VI03CH02-Coffin ARI 10 September 2016 9:14 ANNUAL REVIEWS Further Click here to view this article's online features: • Download figures as PPT slides • Navigate linked references • Download citations The Discovery of Reverse • Explore related articles • Search keywords Transcriptase John M. Coffin1 and Hung Fan2 1Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts 02111; email: john.coffi[email protected] 2Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697 Annu. Rev. Virol. 2016. 3:29–51 Keywords First published online as a Review in Advance on retrovirus, RNA tumor virus, HIV, murine leukemia virus, Rous sarcoma July 22, 2016 virus, Howard Temin, David Baltimore The Annual Review of Virology is online at virology.annualreviews.org Abstract Access provided by Tulane University on 11/25/20. For personal use only. This article’s doi: Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org In 1970 the independent and simultaneous discovery of reverse transcriptase 10.1146/annurev-virology-110615-035556 in retroviruses (then RNA tumor viruses) by David Baltimore and Howard Copyright c 2016 by Annual Reviews. Temin revolutionized molecular biology and laid the foundations for retro- All rights reserved virology and cancer biology. In this historical review we describe the for- mulation of the controversial provirus hypothesis by Temin, which ulti- mately was proven by his discovery of reverse transcriptase in Rous sarcoma virus virions. Baltimore arrived at the same discovery through his studies on replication of RNA-containing viruses, starting with poliovirus and then moving to vesicular stomatitis virus, where he discovered a virion RNA poly- merase. Subsequent studies of reverse transcriptase led to the elucidation of the mechanism of retrovirus replication, the discovery of oncogenes, the advent of molecular cloning, the search for human cancer viruses, and the discovery and treatment of HIV/AIDS. 29 VI03CH02-Coffin ARI 10 September 2016 9:14 INTRODUCTION AND BACKGROUND The year 1970 was a tumultuous one on campuses across the United States. Protests against the Vietnam War, by then in its second decade, escalated in many ways; in Cambridge, Massachusetts, in January, protestors took over and occupied the office of the president of the Massachusetts Institute of Technology (MIT). In May of that year, the invasion of Cambodia by US forces resulted in major escalation of college protests, one of which led to the shooting deaths of four students at the hands of the National Guard at Kent State University in Ohio. In Madison, home of the University of Wisconsin (UW), student demonstrations often closed roads around the campus, and police made frequent use of tear gas. In August, Sterling Hall, situated in the heart of the UW campus and home to the Physics Department and the Army Math Research Center, was bombed by a van loaded with a mixture of ammonium nitrate and fuel oil, which killed a postdoctoral fellow who was working late. Around the same time, in the laboratories of David Baltimore at MIT and Howard Temin at UW, experiments were being done that, although simple in concept and execution, were to have a dramatic effect on embryonic areas of eukaryotic molecular biology. The simultaneous reports (1, 2) of RNA-dependent DNA polymerase—soon renamed reverse transcriptase—in the two laboratories led to rapid conceptual advances in our thinking about virus replication, the genetic basis of cancer, and mechanisms of eukaryotic gene expression. This work also provided an important tool for the development of the remarkable biotechnological advances that would have been considered science fiction at the time, but that we all take for granted today. Finally, the discovery of RT helped to galvanize public support, leading to large increases in funding for cancer research—virology in particular—which, in turn, paved the way for the discovery of new and important human pathogens, such as the human T cell leukemia viruses (HTLVs) and human immunodeficiency virus (HIV), as well as for studies that provided the first insights into fundamental mechanisms of cancer. HISTORY OF RETROVIRUSES A timeline of the discoveries discussed here is presented in the Summary Figure. The first malignancies transmissible by filtered extracts (i.e., viruses) were found in chickens—namely, avian leukosis (actually leukemia) in 1907 (3) and sarcoma in 1911 (4). Peyton Rous, who discovered Rous sarcoma virus (RSV), was awarded the Nobel Prize for this work over 50 years later. Descendants of Rous’s original virus played a key role in later Nobel Prize–winning research, including the Access provided by Tulane University on 11/25/20. For personal use only. Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org elucidation of the origin of oncogenes (5, 6) as well as the discovery of reverse transcriptase. Avian tumor viruses were widely considered to be irrelevant to human cancer until the discovery of similar viruses in mammals, including murine leukemia virus (MLV), murine sarcoma virus (MSV) (7, 8), and mouse mammary tumor virus (9), as well as sarcoma and leukemia viruses in cats (10) and other species. Although the tumor viruses of birds and mammals have similar biological properties and virion morphology, and were both once termed oncoviruses, they are not closely related to one another and are now divided into two genera: Alpharetrovirus comprises the bird viruses, and Gammaretrovirus is a widespread group of viruses found primarily in mammals (11). Until the 1960s, these viruses were primarily studied in whole animals, with disease as the endpoint. Other viruses discovered in the late nineteenth and early twentieth centuries as associated with other diseases—including neurological disorders, immunodeficiency, wasting, and anemia—were later shown to be retroviruses as well. Although limited in power, the early animal studies did yield important observations, including visualization of the virion by electron microscopy (12) and determination that the genomes of these viruses consisted of RNA (13). This finding provided 30 Coffin · Fan VI03CH02-Coffin ARI 10 September 2016 9:14 1907 Discovery of transmissible avian leukosis in chickens 1911 Discovery of transmissible Rous sarcoma virus 1936 Discovery of transmissible mammary tumor virus in mice 1957 Discovery of murine leukemia virus 1958 Development of focus-formation assay for Rous sarcoma virus (Temin & Rubin) 1959 Temin moves to the University of Wisconsin 1960 Formulation of the provirus hypothesis (Temin) 1964 Formulation of the DNA provirus hypothesis (Temin) 1967 Discovery of virion RNA polymerase in vaccinia virus 1968 Baltimore moves to the Massachusetts Institute of Technology 1968 Discovery of virion RNA polymerase in reovirus 1970 Discovery of virion RNA polymerase in vesicular stomatitis virus (Baltimore) 1970 Discovery of virion DNA polymerase in retroviruses (Baltimore; Temin & Mizutani) 1972 Reverse transcription of cellular mRNA into complementary DNA 1973 Development of plasmid DNA cloning 1976 Discovery of cellular proto-oncogenes (c-src) 1981 First clinical report of AIDS 1982 Activation of proto-oncogenes in human cancer (H-ras) 1983 Development of retroviral vectors and packaging cells 1983 Isolation of HIV-1 1986 Development of polymerase chain reactions (PCRs) 1987 Development of first HIV antiretroviral (AZT) 1993 Development of combination suppressive antiviral therapy for HIV 1996 Development of quantitative reverse transcription–polymerase chain reactions (qRT-PCRs) for HIV-1 2001 Development of oncogene-targeted drugs in cancer (trastuzumab, imatinib) 2002 First successful gene therapy with retroviral vectors in humans Access provided by Tulane University on 11/25/20. For personal use only. Summary Figure Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org Timeline depicting the major events in retrovirus history. a contrast with the many DNA-containing viruses that were discovered in the same time frame, starting with the Shope papilloma virus of rabbits in 1933 (14). An important distinction that became apparent in these early studies was the difference between two types of RNA tumor viruses (as they came to be called at the time). One type, now referred to as acute transforming viruses, was represented by the avian and murine sarcoma viruses—of which there were a number of distinct isolates by 1960. These viruses caused relatively rapid tumor formation, with tumors appearing a week or two after inoculation and death of the host following shortly thereafter. The other type, generally called leukemia viruses or nonacute viruses, took much longer (months) to cause disease, often leukemia or lymphoma, after inoculation. This difference was to become crucial in the discovery of viral and cellular oncogenes. www.annualreviews.org • The Discovery of Reverse Transcriptase 31 VI03CH02-Coffin ARI 10 September 2016 9:14 Despite the knowledge gained from animal studies, further understanding of these viruses awaited the development of cell culture models, particularly connective tissue cells (fibroblasts) derived from chicken or mouse embryos. Infection of such cultures with RSV or MSV leads to phenotypic changes reminiscent of cancer cells—change of shape, loss of contact inhibition of growth, and altered metabolism. Cell culture also allowed isolation of transformed cell clones and characterization of the viruses within them. These experiments revealed that the sarcoma