Deleu L, Pujol A, Faisst S, Rommelaere J
Applied Tumor Virology, Abteilung F0100 and Institut National de la Santé et de la Recherche Médicale U375, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany.
J Virol. 1999 May;73(5):3877-85. doi: 10.1128/JVI.73.5.3877-3885.1999.
Autonomous parvoviruses are tightly dependent on host cell factors for various steps of their life cycle. In particular, DNA replication and gene expression of the prototype strain of the minute virus of mice (MVMp) are closely linked to the onset of host cell DNA replication, pointing to the involvement of an S-phase-specific cellular factor(s) in parvovirus multiplication. The viral nonstructural protein NS-1 is absolutely required for parvovirus DNA replication and is able to transcriptionally regulate parvoviral and heterologous promoters. We previously showed that the promoter P4, which directs the transcription unit encoding the NS proteins, is activated at the onset of S phase. This activation is dependent on an E2F motif in the proximal region of promoter P4. An infectious MVM DNA clone was mutated in the E2F motif of P4. The wild type and the E2F mutant derivative were tested for their ability to produce progeny viruses after transfection of permissive cells. In the context of the whole MVMp genome, the E2F mutation abolished P4 induction in S phase and inactivated the infectious molecular clone, which failed to become amplified and generate progeny particles. The virus could be rescued when NS proteins were supplied in trans, showing that P4 hyperactivity in S is needed to reach a level of NS-1 expression that is sufficient to drive the viral replication cycle. These data show that E2F-mediated P4 activation at the early S phase is a limiting factor for parvovirus production. The primary barrier to parvovirus gene expression in G1 is thought to be promoter formation rather than activation, due to the poor conversion of the parental single-strand genome to a duplex form. The S dependence of P4 activation may therefore be a sign of the virus adaptation to life in the S-phase host cell. If the conversion block in G1 were to be leaky, the S induction of promoter P4 could be envisioned as a safeguard against the production of toxic NS proteins until cells reach the S phase and provide the full machinery for parvovirus replication.
自主性细小病毒在其生命周期的各个阶段都紧密依赖宿主细胞因子。特别是，小鼠微小病毒原型株（MVMp）的DNA复制和基因表达与宿主细胞DNA复制的起始密切相关，这表明一种S期特异性细胞因子参与了细小病毒的增殖。细小病毒DNA复制绝对需要病毒非结构蛋白NS-1，并且它能够转录调控细小病毒和异源启动子。我们先前表明，指导编码NS蛋白转录单元的启动子P4在S期开始时被激活。这种激活依赖于启动子P4近端区域的一个E2F基序。一个有感染性的MVM DNA克隆在P4的E2F基序中发生了突变。在转染允许细胞后，测试了野生型和E2F突变衍生物产生子代病毒的能力。在整个MVMp基因组的背景下，E2F突变消除了S期对P4的诱导，并使有感染性的分子克隆失活，该克隆无法扩增并产生子代颗粒。当通过反式提供NS蛋白时，病毒可以被拯救，这表明S期P4的高活性是达到足以驱动病毒复制周期的NS-1表达水平所必需的。这些数据表明，E2F介导的早期S期P4激活是细小病毒产生的一个限制因素。由于亲代单链基因组向双链形式的转化率低，G1期细小病毒基因表达的主要障碍被认为是启动子形成而非激活。因此，P4激活对S期的依赖性可能是病毒适应在S期宿主细胞中生存的一个标志。如果G1期的转化阻滞是渗漏性的，那么可以设想启动子P4的S期诱导是一种防止产生有毒NS蛋白的保障措施，直到细胞进入S期并提供细小病毒复制的完整机制。
