Department of Plant Pathology, Ohio Agricultural Research and Development Center, The Ohio State Universitygrid.261331.4, Wooster, Ohio, USA.
J Virol. 2021 Aug 25;95(18):e0016921. doi: 10.1128/JVI.00169-21.
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 . 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 . 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. 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.
病毒来源的长非编码 RNA（lncRNA）在许多正链（+）RNA 病毒感染的细胞中积累，以增强病毒感染力。它们的生物发生主要利用宿主细胞的外切核酸酶，这些酶沿着 5' 到 3' 的方向降解病毒基因组或亚基因组 RNA，直到被明确的 RNA 结构阻止。在这里，我们报告了一种由新型复制依赖性机制产生的病毒 lncRNA。这种 lncRNA 对应于芜菁皱缩病毒（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 依赖性 RNA 聚合酶（RdRp），也称为 p88，过表达时，ttsgR 在 TCV 感染的 Nicotiana benthamiana 细胞中高水平积累。以 RdRp 功能依赖性的方式产生了（+）和（-）链形式的 ttsgR。引人注目的是，只要提供 TCV 编码的复制蛋白 p28 和 p88，即使模板短至 ttsgR 本身，也足以编程 ttsgR 扩增。与复制起源一致，ttsgR 的积累需要 5' 端 carmovirus 保守序列（CCS），这是与 TCV 在系统发育上相关的许多病毒的基因组和亚基因组 RNA 共有的序列基序。更重要的是，在 TCV 基因组的其他位置引入新的 CCS 基序足以导致另一种 lncRNA 的出现。最后，通过突变其 5' CCS 消除 ttsgR 会导致 TCV 突变体在竞争中无法与野生型 TCV 竞争。总的来说，我们的结果揭示了病毒 lncRNA 生物发生的复制依赖性机制，这表明可能有多种机制（单独或组合）负责病毒 lncRNA 的产生。许多正链（+）RNA 病毒在细胞感染过程中产生长非编码 RNA（lncRNA），并利用这些 lncRNA 来对抗抗病毒防御，以及协调病毒蛋白的翻译。大多数病毒 lncRNA 是由较长病毒 RNA 在稳定的二级结构处停滞时沿着 5' 到 3' 的降解产生的。在这里，我们报告了一种由芜菁皱缩病毒（TCV）复制机制产生的病毒 lncRNA。这种 lncRNA 被命名为 ttsgR，与 TCV 基因组和亚基因组 RNA 具有相同的末端特征，并且在适度过表达的 TCV RNA 依赖性 RNA 聚合酶（RdRp）存在下过度积累。此外，来自非病毒来源的 TCV 复制蛋白（p28 和 RdRp）很容易复制与 ttsgR 大小相似的模板。总之，本研究建立了一种揭示低丰度病毒 lncRNA 的方法，并对复制的 TCV lncRNA 进行了特征描述。对人类致病性（+）RNA 病毒的类似研究可能会产生新的治疗靶点。
