The Discovery of Reverse Transcriptase

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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 formulation of the controversial provirus hypothesis by Temin, which ultimately 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 polymerase. 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.

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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

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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.

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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 viruses often consisted of mixtures of two viruses: one that was capable of infection and transformation but defective for late steps in replication, and a second one, referred to as a helper virus, that was competent for replication but had no morphological effect on infected cells. The use of cell culture also allowed preparation of relatively pure virions and biochemical characterization of their structure. In particular, the genome was found to consist of single-stranded RNA sedimenting at about 70S in sucrose gradients (15), which was reduced to about 35S by denaturing treatment, consistent with a composition of 2 or 3 (later shown to be 2) subunits of about 10 kb each. The subunits were later shown to be identical in sequence (16), making these the only known viruses with diploid genomes. The 1950s and 1960s were, to some, the golden era of molecular biology, beginning with the discovery of the double helical structure of DNA and the development of extremely simple yet powerful approaches often based on little more than counting bacterial colonies or bacteriophage plaques on nutrient agar plates (17). Combined with the tools of nucleic acid chemistry, these methods, in the right hands, led to the fundamental elucidation of the structure of genes, the mechanisms of their regulation, and the means by which genetic information is expressed, eventually in the form of structural and regulatory proteins and enzymes; virtually all of these findings resulted from studies in bacteria. This remarkable period of discovery led to two guiding principles. First was the central dogma of molecular biology—that cellular genetic information is encoded and stored in the form of DNA and flows from there to RNA and then to protein in irreversible steps (18). In particular, although there was no fundamental biochemical barrier preventing copying of RNA into DNA, the possibility was widely thought of as outlandish—if it was thought of at all. Second was the fundamental unity of biology. “What’s true for E. coli is true for the elephant” was the mantra of the time. Both of these ideas were to change suddenly in the middle of 1970.

HOWARD TEMIN’S DISCOVERY OF REVERSE TRANSCRIPTASE Howard Temin exhibited a strong interest in biology from his early days as a student, when his 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 interest was focused by summers in a special biology program at the Jackson Laboratory in Bar Harbor, Maine (19). There, following his graduation from Swarthmore College in 1955, he was a counselor for a group of students including David Baltimore, who was about to enter Swarthmore (Figure 1). Temin subsequently entered graduate school at the California Institute of Technology (Caltech) and eventually joined the laboratory of Renato Dulbecco, where he worked closely with Harry Rubin, a postdoctoral fellow in the same group. At the time, Caltech was home to some of the pioneers of molecular biology, including members of the so-called phage group, such as Max Delbruck¨ and Matthew Meselson (17). Dulbecco, who was to share the 1975 Nobel Prize with Temin and Baltimore, played a major role in developing quantitative animal virology by establishing the ﬁrst plaque assays for animal viruses such as poliovirus (20) and by establishing oncogenic transformation of tissue culture cells after infection with DNA tumor viruses (21). For DNA tumor viruses he elucidated the nature of the virus-host interaction, inferring that integration of viral DNA into the host cell genome is a mechanism to confer permanent genetic change to cells even when they are unable to support complete virus replication.

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Figure 1 The Jackson Laboratory summer camp, class of 1955. Baltimore is at the upper left and Temin at the far right. Reproduced with permission from the Jackson Laboratory Archives.

In this laboratory context, Temin realized the importance of establishing quantitative cell culture systems to assay and analyze infection by RNA tumor viruses. Building on the original observations that RSV inoculation of embryonated chicken eggs led to the formation of visible minitumors, or pocks, on internal membranes (22), and that infection of chicken embryos led to morphological transformation of chicken embryo fibroblast cultures, Temin and Rubin developed the first focus-forming assay (23) for RSV, which allowed not only accurate quantification of the virus but also isolation, growth, and characterization of single infected cells. The last papers from Temin’s predoctoral work (24) were to have a profound effect on his 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 subsequent career. In them, he reported that different RSV strains induced differing shapes— spherical (round) or elongated (fusiform) morphology—in the cells they infected and transformed. This difference was determined by the genetics of the virus, not the cell. How, he wondered in the discussion, could an RNA virus cause such a permanent change? “There are two means by which RSV could operate: either it could contribute genetic information directly to the cell to enable it to become tumorous, or it could activate a tumorous state of the cell” (24, p. 197). Although he was circumspect in his interpretation, it was clear that he favored the first explanation, and he would spend the next ten years attempting to prove it. On the strength of these studies, and following a brief postdoctoral period in the same laboratory, Temin was recruited in 1959 to the McArdle Laboratory for Cancer Research at UW, where he focused on developing and then proving the provirus hypothesis, as the idea that RSV contributes genetic information to the cell came to be known. This hypothesis met with an increas- ingly skeptical scientific audience [famously including Harry Rubin as one of the more outspoken critics (25)]. In the initial experiments, Temin used inhibitors of DNA and RNA synthesis to

