Laco G S, Kent S B, Beachy R N
Division of Biology and Biomedical Sciences, Washington University, St Louis, Missouri 63110, USA.
Virology. 1995 Apr 1;208(1):207-14. doi: 10.1006/viro.1995.1144.
Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and member of the badnavirus subgroup. Open reading frame (ORF) 3 encodes the viral capsid protein, protease (PR), and reverse transcriptase (RT). A DNA fragment of ORF 3 that contains PR and RT sequences was previously expressed in insect cells to produce the PR/RT polyprotein that was processed to yield p62 and p55. p62 and p55 share common N-terminal amino acid sequences and exhibit reverse transcriptase activity. Mass spectrometry was employed to determine the precise molecular weight of the p62 and p55 proteins and enabled determination of the C-termini for both proteins. ORFs encoding either p62 or p55 were constructed and expressed in insect cells using the baculoviruses 62R-BBac and 55R-BBac, respectively. The recombinant p62R and p55R proteins were purified separately and shown to have the same enzymatic activities as previously reported for the processed p62 and p55. The putative active site of the PR was mutated (mpr), and the resulting mpr/RT ORF was expressed in insect cells using the baculovirus mpr/RT-BBac. The mpr/RT polyprotein was not processed in insect cells, resulting in the accumulation of the approximately 87-kDa mpr/RT polyprotein. This study further extends the understanding of p62 and p55 and clarifies the role of the RTBV PR in processing of the RT.
水稻东格鲁杆状病毒（RTBV）是一种植物副逆转录病毒，属于杆状DNA病毒亚组。开放阅读框（ORF）3编码病毒衣壳蛋白、蛋白酶（PR）和逆转录酶（RT）。先前在昆虫细胞中表达了包含PR和RT序列的ORF 3 DNA片段，以产生PR/RT多蛋白，该多蛋白经加工后产生p62和p55。p62和p55具有共同的N端氨基酸序列，并表现出逆转录酶活性。采用质谱法测定p62和p55蛋白的精确分子量，并确定这两种蛋白的C端。分别使用杆状病毒62R-BBac和55R-BBac构建了编码p62或p55的ORF，并在昆虫细胞中表达。重组p62R和p55R蛋白分别纯化，结果表明它们具有与先前报道的加工后的p62和p55相同的酶活性。对PR的假定活性位点进行了突变（mpr），并使用杆状病毒mpr/RT-BBac在昆虫细胞中表达了产生的mpr/RT ORF。mpr/RT多蛋白在昆虫细胞中未被加工，导致约87 kDa的mpr/RT多蛋白积累。本研究进一步扩展了对p62和p55的认识，并阐明了RTBV PR在RT加工中的作用。
