Warter Lucile, Cohen Lisette, Benureau Yann, Chavez Deborah, Yang Yan, Bodola Francis, Lemon Stanley M, Traboni Cinzia, Lanford Robert E, Martin Annette
Institut Pasteur, Unité de Génétique Moléculaire des Virus à ARN, CNRS URA 3015, Université Paris Diderot - Paris 7 EA 302, Paris, France.
PLoS One. 2009;4(2):e4419. doi: 10.1371/journal.pone.0004419. Epub 2009 Feb 10.
GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC)) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC) genome (within the 3'NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC) RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.
GB病毒B（GBV-B）与丙型肝炎病毒（HCV）密切相关，可感染小型非人类灵长类动物，因此是研究HCV的一种有价值的替代物。尽管存在显著差异，但这些病毒的5'非翻译RNA（NTR）折叠成四个相似的结构域（I-IV），其中结构域II-III-IV构成病毒内部核糖体进入位点（IRES）。我们之前报道了在结构域III（IRES的一个重要片段）中含有HCV序列的嵌合GBV-B（vGB/III(HC)）在体内的拯救。我们在此表明，在绢毛猴肝内接种合成RNA后，vGB/III(HC)基因组内鉴定出的三个突变（在3'NTR内、多聚（U）序列上游以及NS5A编码序列内）对于产生这种嵌合病毒是必要且充分的，因此显然补偿了结构域III中HCV序列的存在。为了评估这些补偿性突变的潜在机制，并确定参与基因组复制的5'NTR亚结构域是否以病毒特异性方式发挥作用，我们构建并评估了一系列嵌合亚基因组GBV-B复制子，其中各种5'NTR亚结构域被其HCV同源物取代。GBV-B 5'NTR的结构域I和II不能被HCV序列取代，这表明它们包含必需的、病毒特异性的RNA复制元件。相比之下，在细胞培养中，结构域III可以被替换，且基因组复制能力仅有最小程度的损失。在体内拯救相关嵌合病毒所需的3'NTR和NS5A突变对亚基因组GBneoD/III(HC) RNA在体外的复制没有影响。数据表明，结构域III嵌合病毒在体内的适应性取决于5'NTR、3'NTR和NS5A在病毒生命周期中基因组复制后的一个步骤（很可能是在病毒颗粒组装期间）的协同相互作用。这种机制可能对所有丙型肝炎病毒都是共同的。
