Scholthof K B, Scholthof H B, Jackson A O
Department of Plant Biology, University of California, Berkeley 94720, USA.
Virology. 1995 Apr 1;208(1):365-9. doi: 10.1006/viro.1995.1162.
Two open reading frames at the 5'-end of the tomato bushy stunt virus genomic RNA are predicted to encode a 33-kDa (p33) protein and its 92-kDa (p92) readthrough product. From amino acid sequence comparisons with other small single-stranded RNA viruses, these proteins resemble viral components of the replicase-transcriptase complex. To investigate the accumulation of these proteins in the infected cell, two chimeric proteins were produced that expressed either a portion of p33 or the carboxy-terminal "half" of p92 fused with glutathione S-transferase, and polyclonal ascites fluids specific to p33 or p92 were elicited in mice. As expected, the anti-p33 antibody recognized p33 and the p92 readthrough protein, but the anti-p92 antibody was specific for p92. Immunoblot analyses revealed that at an early stage of infection both proteins were associated with the membrane fractions isolated from virus-infected plants, but later in the infection, prior to collapse of the tissues, these proteins were also associated with the cytoplasmic fraction. At all time points in plants and protoplasts p33 was about 20-fold more abundant than p92. A series of mutations derived from an infectious cDNA clone demonstrated that both the p33 and the p92 proteins were required for replication in protoplasts and the ratio of the two proteins was maintained in the replication-competent mutants. The wild-type amber (UAG) and in vitro-generated ochre (UAA) readthrough codon derivatives replicated in protoplasts. However, the tyrosine mutants (UAC or UAU) that were predicted to express only p92 were not viable in protoplasts.
番茄丛矮病毒基因组RNA 5′端的两个开放阅读框预计编码一个33 kDa（p33）蛋白及其92 kDa（p92）通读产物。通过与其他小单链RNA病毒的氨基酸序列比较，这些蛋白类似于复制酶-转录酶复合体的病毒组分。为了研究这些蛋白在受感染细胞中的积累情况，制备了两种嵌合蛋白，它们分别表达与谷胱甘肽S-转移酶融合的p33的一部分或p92的羧基末端“一半”，并在小鼠体内诱导产生了针对p33或p92的多克隆腹水。正如预期的那样，抗p33抗体识别p33和p92通读蛋白，但抗p92抗体对p92具有特异性。免疫印迹分析表明，在感染早期，这两种蛋白都与从病毒感染植物中分离的膜组分相关，但在感染后期，在组织崩溃之前，这些蛋白也与细胞质组分相关。在植物和原生质体的所有时间点，p33的丰度都比p92高约20倍。一系列源自感染性cDNA克隆的突变表明，p33和p92蛋白都是原生质体复制所必需的，并且在具有复制能力的突变体中这两种蛋白的比例得以维持。野生型琥珀色（UAG）和体外产生的赭石色（UAA）通读密码子衍生物在原生质体中复制。然而，预计仅表达p92的酪氨酸突变体（UAC或UAU）在原生质体中无法存活。
