Alazard-Dany Nathalie, Nicolas Armel, Ploquin Aurélie, Strasser Regina, Greco Anna, Epstein Alberto L, Fraefel Cornel, Salvetti Anna
INSERM U758, Lyon, France.
PLoS Pathog. 2009 Mar;5(3):e1000340. doi: 10.1371/journal.ppat.1000340. Epub 2009 Mar 13.
The human parvovirus Adeno-Associated Virus (AAV) type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1); whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP) complex (UL5/8/52) and the single-stranded DNA-Binding Protein (ICP8) were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42) was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1-induced AAV replication.
人细小病毒2型腺相关病毒（AAV）仅能在与辅助病毒（如腺病毒或1型单纯疱疹病毒（HSV-1））共同感染的细胞中复制；而在没有辅助病毒的情况下，它会建立潜伏感染。先前的研究表明，三元HSV-1解旋酶/引发酶（HP）复合物（UL5/8/52）和单链DNA结合蛋白（ICP8）足以在转染细胞中诱导AAV-2复制。我们独立表明，在潜伏性AAV-2感染的情况下，HSV-1 ICP0蛋白能够激活rep基因表达。本研究旨在整合这些观察结果，并进一步探索HSV-1其他蛋白在AAV早期复制步骤（即rep基因表达和AAV DNA复制）中的需求。使用模拟AAV潜伏的细胞模型和编码各种HSV-1基因集的复合构建体，我们首先证实了ICP0对rep基因表达的作用，并证明了ICP4以及程度较轻的ICP22的协同作用。相反，ICP27表现出抑制作用。其次，我们的分析表明，ICP0、ICP4和ICP22对rep基因表达的作用对于与HP复合物和ICP8一起启动AAV DNA复制至关重要。第三，也是最重要的，我们证明了HSV-1 DNA聚合酶复合物（UL30/UL42）对于在转染细胞中将AAV DNA复制提高到显著水平至关重要，并且其催化活性参与了这一过程。总之，这项工作代表了第一项全面研究，概括了HSV-1诱导的AAV复制过程中发生的一系列早期事件。
