In Vitro Synthesis of Long Reverse Transcription Products from Genomic RNA of Human Immunodeficiency Virus

Abstract

The retroviral reverse transcription reaction normally occurs in capsid-like structures in the cytoplasm of infected cells. Reverse transcription can also be carried out in vitro in totally reconstituted reactions with purified enzymes and model RNA templates. However, in this case fully synthesized DNAs are rarely generated from genomic RNA. This could be because the capsid creates an extremely concentrated and specific environment that cannot be completely reproduced in vitro. An in vitro system that closely mimics replication and that can be easily manipulated would enhance our understanding of the replication process. In this thesis report, in vitro reaction conditions that allowed efficient synthesis of DNA products up to 4 kb from genomic RNA segments of Human Immunodeficiency Virus (HIV) were generated. The reactions required high amounts of HIV reverse transcriptase enzyme (RT) and nucleocapsid protein (NC) sufficient to completely coat the RNA template in the reaction. Synthesis of long DNA products required the formation of high molecular weight aggregates with nucleic acids, RT and NC. Removal of the dimerization region did not affect synthesis of long DNA products in vitro. Processivity of RT does not play a role in the synthesis of long DNA products. NC finger mutants lacking either finger or with the finger positions switched were all effective in synthesizing long DNA products suggesting that the aggregation/condensation activity but not the unwinding activity of NC is required for the synthesis of long DNAs in vitro. These results taken together, we propose that high molecular weight aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC into a smaller area, thereby mimicking the role of the capsid environment within the host cell.

In addition, strand transfer assays indicate that strand transfer is the molecular mechanism involved in the synthesis of long DNAs and the rate of transfer (cross-over events per nucleotide synthesized) is higher than that found in tissue culture-based recombination assays. An in vitro system that closely mimics what occurs in the cell could be used to screen inhibitors on RT, NC and recombination.