寻找丁型肝炎病毒RNA中的核输出元件。

Searching for nuclear export elements in hepatitis D virus RNA.

作者信息

Freitas Natália, Cunha Celso

机构信息

Natália Freitas, Department of Microbiology, Molecular Genetics, and Immunology, Kansas University Medical Center, Rainbow Boulevard, Kansa, KS 66160, United States.

出版信息

World J Virol. 2013 Aug 12;2(3):123-35. doi: 10.5501/wjv.v2.i3.123.

DOI:10.5501/wjv.v2.i3.123

PMID:24255883

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3832856/

Abstract

AIM

To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export.

METHODS

We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export.

RESULTS

Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway.

CONCLUSION

A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.

摘要

目的

探寻丁型肝炎病毒（HDV）基因组和反基因组RNA中能够促进核输出的顺式元件的存在情况。

方法

我们利用了基于质粒pDM138的一个特征明确的氯霉素乙酰转移酶报告系统。将对应于不同HDV基因组和反基因组RNA序列的20个cDNA片段插入质粒pDM138中，并用于Huh7细胞的转染实验。然后通过实时聚合酶链反应和Northern印迹法测定核组分和细胞质组分中HDV RNA的相对含量。使用网络界面进一步预测显示出核输出能力的RNA序列的二级结构。最后，评估对雷帕霉素B的敏感性，以研究参与HDV RNA核输出的可能细胞途径。

结果

对基因组RNA序列的分析未能鉴定出明确的核输出元件。然而，发现两个区域能够促进报告mRNA的输出，其效率高于阴性对照，尽管低于阳性对照。这些区域分别对应于核苷酸266 - 489和584 - 920。此外，在分析反基因组RNA序列时，在位置214 - 417发现了一个核输出元件。HDV反基因组RNA的核输出元件介导的输出对雷帕霉素B敏感，表明CRM1可能在该转运途径中发挥作用。