Molecular characterization of totiviruses in Xanthophyllomyces dendrorhous.
Molecular characterization of totiviruses in Xanthophyllomyces dendrorhous.
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DOI：
10.1186/1743-422x-9-140
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发表时间：
2012-07-28
期刊：
影响因子：
4.8
通讯作者：
Cifuentes V
中科院分区：
文献类型：
--
作者：
Baeza M;Bravo N;Sanhueza M;Flores O;Villarreal P;Cifuentes V
在树状红酵母中发现了染色体外dsRNA元件，其数量和大小在不同地理来源的菌株之间有很大的差异。有关病毒样颗粒的包埋和dsRNA固化的研究表明，dsRNAs有的是辅助病毒，有的是卫星病毒。然而，这些dsRNAs的核苷酸序列和功能仍不清楚。本工作测定了华支睾吸虫UCD67-385株的4个dsRNAs的核苷酸序列，并提出了它们的同源性和基因组结构。在此基础上，对不同品系的树突圆线虫的dsRNA进行了分析。测定了树突圆线虫UCD 67-385的L1、L2、S1和S2dsRNA的全序列，发现了L1dsRNA的两个序列(L1a和L1B)。在S1和S2dsRNAs中都发现了几个ORF，但与数据库相比没有发现任何同源性。相反，在每个L1a、L1B和L2dsRNAs中发现了两个ORF，其推导的氨基酸序列与Totiviridae家族的一个主要衣壳蛋白(5‘-ORF)和一个依赖RNA的RNA聚合酶(3’-ORF)同源。这些dsRNA的基因组结构具有TotiVirus的特征，有两个重叠的ORF(−1框架中的3‘-ORF相对于5’-ORF)，在重叠的区域有一个光滑的位置和一个假结。这些结构对于通过−1核糖体框架转移合成病毒聚合酶作为与病毒衣壳蛋白的融合蛋白是必不可少的。在RNase保护分析中，所分析的四个树枝杆菌菌株中的dsRNAs都被保护而不被酶消化。RT-PCR分析表明，与菌株UCD 67-385相似，菌株VKM Y-2059、UCD 67-202和VKM Y-2786中L1A和L1B dsRNAs共存。此外，用两步RT-qPCR测定L1dsRNA的相对数量表明，S2 dsRNA固化菌株的L1a/L1B比其亲本菌株增加了40倍。从松材线虫UCD67-385菌株中分离到3种托蒂病毒，分别命名为XDV-L1A、XDV-L1B和XDV-L2。XDV-L1A和XDV-L1B病毒也在其他三个树突线虫株中发现。我们的结果表明，UCD67-385株较小的dsRNAs(命名为XdRm-S1和XdRm-S2)是卫星病毒，特别是XdRm-S2是XDV-L1a的卫星。
Occurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation in viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNAs are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67–385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed. The complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67–385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5’-ORF) and a RNA-dependent RNA polymerase (3’-ORF) belonging to the Totiviridae family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3’-ORF in the −1 frame with respect to the 5’-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through −1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67–385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67–202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain. Three totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67–385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67–385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.