没有复制起点的情况下进行复制，并且我们提出了其扩增模型。与其他人的工作一致但采用了不同的方法，我们为游离型在HIV持续存在中的潜在作用提供了支持。我们的数据突出了HIV复制的生物学复杂性以及游离型对HIV持续存在的潜在贡献。
