Reverse transcriptase

SIGNIFICANCE: Reverse transcriptases (RTs) are enzymes that allow retroviruses and some other viruses to infect eukaryotic cells by turning their RNA genomes to DNA, enabling the host cell to use the DNA to make new virus particles. This process, known as reverse transcription, is an important part of how retroviruses operate and cause disease. Retroviral DNA, often dormant for years before new virus particles are released, can be oncogenic, giving infected cells high incidences of cancer. Purified RTs are used to make RNA into DNA for biotechnology.

Genetic Information Flow and Retroviruses

The central dogma of molecular genetics states that information flow is from DNA to RNA to proteins. RNA polymerase transcribes RNA using a DNA template. For structural genes, the transcribed RNA is a messenger RNA (mRNA), which is used by ribosomes to produce a protein. To maintain and reproduce its DNA, an organism uses RNA to make DNA, via DNA polymerase. It was long believed by geneticists that there were no exceptions to the central dogma.

However, it was discovered that some viruses, particularly retroviruses, possess RNA genomes with genetic information flow from RNA to DNA and back, before translation. Retroviruses have been isolated from cancers and cancer tissue cultures from birds, rodents, primates, and humans, and some retroviruses cause a high incidence of certain cancers. Particularly infectious retroviruses affecting humans include human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), and the leukemia-causing human T-cell lymphotrophic virus I (HTLV-I).

Flow of retroviral genetic information from RNA to DNA was proposed in 1964 by Howard Temin (1934–1994) for Rous sarcoma virus. Rous sarcoma virus causes tumors in birds. Temin’s hypothesis was based on effects of nucleic acid synthesis inhibitors on replication of the virus. First, the process was inhibited by actinomycin D, an inhibitor of DNA-dependent RNA synthesis. Furthermore, DNA synthesis inhibition by cytosine arabinoside, early after infection, stopped viral replication. Therefore, a DNA intermediate seemed involved in viral replication. The expected process was termed reverse transcription because RNA becomes DNA instead of DNA becoming RNA. Temin, along with David Baltimore, jointly received the Nobel Prize in Physiology or Medicine in 1975 for independently discovering reverse transcriptase (RT), the enzyme involved in the reverse transcription process.

RT Discovery and Properties

Retrovirus infection begins with injection of RT and single-stranded RNA into host cells. RT (an RNA-dependent DNA polymerase) causes biosynthesis of viral DNA using an RNA template from the retrovirus HIV (human immunodeficiency virus), the causative agent of acquired immunodeficiency syndrome (AIDS). RTs have been purified from many retroviruses. Avian, murine, and human RTs have been studied most. All have ribonuclease H (RNase H) activity on the same protein as polymerase activity. Ribonuclease H degrades RNA strands of DNA-RNA hybrids. A nuclease that degrades DNA is later involved in retrovirus DNA integration into host cell DNA. Most biochemical properties of purified RTs are common to them and other DNA polymerases. For example, all require the following for DNA synthesis: a primer on which synthesis begins, a template that is copied, and a supply of the four dNTPs.

An RT converts a retroviral single-stranded RNA genome to “integrated double-stranded DNA” as follows: First a hybrid (DNA-RNA) duplex is made from viral RNA, as an antiparallel DNA strand is produced. The RNA-directed DNA polymerase activity of RT is primed by host cell transfer RNA, which binds to the viral RNA. Then, the viral RNA strand is destroyed by RNase H, and the first DNA strand now becomes the template for synthesis of a second antiparallel DNA strand. Resultant duplex DNA is next integrated into a host cell chromosome, where it is immediately used to make virus particles or, alternatively, it takes up residence in the host cell’s genome, remaining unused—often for years—until it is activated and causes cancer or production of new viruses.

Importance of Reverse Transcriptases

RTs can use almost any RNA template for DNA synthesis. Low RT template specificity allows RT to be used to make DNA copies of a wide variety of RNAs in vitro. This has been very useful in molecular biology, especially in production of exact DNA copies of purified RNAs, a process known as reverse transcription-polymerase chain reaction (RT-PCR). Once the copies are made by RT, they can be cloned into bacterial expression vectors, where mass quantities of the gene product can be produced. It has also been shown that RT activity takes part in making telomeres (protective chromosome ends). Telomere formation and maintenance are essential cell processes, related to life span and deemed important to understanding cancer.

RTs are also important in treatment of acquired immunodeficiency syndrome (AIDS). The drugs most useful for AIDS treatment are RT inhibitors such as zidovudine, didanosine, zalcitabine, and stavudine. RTs are also associated with the difficulty in maintaining successful long-term AIDS treatment, due to rapid development of resistant HIV in individual AIDS patients. The resistance is postulated to be due to RT’s lack of a proofreading component. Inadequate proofreading in sequential replication of HIV viral particles from generation to generation is believed to cause the rapid mutation of the viral genome.

Scientists continue to find new ways to develop drugs and treatments for various conditions using RTs. It was estimated that in 2021, the market for RTs was $286.19 million in US currency. It is predicted that this market could reach nearly $383 million by 2030.

Key terms

• deoxyribonucleoside triphosphate (dNTP)one of four monomers (dATP, dCTP, dGTP, dTTP) incorporated into DNA
• DNA polymerasean enzyme that catalyzes the formation of a DNA strand using a template DNA or RNA molecule as a guide
• primerA short piece of single-stranded DNA that can hybridize to denatured DNA and provide a start point for extension by a DNA polymerase
• proofreading activityenzyme activity in DNA polymerase that fixes errors made in copying templates
• retrovirusesviruses that possess RNA genomes with genetic information that flows from RNA to host DNA via reverse transcriptases

Bibliography

Coffin, John M., and Hung Fan. "The Discovery of Reverse Transcriptase." Annual Review of Virology, vol. 3, September 2016, pp. 29–51. doi:10.1146/annurev-virology-110615-035556. Accessed 3 Sept. 2024.

Everts, Sarah. "Biologists Evolve a Reverse Transcriptase that Can Proofread." Chemical & Engineering News, 23 June 2016, cen.acs.org/articles/94/i26/Biologists-evolve-reverse-transcriptase-proofread.html. Accessed 3 Sept. 2024.

Goff, S. P. “Retroviral Reverse Transcriptase: Synthesis, Structure, and Function.” Journal of Acquired Immune Deficiency Diseases 3, no. 8 (1990): 817-831.

Joklik, Wolfgang K., ed. Microbiology: A Centenary Perspective. Washington, D.C.: ASM Press, 1999.

Litvack, Simon. Retroviral Reverse Transcriptases. Austin, Tex.: R. G. Landes, 1996.

Morel, Gérard, and Mireille Raccurt. “Reverse Transcription.” In PCR/RT-PCR In Situ Light and Electron Microscopy. Boca Raton, Fla.: CRC Press, 2003.

O’Connell, Joe, ed. RT-PCR Protocols. Totowa, N.J.: Humana Press, 2002.

"Reverse Transcriptase Market Report Overview." Business Research Insights, 2024, www.businessresearchinsights.com/market-reports/reverse-transcriptase-market-102319. Accessed 3 Sept. 2024.

Shippen-Lentz, D., and E. H. Blackburn. “Functional Evidence for an RNA Template in Telomerase.” Science 247, no. 4942 (February 2, 1990): 546-552.

Skowron, Gail, and Richard Ogden, eds. Reverse Transcriptase Inhibitors in HIV/AIDS Therapy. Totowa, N.J.: Humana Press, 2006.

Varmus, Harold. “Retroviruses.” Science 240, no. 4858 (June 6, 1988): 1427-1435.