Characterization of Active Reverse Transcriptase and Nucleoprotein Complexes of the Yeast Retrotransposon Ty3 in Vitro

Gaël Cristofari,Caroline Gabus,Damien Ficheux,Marion Bona,Stuart F.J Le Grice,Jean-Luc Darlix

doi:10.1074/jbc.274.51.36643

Abstract

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.

Highlights

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for human immunodeficiency virus type 1 (HIV-1) replication and Ty3 transposition, respectively
Ty3 RT Is Active on Ty3 Nucleoprotein Complexes Formed in Vitro—We have previously shown that the addition of MuLV RT and dNTPs to Ty3 RNA:tRNA:NCp9 complexes resulted in the synthesis of strong stop cDNA (ss-cDNA), the initial product of reverse transcription
The ubiquitous nature of the RT and NC protein among retroviruses and retrotransposons such as yeast Ty3 and Drosophila Copia prompted us to analyze the reverse transcription process in Ty3 and compare it with that in HIV-1, a distantly related long terminal repeat-containing retroelement belonging to the lentivirus family [12]

Summary

Introduction

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. We show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7. HIV-1 NCp7 is a small basic protein possessing two CCHC motif zinc fingers [12,13,14] that appears to function as an RNA/DNA binding and annealing protein chaperone, promoting specific reverse transcription to generate a complete double-stranded cDNA copy with two long terminal repeats [15,16,17,18,19,20,21,22,23]. Interactions between NCp7 and RT appear to promote the recruitment of RT in these nucleoprotein complexes, resulting in the initiation of cDNA synthesis with subsequent elongation [26], corresponding to the early phases of viral DNA synthesis [3, 16]

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Results

Conclusion