ANALYSIS OF THE PROTEOLYTIC PROCESSING AND ACTIVATION OF THE RICE TUNGRO BACILLIFORM VIRUS REVERSE-TRANSCRIPTASE

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and mem ber 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 previous ly 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 a mino acid sequences and exhibit reverse transcriptase activity. Mass s pectrometry was employed to determine the precise molecular weight of the p62 and p55 proteins and enabled determination of the C-termini fo r both proteins. ORFs encoding either p62 or p55 were constructed and expressed in insect cells using the baculoviruses 62R-BBac and 55R-BBa c, respectively. The recombinant p62R and p55R proteins were purified separately and shown to have the same enzymatic activities as previous ly 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 polyprot ein was not processed in insect cells, resulting in the accumulation o f the similar to 87-kDa mpr/RT polyprotein. This study further extends the understanding of p62 and p55 and clarifies the role of the RTBV P R in processing of the RT. (C) 1995 Academic Press, Inc.