RT

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Introduction

1970 was a tumultuous year on campuses across the United States. Around the same time, experiments that were about to have a dramatic effect on the embryonic field of eukaryotic molecular biology were underway in the labs of David Baltimore at MIT and Howard Temin at UW. They also reported on the RNA-dependent DNA polymerase (soon renamed reverse transcriptase, RT), a discovery that would later influence research on viral replication, the genetic basis of cancer, and the mechanisms of eukaryotic gene expression, and it provided an indispensable tool for the development of biotechnology, which was still relatively backward at that time. Notably, the discovery of RT led to substantial funding for cancer research, especially virology, which paved the way for the discovery of new important human pathogens, such as the human T-cell leukemia viruses (HTLVs) and human immunodeficiency virus (HIV).

Figure 1. Structures of Representative Reverse Transcriptases (RTs) (Martín-Alonso, S.; et al. 2020)

History of Retroviruses

Avian leukosis (actually leukemia) was discovered in 1907, the first malignancies transmissible by filtered extracts (ie, viruses). Peyton Rous won the Nobel Prize for his work on the discovery of Rous Sarcoma Virus (RSV) in 1911. Other viruses discovered in the late 19th and early 20th centuries linked to other diseases—including neurological disorders, immunodeficiency, wasting, and anemia—turned out to be retroviruses as well. Experimental capabilities were limited at the time, but early animal studies did yield important observations, such as the visualization of virus particles by electron microscopy.

The differences between the two types of RNA tumor viruses (as they were called at the time) were gradually discovered. The first is now called acute transforming virus, represented by avian and murine sarcoma virus. These viruses trigger tumor formation in a relatively rapid time, usually killing the host after a week or two. The other, called a non-acute virus, takes longer to cause disease, usually leukemia or lymphoma.

The 1950s and 1960s were the golden age of molecular biology, during which the double helix structure of DNA was discovered and many extremely simple but powerful methods were developed. Combined with the tools of nucleic acid chemistry, these methods provide a fundamental understanding of gene structure, its regulatory mechanisms, and the means genetic information is expressed. Almost all these discoveries resulted from the studies of bacteria and led to the central dogma of molecular biology — that a cell's genetic information is encoded and stored in the form of DNA and flows irreversibly to RNA and then to proteins. Second was the fundamental unity of biology, "what’s true for E. coli is true for elephant" was the mantra at the time. But both of these beliefs, held at the time, changed suddenly in the mid-1970s.

Reverse Transcriptase in the Discovery of Oncogenes

Retroviral RT plays an important role in the discovery of oncogenes. The presence of RT in virions can be exploited to make molecular probes of virus-related sequences, which are useful for monitoring viral replication. In RT reactions (replicating native viral RNA in virions or copying mRNA with purified RT), actinomycin D does not interfere with DNA synthesis from RNA templates, but it prevents DNA replication. Therefore, in the presence of actinomycin D, the resulting product is single-stranded DNA complementary to the template RNA; when actinomycin D is absent, the final product is double-stranded DNA. Probes made in this way were used days before molecular cloning to discover the cellular origin of viral oncogenes.

Reverse Transcriptase and Telomerase

Francis Crick wrote a review at the time stating that RT's activities were not really incompatible with the central dogma. RT was initially thought to be a viral enzyme unique to retroviruses and pararetroviruses, and later reverse transcription was found to be the basis of cellular telomerase. Telomerase is a multiprotein complex responsible for maintaining chromosome ends (telomeres) by producing tandem copies of hexanucleotide repeats. The key components of telomerase are telomerase reverse transcriptase (TERT) and telomerase RNA. Telomerase generates telomeric repeats by cyclically extending the lagging strand of telomeric DNA 6 nt at a time, and TERT uses telomerase RNA as a template. Therefore, fundamental processes in cells all involve the use of RT. It is possible that cellular RTs were originally derived from viral RTs and vice versa.