De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus
De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus
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DOI：
10.1128/jvi.74.2.851-863.2000
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发表时间：
2000-01-01
影响因子：
5.4
通讯作者：
Colonno, RJ
中科院分区：
文献类型：
--
作者：
Luo, GX;Hamatake, RK;Colonno, RJ
丙型肝炎病毒(HCV)NS5B蛋白具有依赖于RNA的RNA聚合酶(RdRp)活性，RdRp是病毒RNA基因组复制的主要功能。为了进一步鉴定RdRp的活性，从重组杆状病毒中表达了NS5B蛋白，纯化到接近均一的水平，并检测了它们在体外合成RNA的能力。因此，在一些独立的分离物中鉴定出一个高活性的NS5B RdRp(Lb-42)，它含有一个新产生的终止密码子导致的18个氨基酸的C-末端截短。截短的NS5B的RdRp活性与全长蛋白的活性相当，在Mn2+存在下的活性是在Mg2+存在下的20倍。当以384个核苷酸的RNA为模板时，Lb-42合成了两个主要的RNA产物。一种是与输入RNA模板(单体)大小相同的互补RNA，另一种是发夹状二聚体RNA，通过“复制”机制合成。大量实验证据表明，该RNA单体是由NS5B从头引发合成的，而不是通过末端转移酶活性合成的。合成RNA单体需要所有四个核苷酸。RNA单体产物被证实是从头合成RNA的结果，因为由单体RNA经RNaseH消化产生了两个预期的RNA产物。此外，在3‘端加入终止链虫草素对RNA模板进行修饰并不影响RNA单体的合成，但消除了自启动发夹二聚体RNA的合成。此外，用Lb-42 NS5B在聚(C)和聚(U)均聚物模板上合成RNA时，不需要高浓度(大于或等于50mM)的GTP和ATP的寡核苷酸引物，进一步支持从头启动机制。这些发现表明，丙型肝炎病毒NS5B能够启动RNA从头合成。
Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (lb-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by lb-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by lb-42 NS5B did not require the oligonucleotide primer at high concentrations (greater than or equal to 50 mu M) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.