Behrens S E, Grassmann C W, Thiel H J, Meyers G, Tautz N
Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität Giessen, Germany.
J Virol. 1998 Mar;72(3):2364-72. doi: 10.1128/JVI.72.3.2364-2372.1998.
As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of the Flaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5' and 3' untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3' end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5' third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5' end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.
作为确定牛病毒性腹泻病毒（BVDV，黄病毒科成员，具有正链RNA基因组）复制所需条件的初步方法，通过对多种BVDV cDNA构建体进行体外转录产生全长基因组RNA和亚基因组RNA，并将其转染到真核宿主细胞中。通过核糖核酸酶保护法直接测量RNA复制，或通过监测病毒蛋白的合成来进行测量。当最初应用全长BVDV cRNA时，在转染后于宿主细胞的细胞质中检测到负链RNA中间体以及子代正链RNA的合成。与负链RNA中间体相比，合成了过量的正链RNA。令人惊讶的是，发现一种亚基因组RNA分子DI9c，它对应于先前表征的缺陷干扰颗粒，在没有辅助病毒的情况下也能支持RNA复制的两个步骤，因此起到自主复制子的作用。DI9c包含BVDV基因组的5'和3'非翻译区以及自蛋白酶Npro和非结构蛋白NS3、NS4A、NS4B、NS5A和NS5B的编码区。最有趣的是，由此确定NS2多肽对于RNA复制是非必需的。正如预期的那样，基因组3'端的缺失以及病毒编码的丝氨酸蛋白酶催化功能的丧失导致DI9c分子无法复制。整个Npro基因的缺失也破坏了DI9c分子的复制能力。另一方面，其中Npro基因的5'三分之一与泛素基因融合的DI9c衍生物，允许NS3在反式中进行蛋白水解释放，结果证明具有复制能力。这些结果表明，位于开放阅读框5'端的RNA序列在BVDV复制过程中发挥着重要作用。发现DI9c和DI9c衍生物的复制不限于牛源宿主细胞，这表明作为病毒复制机制潜在组成部分的细胞因子在不同哺乳动物细胞之间高度保守。我们的数据为快速鉴定和表征参与瘟病毒生命周期的病毒因子和基因组元件迈出了重要一步。讨论了其对其他黄病毒科病毒，特别是与BVDV相关的人类丙型肝炎病毒的影响。
