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HIV-1 Reverse Transcriptase

Reverse Transcriptase (RT), also known as RNA-Directed DNA Polymerase, is an enzyme used by retroviruses for the catalysis of the transcription of retrovirus RNA into DNA, which is the reverse of normal cellular transcription (DNA to RNA). HIV-1 RT is the reverse transcriptase used by the Human Immunodeficiency Virus and is what makes the virus so infectious once in the body. The discovery of HIV-1 RT was a landmark discovery, and the first drugs offered to people living with HIV-1 were inhibitors of HIV-1 RT (5).

Function

HIV is a retrovirus, which means that it only carries single stranded RNA, and so relies on reverse transcription to propagate itself to new cells (1). HIV reverse transcriptase (RT) is a heterodimer consisting of a p51 and a p66 subunit, the latter of which contains catalytically active DNA polymerase and RNase H domains, essentially functioning as a catalyst for the reverse transcription reaction (2). The enzyme RT is responsible for turning the infectious single-stranded ssRNA genome into double-stranded dsDNA provirus to integrate the virus into a new host cell (3). HIV integrase then integrates it into the host chromosome. The retro transcription process begins when the HIV particle fuses with the membrane of the host due to interactions between the envelope of the glycoprotein and coreceptors on the surface of the host cell (4). The infectious content of the HIV particle is then released into the cytoplasm of the host cell. Here the ssRNA serves as a template for HIV RT to form the dsDNA provirus. The dsDNA is then imported into the nucleus, which is then integrated into the host chromosome by another enzyme, HIV integrase. Once the dsDNA is integrated into the nucleus it makes modified mRNA that code for viral proteins and new ssRNA gnomes that combine to form new viral HIV particles (3). The process then repeats as the new viral particles target uninfected host cells.

The actual process of HIV reverse transcription begins when the host cell tRNALsy3 hybridizes at the 5’-end of the (+) strand on the RNA genome. tRNALys3 is then used as a primer for the (-) strand DNA synthesis. DNA synthesis continues until the 5’ end of the RNA strand. The next step in the process involves RNase hydrolysis of the RNA/DNA hybrid to determine the (-) strand strong stop DNA by exposing the ssDNA product. The (-) strand DNA is then lengthened further and hybridized with the R region at the 3’-end of the ssRNA strand to transfer it. DNA synthesis continues when the RNase H function cleaves the RNA strand in the RNA/DNA hybrid at multiple different points, leaving two specific sections undamaged. These sections are cPPT and 3’PPt, both of which are resistant to the RNase H cleavage. DNA synthesis for the (-) strand is initiated again, only this time using the PPT strand fragments as primers. Hydrolysis of the PPT segments and the junction of the tRNA/DNA hybrid is then initiated by the RNase H, which frees up the PBS sequence of the (+) strand DNA. The PBS sequence from the (+) strand of DNA then anneals to the PBS on the (-) strand DNA. The DNA synthesis then continues, using strand displacement synthesis to get a linear dsDNA with long terminal repeats on both ends as the product (3).

Disease

Note: We plan to add a figure here from https://www.britannica.com/science/reverse-transcriptase

The HIV-1 virus infects white blood cells, or helper T cells. The results in the production of more virus, and eventually cell death and AIDS using the process of reverse transcription (5).

Relevance

The finding of HIV-1 RT was monumental for offering treatment to individuals living with HIV-1. After its discovery, the development of nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are responsible for inhibition of different mechanisms of reverse transcription (6).

"Of the 26 drugs currently approved to treat HIV-1 infections, 14 are RT inhibitors" (4).

Structural highlights

HIV reverse transcriptase is a heterodimer with chains of 66 kDA and 51 kDA. P66 chain is proteolytically processed by HIV protease to form the smaller unit of the heterodimer. HIV-1 reverse transcriptase produces a homodimer p66/p66 and a heterodimer p66/p51 when expressed in bacteria. Ribonucleases H (RNase H) are enzymes that cleavage the RNA of DNA/RNA hybrid. In reverse transcriptase the hydrolysis of an RNA strand in a DNA/RNA hybrid generates 5' phosphate and 3’ hydroxyl ends.

References

Note: We will adjust the references to go in sequential order once we are finished with references, as we plan to add more information with more references looking forward.

(1) Kati, W.M.; Johnson, K.A.; Jerver, L.F.; Anderson, K.F. Mechanism and Fidelity of HIV Reverse Transcriptase. J. Biol. Chem. 1992, 267(36), 25988-25997

(2) Abbondanzieri, E. A.; Bokinsky, G.; Rausch, J. W.; Zhang, J. X.; Le Grice, S. F.; Zhuang, X. Dynamic Binding Orientations Direct Activity of HIV Reverse Transcriptase. Nat. 2008, 453(7192), 184–189.

(3) Esposito, F.; Corona, A.; Tramontano, E. HIV-1 Reverse Transcriptase Still Remains a New Drug Target: Structure, Function, Classical Inhibitors, and New Inhibitors with Innovative Mechanisms of Actions. Mol. Biol. Int. 2012, 2012, 1–23.

(4) Hu, W. S.; Hughes, S. HIV-1 Reverse Transcription. Cold Spring Harb. Prospect. Med. 2012 2(10) doi: 10.1101/cshperspect.a006882

(5) Morier, D. Reverse transcriptase. https://www.britannica.com/science/reverse-transcriptase (accessed Nov 14, 2022).

(6) Singh, A. K.; Das, K. Insights into HIV-1 Reverse Transcriptase (RT) Inhibition and Drug Resistance from Thirty Years of Structural Studies. Viruses 2022, 14 (5), 1027.

(7) Jacobo-Molina, A.; Arnold, E. Perspectives in Biochemistry: HIV Reverse Transcriptase Structure-Function Relationships. Biochem. 1991, 30 (26), 6351-6361.