Dynamic binding orientations direct activity of HIV reverse transcriptase

Abstract

The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates. The human immunodeficiency virus encodes an essential protein called reverse transcriptase, a primary drug target for the treatment of AIDS. It has two activities: synthesis of DNA using the viral RNA genome as a template, and cleavage of a DNA-RNA hybrid. Each activity requires the enzyme to bind to both DNA and RNA. But how does it know which activity to perform on which substrate? The use of single-molecule technology shows that the enzyme binds to RNA and DNA in different orientations, and can flip between these orientations and activities without parting from the substrate. The flipping dynamics are strongly regulated by non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drug, suggesting a mechanism by which these drugs inhibit viral replication. HIV-1 reverse transcriptase (RT) is an important drug target. RT has two activities, DNA synthesis, and cleavage of a DNA–RNA hybrid. These activities require that RT bind to both DNA and RNA. Single-molecule technology is used to show that the enzyme binds to RNA and DNA in different orientations, and provide insight into how it can flip between these orientations and activities without dissociating from the substrate.