Becher P, Orlich M, Thiel H J
Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität, D-35392 Giessen, Germany.
J Virol. 1998 Nov;72(11):8697-704. doi: 10.1128/JVI.72.11.8697-8704.1998.
Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3' part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5' region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.
致细胞病变（cp）的牛病毒性腹泻病毒（BVDV）毒株CP Rit是一种广泛用于疫苗接种的温度敏感型毒株，其分子特征显示，病毒基因组RNA约15.2 kb长，比非致细胞病变（noncp）的BVDV毒株的基因组RNA长约2.9 kb。对基因组部分区域进行分子克隆和核苷酸测序后，鉴定出编码非结构蛋白NS3、NS4A和部分NS4B的基因组区域存在重复。此外，在NS3基因的第二个拷贝的直接上游发现了一个非病毒序列。该插入序列的3'部分编码一个N端截短的泛素单体。这很值得注意，因为所有已描述的带有泛素编码序列的cp BVDV毒株都至少含有一个完整的泛素单体。该非病毒序列的5'区域与迄今在cp BVDV毒株中鉴定出的细胞序列没有任何同源性。数据库搜索显示，这个第二个细胞插入片段编码核糖体蛋白S27a的一部分。进一步的分析包括对细胞重组伙伴进行分子克隆和核苷酸测序。序列比较强烈表明，CP Rit基因组中发现的S27a和泛素编码序列均源自编码结构为NH2-泛素-S27a-COOH的杂合蛋白的牛mRNA。通过瞬时表达试验研究了编码NS4B N端部分、两个细胞插入片段和NS3的基因组区域中的多蛋白加工过程。各自的分析表明，源自S27a的多肽与截短的泛素一起作为加工信号产生NS3，而单独的截短泛素则不能介导切割。由于NS3的表达与BVDV的cp表型严格相关，导致NS3表达的基因组组织改变很可能代表了CP Rit细胞病变性的遗传基础。
