Systems To Establish Bunyavirus Genome Replication in the Absence of Transcription
Systems To Establish Bunyavirus Genome Replication in the Absence of Transcription
复制标题
DOI：
10.1128/jvi.00371-13
复制
发表时间：
2013-07-01
影响因子：
5.4
通讯作者：
Weber, Friedemann
中科院分区：
文献类型：
--
作者：
Klemm, Carolin;Reguera, Juan;Weber, Friedemann
布尼亚病毒的L聚合酶复制和转录病毒基因组。虽然复制产物是未封顶基因组RNA的忠实副本，但转录产物包含从宿主细胞mRNAs上切割的封端5‘延伸。对于La Crosse病毒，负责裂解宿主细胞基因的核酸酶位于L蛋白的N端，活性部位由5个保守氨基酸(H34、D52、D79、D92和K94)包围两个Mn2+离子(J.Rguera，F.Weber和S.Cusack，PLoS pathog)。6：E1001101,2010)。在这里，我们介绍了反向遗传学系统和L突变体，使我们能够在没有转录的情况下研究本亚病毒的基因组复制。转录用由病毒聚合酶L、核衣壳蛋白N、负义微型基因组和抗病毒蛋白激酶R的显性-负性突变体(PKR Delta E7)组成的增强型微型基因组系统进行转录评估。5个关键氨基酸的突变使转录活性显著降低，而H34K、D79A、D92A和K94A突变株L在转录中几乎完全沉默。通过将后代微基因组包装成病毒样颗粒(VLP)来检测L突变体的复制活性。除K94A外，所有突变体L蛋白均保持完全复制活性。为了检验我们的结果的更广泛的适用性，我们将突变D79A(D111A)的同源基因引入裂谷热病毒的L序列中。至于LACV D79A，RVFV D111a不能转录，但完全具有复制活性。因此，我们获得了特异转录缺陷的LACV和RVFV L聚合酶的突变体。现在可以利用方便的反向遗传学系统在没有转录的情况下研究由布尼亚病毒聚合酶进行的基因组复制。
The L polymerase of bunyaviruses replicates and transcribes the viral genome. While replication products are faithful copies of the uncapped genomic RNA, transcription products contain capped 5' extensions which had been cleaved from host cell mRNAs. For La Crosse virus (LACV; genus Orthobunyavirus), the nuclease responsible for host cell mRNA cleavage is located at the N terminus of the L protein, with an active site of five conserved amino acids (H34, D52, D79, D92, and K94) surrounding two Mn2+ ions (J. Reguera, F. Weber, and S. Cusack, PLoS Pathog. 6:e1001101, 2010). Here, we present reverse genetics systems and L mutants enabling us to study bunyaviral genome replication in the absence of transcription. Transcription was evaluated with an enhanced minigenome system consisting of the viral polymerase L, nucleocapsid protein N, a negative-sense minigenome, and-to alleviate antiviral host responses-a dominant-negative mutant (PKR Delta E7) of the antiviral kinase protein kinase R (PKR). The transcriptional activity was strongly reduced by mutation of any of the five key amino acids, and the H34K, D79A, D92A, and K94A LACV L mutants were almost entirely silent in transcription. The replication activity of the L mutants was measured by packaging of progeny minigenomes into virus-like particles (VLPs). All mutant L proteins except K94A retained full replication activity. To test the broader applicability of our results, we introduced the homolog of mutation D79A (D111A) into the L sequence of Rift Valley fever virus (RVFV; genus Phlebovirus). As for LACV D79A, the RVFV D111A was incapable of transcription but fully active in replication. Thus, we generated mutants of LACV and RVFV L polymerases that are specifically deficient in transcription. Genome replication by bunyavirus polymerases can now be studied in the absence of transcription using convenient reverse genetics systems.