Cis-Allosteric Regulation of HIV-1 Reverse Transcriptase by Integrase

• Published in: Viruses Vol. 15; no. 1; p. 31
• Main Authors: Masuda, Takao, Kotani, Osamu, Yokoyama, Masaru, Abe, Yuya, Kawai, Gota, Sato, Hironori
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 21.12.2022; MDPI
• Subjects: Allosteric properties; Allosteric Regulation; cell free system; Chromatography; Complementary DNA; E coli; Enzymatic activity; enzyme activity; HIV; HIV Integrase - metabolism; HIV Reverse Transcriptase - genetics; HIV Reverse Transcriptase - metabolism; HIV-1; Human immunodeficiency virus; Integrase; integrases; integration; Intermediates; Life sciences; molecular dynamics; pol; protease; proteinases; Proteins; Retroviridae; reverse transcriptase; Reverse Transcriptase Inhibitors - pharmacology; Reverse transcription; RNA-directed DNA polymerase; Simulation; United States > US; Japan; Germany; HIV-1; protease; reverse transcriptase; integration; deoxyribonucleoside triphosphates; molecular dynamics; pol; integrase
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v15010031

Reverse transcriptase (RT) and integrase (IN) are encoded tandemly in the pol genes of retroviruses. We reported recently that HIV-1 RT and IN need to be supplied as the pol precursor intermediates, in which RT and IN are in fusion form (RTIN) to exert efficient reverse transcription in the context of HIV-1 replication. The mechanism underlying RTIN’s effect, however, remains to be elucidated. In this study, we examined the effect of IN fusion on RT during reverse transcription by an in vitro cell-free assay, using recombinant HIV-1 RTIN (rRTIN). We found that, compared to recombinant RT (rRT), rRTIN generated significantly higher cDNAs under physiological concentrations of dNTPs (less than 10 μM), suggesting increased affinity of RTIN to dNTPs. Importantly, the cleavage of RTIN with HIV-1 protease reduced cDNA levels at a low dose of dNTPs. Similarly, sensitivities against RT inhibitors were significantly altered in RTIN form. Finally, analysis of molecular dynamics simulations of RT and RTIN suggested that IN can influence the structural dynamics of the RT active center and the inhibitor binding pockets in cis. Thus, we demonstrated, for the first time, the cis-allosteric regulatory roles of IN in RT structure and enzymatic activity.