IIMCB scientists have characterized a novel mechanism of action of reverse transcriptase

Proteins are the basic building blocks of every organism. In the first step of protein synthesis, an enzyme called polymerase copies the gene's DNA sequence into RNA. The reverse process of synthesizing DNA from RNA is called reverse transcription and is used by retroviruses, e.g., HIV-1, which causes AIDS. During reverse transcription, the viral genome encoded by RNA is converted into double-stranded DNA, which is then integrated into the host genome. The enzymes that catalyze the reverse transcription process are called reverse transcriptases (RTs). They contain two domains: polymerase and RNase H. Polymerase is responsible for the synthesis of DNA on DNA or RNA template. RNase H degrades viral RNA strand of RNA / DNA intermediates.

Retroviruses are divided into two subfamilies Orthoretrovirinae, which includes HIV-1 virus and Spumaretrovirinae (Spumavirus), with simian foamy virus (SFV). Spumaviruses, also known as foamy viruses (FV), have a unique feature among retroviruses: they do not cause any disease. This makes these viruses useful for gene therapy. Moreover, they are the oldest group of retroviruses and have coexisted with their host for over 100 million years, which may have contributed to their non-pathogenicity. Foamy viral reverse transcriptases are unique because they contain an additional protease domain located at the N-terminus.

The scientists from the Laboratory of Protein Structure solved the first 3D structures of PR-RT proteins from FV. Using protein crystallography methods, two structures in which a nucleic acid is present (double-stranded DNA or RNA/DNA hybrid) have been solved. This allowed the description of the substrate binding mechanism of spumaviral reverse transcriptases. Additionally, structural and biochemical studies have shown that the protein adopts monomeric configuration in the presence of RNA/DNA hybrid, and an asymmetric dimer form in the presence of dsDNA. This is different from other reverse transcriptases characterized to date which function as dimers (i. e. HIV-1 enzyme) or monomers.

A structure of the FV PR-RT and dsDNA complex was also solved using a revolutionary technique of high-resolution cryo-electron microscopy (cryo-EM), which additionally confirmed the protein dimerization in the presence of this type of substrate. This is the first cryo-EM structure published by the scientists from the Laboratory of Protein Structure.

The work has been published in the Journal of Virology. Full article:
Nowacka M, Nowak E, Czarnocki-Cieciura M, Jackiewicz J, Skowronek K, Szczepanowski RH, Wöhrl BM, Nowotny M. 2021. Structures of substrate complexes of foamy viral protease-reverse transcriptase. J Virol 95:e00848-21.

Fig

Atomic model fitted into cryo-EM reconstruction of foamy viral PR-RT-dsDNA complex and crystals of foamy viral PR-RT bound to RNA/DNA hybrid.