Replication-Dependent Biogenesis of Turnip Crinkle Virus Long Noncoding RNAs
Replication-Dependent Biogenesis of Turnip Crinkle Virus Long Noncoding RNAs
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DOI：
10.1128/jvi.00169-21
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发表时间：
2021-05
影响因子：
5.4
通讯作者：
Shaoyan Zhang;Rong Sun;Camila Perdoncini Carvalho;Junping Han;Limin Zheng;F. Qu
中科院分区：
文献类型：
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作者：
Shaoyan Zhang;Rong Sun;Camila Perdoncini Carvalho;Junping Han;Limin Zheng;F. Qu
许多正链(+)RNA病毒在细胞感染过程中产生长的非编码RNA(LncRNAs)，并动员这些lncRNAs对抗病毒防御，以及协调病毒蛋白的翻译。大多数病毒lncRNA产生于较长的病毒RNA在稳定的二级结构上停滞不前的5‘到3’降解。摘要病毒来源的长非编码RNA(LncRNAs)在许多正链(+)RNA病毒感染的细胞中积聚，以增强病毒的感染性。它们的生物发生主要利用宿主细胞的外切核糖核酸酶，这些核糖核酸酶在5‘-3’方向降解病毒基因组或亚基因组RNA，直到被明确定义的RNA结构停滞。在这里，我们报告了一种病毒lncRNA，它是由一种新的复制依赖机制产生的。这个lncRNA对应于萝卜皱缩病毒(TCV)基因组的最后283个核苷酸，因此被命名为微小TCV亚基因组RNA(TtsgR)。当TCV编码的RNA依赖的RNA聚合酶(RdRp)，也被称为P88过表达时，TCV感染的本底烟草细胞中ttsgR积累到高水平。(+)和(−)链形式的ttsgR都是以依赖于RdRp功能的方式产生的。值得注意的是，只要TCV编码的复制蛋白P28和P88以反式形式提供，与ttsgR本身一样短的模板就足以编程ttsgR扩增。与其复制起源一致，ttsgR的积累需要一个5‘末端CarmoVirus Consensus Sequence(CCS)，这是许多与TCV有亲缘关系的病毒的基因组和亚基因组RNA共享的序列基序。更重要的是，在TCV基因组的其他地方引入一个新的CCS基序就足以导致另一个lncRNA的出现。最后，通过突变ttsgR的5‘CCS获得了一个不能与野生型TCV竞争的拟南芥TCV突变体。总之，我们的结果揭示了病毒lncRNA的生物发生依赖于复制的机制，从而表明多种机制，单独或联合，可能对病毒lncRNA的产生负责。重要性许多正链(+)RNA病毒在细胞感染过程中产生长的非编码RNA(LncRNAs)，并动员这些lncRNAs对抗病毒防御，以及协调病毒蛋白的翻译。大多数病毒lncRNA产生于较长的病毒RNA在稳定的二级结构上停滞不前的5‘到3’降解。在这里，我们报告了一种病毒lncRNA，它是由萝卜皱缩病毒(TCV)的复制机制产生的。这种被命名为ttsgR的lncRNA与TCV基因组和亚基因组RNA具有相同的末端特征，并在适度过表达的TCV RNA依赖RNA聚合酶(RdRp)的存在下过度积累。此外，与ttsgR大小相似的模板很容易被非病毒源提供的TCV复制蛋白(p28和RdRp)复制。综上所述，本研究建立了一种发现低丰度病毒lncRNA的方法，并鉴定了一种可复制的TCV lncRNA。对人类致病(+)RNA病毒的类似研究可能会产生新的治疗靶点。
Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5′-to-3′ degradation of longer viral RNAs being stalled at stable secondary structures. ABSTRACT Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5′-to-3′ direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (−) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans. Consistent with its replicational origin, ttsgR accumulation required a 5′ terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5′ CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis. Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5′-to-3′ degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets.