Scholthof H B, Jackson A O
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843, USA.
Virology. 1997 Oct 13;237(1):56-65. doi: 10.1006/viro.1997.8754.
Tomato bushy stunt virus (TBSV) is a small isometric virus that contains a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open reading frame (ORF) of 207 nucleotides, designated pX, which resides at the 3' end of the genome. Bioassays showed that deletions or additions of nucleotides at the 5' end of the pX gene that were designed to disrupt the ORF, or site-specific inactivation of its start codon, all gave rise to TBSV mutants which were unable to accumulate to detectable levels in cucumber or Nicotiana benthamiana protoplasts. Although these results suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect. However, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumulation. Therefore, we hypothesize that cis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of the cis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mutations that prohibited accumulation of TBSV RNA in cucumber or N. benthamiana, failed to interfere substantially with replication in Chenopodium quinoa protoplasts or plants. Irrespective of the host, the cis-acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replication in trans. In addition, the pX gene was not essential for in vitro translation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequences. However, it is possible that very early in the replication cycle of genomic RNA in vivo, the pX gene cis-acting element is essential for some other unidentified function which involves interaction with one or more host components whose composition varies slightly between different plants.
番茄丛矮病毒（TBSV）是一种小型等轴病毒，其单链RNA基因组包含五个主要基因。在本研究中，我们分析了基因组3'端一个额外的207个核苷酸的小的第六开放阅读框（ORF）（命名为pX）的重要性。生物测定表明，在pX基因5'端设计的用于破坏ORF的核苷酸缺失或添加，或其起始密码子的位点特异性失活，均产生了无法在黄瓜或本氏烟草原生质体中积累到可检测水平的TBSV突变体。尽管这些结果表明假定的pX蛋白具有一定作用，但在pX基因中引入提前终止密码子并没有强烈的负面影响。然而，一个影响相同核苷酸但不改变预测氨基酸序列的类似突变极大地降低了RNA积累。因此，我们推测pX基因内的顺式作用RNA序列而非预测的蛋白质影响基因组积累。顺式作用pX ORF序列的需求似乎依赖于宿主，因为比较显示，禁止TBSV RNA在黄瓜或本氏烟草中积累的pX基因微小突变，并未对藜麦原生质体或植物中的复制产生实质性干扰。无论宿主如何，顺式作用pX基因序列对于需要辅助TBSV进行反式复制的复制酶缺陷型RNA是可有可无的。此外，pX基因对于从基因组RNA体外翻译复制酶蛋白并非必需。这些结果表明，复制酶蛋白的翻译和聚合酶活性均不需要pX基因序列。然而，在体内基因组RNA复制周期的早期阶段，pX基因顺式作用元件对于某些其他未确定的功能可能是必不可少的，这些功能涉及与一种或多种宿主成分的相互作用，而这些宿主成分在不同植物之间略有差异。
