Gareus R, Gigler A, Hemauer A, Leruez-Ville M, Morinet F, Wolf H, Modrow S
Institut für Medizinische Mikrobiologie und Hygiene, Universität Regensburg, Germany.
J Virol. 1998 Jan;72(1):609-16. doi: 10.1128/JVI.72.1.609-616.1998.
Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans-activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photinus pyralis). After transfection into HeLa, CEM, BJAB, and K562 cells, the p6 promoter was found to be highly active. When introduced into the erythroid cell line K562, p6-controlled transcription exceeded that of the simian virus 40 promoter-enhancer used as a control by more than 25-fold. Sequence elements relevant for promoter activity mapped to the regions from nucleotides (nt) 100 to 190 and 233 to 298. Also, the segment from nt 343 to 400 downstream of the TATA box was important for transcriptional activity in HeLa and K562 cells. By transfecting the promoter-luciferase constructs into a HeLa cell line stably carrying the viral NS1 gene under the control of an inducible promoter, transcriptional activity mediated by the p6 promoter rose significantly after induction of NS1 expression. The region from nt 100 to 160 proved to be essential for NS1-mediated transcriptional activation. Furthermore, NS1-mediated transactivation was dependent on the presence of two GC-rich elements arranged in tandem upstream of the TATA box. These data indicate that NS1-mediated p6 transactivation is dependent on a multicomponent complex combining NS1 with ATF, NF-kappaB/c-Rel, and GC-box binding cellular factors.
细小病毒B19感染与多种临床表现相关，从无症状到严重症状不等。该病毒在红系祖细胞中显示出极强的复制嗜性，这可能是由于B19病毒基因组唯一的功能性启动子（p6）的活性，它与细胞和细胞周期特异性因子以及反式激活蛋白NS1共同作用。如本文所述，来自多个B19病毒分离株的p6启动子序列被证明高度保守。此外，突变并未影响转录因子的任何潜在结合位点。仅在来自持续性感染患者或慢性关节炎患者的B19病毒分离株中鉴定出第223位碱基的一个变异。为了确定启动子活性并表征调控元件，将跨越整个p6启动子及其亚片段的序列引入到荧光素酶基因（来自萤火虫）上游的真核表达载体中。转染到HeLa、CEM、BJAB和K562细胞后，发现p6启动子具有高活性。当引入红系细胞系K562时，p6控制的转录比用作对照的猿猴病毒40启动子 - 增强子的转录高出25倍以上。与启动子活性相关的序列元件定位于核苷酸（nt）100至190以及233至298区域。此外，TATA框下游nt 343至400的片段对于HeLa和K562细胞中的转录活性也很重要。通过将启动子 - 荧光素酶构建体转染到在可诱导启动子控制下稳定携带病毒NS1基因的HeLa细胞系中，在诱导NS1表达后，p6启动子介导的转录活性显著升高。nt 100至160区域被证明对于NS1介导的转录激活至关重要。此外，NS1介导的反式激活依赖于TATA框上游串联排列的两个富含GC的元件的存在。这些数据表明，NS1介导的p6反式激活依赖于将NS1与ATF、NF-κB/c-Rel以及与GC框结合的细胞因子结合的多组分复合物。