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more directly probe the mechanism of virus replication. He found that actinomycin D, which intercalates into double-stranded DNA and blocks DNA-directed, but not RNA-directed, RNA synthesis, could reversibly block the production of virus RNA by previously infected cells, consistent with RNA being transcribed from DNA (26). Unexpectedly, however, it also blocked infection if added to cells immediately after the virus, an observation that we explain below. Amethopterin (methotrexate), which inhibits cellular DNA synthesis by blocking formation of thymidine, also blocked infection early in the process but had no effect on virus production by previously infected cells (27). Unfortunately, inhibitor experiments like these, though consistent with the provirus hypothesis, were far from definitive, leaving much room for alternative explanations such as indirect effects on the metabolism of the infected cell. To shore up his conclusions, Temin then turned to the recently developed technique of molecular hybridization. He found that a small fraction of 3H-labeled virion RNA (prepared by growing virus in the presence of 3H-uridine) was converted to an RNase-resistant form by annealing with DNA from RSV-infected cells, to a greater extent than it was after annealing with DNA from uninfected cells (27). On the basis of the formation of more RNase-resistant counts in the reactions with infected than with uninfected cells, Temin proposed the provirus hypothesis in its final form: that the single-stranded viral RNA was somehow copied into DNA early after infection, and that this DNA was subsequently covalently joined (integrated) into the cell’s genomic DNA, where it served as the template for viral RNA synthesis. Unfortunately, again, although Temin stood steadfastly by his ideas, these experiments failed to convince many others. The amount of radioactivity detectable in the positive experiments was generally around 1% of input, or on the order of 10 cpm—too small to change anyone’s mind— and there was a detectable background hybridization in reactions with uninfected cell DNA, later shown to be due to the presence of closely related inherited (endogenous) proviruses found in nearly all chickens (see below) (28). These results were also greeted with (more or less) polite silence. Indeed, an informal poll in the late 1960s revealed that only about half of Temin’s own laboratory was convinced of the correctness of the idea. Discouraged but not defeated, Temin turned much of his attention to other types of studies, such as experiments to understand the reason for the different growth properties of RSV- transformed and normal cells (29). Several studies performed in the late 1960s, however, provided strong additional support for the provirus hypothesis. In the first, Jan Svoboda, working in Prague under the rather stressful conditions of the Soviet occupation, had been studying tumors induced in rats by a completely replication-competent strain of RSV. He found that a particular tumor cell line, called XC, could be carried for many generations from animal to animal as a tumor or in cell 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 culture without producing virus. However, injection of XC cells into chicks, or cocultivation with chick embryo cell cultures—particularly in the presence of inactivated Sendai virus, which effi- ciently mediates cell-cell fusion—led to the production of large amounts of infectious virus (30). This result strongly implied that the XC cells carried the virus information in a stably inherited form (e.g., DNA integrated into that of the host), even though the cells could not produce new virions in the absence of some chicken cell–specific factor. The second experiment, performed by David Boettiger, a graduate student in the Temin laboratory (31), took advantage of the fact that the thymidine analog BrdU at low concentrations has little effect on cells, but BrdU-containing DNA is much more sensitive to damage by exposure to visible light than is unmodified DNA. When nondividing (stationary) cells, which are not synthesizing DNA and are insensitive to BrdU, were treated with the analog immediately after RSV infection, focus formation declined strongly as a function of the time of exposure. This result was the most compelling evidence to date for the copying of viral genetic information into DNA. (Temin presented this experiment at a Gordon Conference in the summer of 1969, and

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one person in the audience who found the result convincing was David Baltimore.) The resulting paper was submitted to Nature in March of 1970, but unfortunately, the reviewers were skeptical. It did not appear until November of that year, five months after the reverse transcriptase papers, after everyone was already convinced. Finally, experiments performed by Satoshi Mizutani, a newly recruited postdoctoral fellow, showed that, at early times, RSV infection was insensitive to treatment with cycloheximide, an inhibitor of protein synthesis, implying that the necessary enzyme was already present in the cells or virions and did not need to be synthesized by translation of incoming RNA (32). In early 1970, prompted by these findings as well as observations of polymerases in virions of poxviruses (33), reoviruses (34, 35), and vesicular stomatitis virus (VSV) (36; this report came from Baltimore and colleagues), Temin arrived at the same insight: that the enzyme necessary for RSV DNA synthesis might not be present in cells prior to the time of infection, but rather, it might pre- exist in the virions ready to do the deed as soon as the virus entered the cell. Fortunately, Mizutani was well trained in the enzymology of nucleic acid polymerases, so once the experiment was con- ceived, it was quite easy to carry it out given a source of purified virus, which was easily prepared in the laboratory. In the basic experiment (Figure 2), RSV virions were incubated with a nonionic detergent (Nonidet P-40, borrowed from one of the present authors), salts (Mg2+ and NaCl), and deoxynucleotide triphosphates, one of which was radioactively labeled, all in a suitable buffer. Syn- thesis of DNA was then monitored by incorporation of the label into trichloroacetic acid–insoluble material (2). That the product was DNA was shown by its insensitivity to ribonuclease or NaOH treatment. That the template was RNA was shown by the sensitivity of the reaction to pretreat- ment with RNase but not DNase. As Temin and Mizutani stated in their Nature paper, the results of these simple experiments “would have strong implications for theories of viral carcinogenesis and, possibly, for theories of information transfer in other biological systems” (2, p. 1213). Temin presented these results for the first time at the Tenth International Cancer Congress, held in Houston in May 1970 (32). To Temin’s great delight, Harry Rubin spoke first in the same session (25), presenting a long list of arguments against the provirus theory, which Temin proceeded to demolish in his presentation a few minutes later. The presentation, however, did not win instant acceptance; an editorial writer in Nature called it “tantalizingly incomplete” (37, p. 1003). Upon returning to Madison, Temin received a call from Baltimore with the news of his independent discovery of the same enzyme in MLV virions. Even worse news, Baltimore had already submitted his paper to Nature. Temin was moving more deliberately, but he picked up speed following this conversation, and the two papers were published back to back in the June 27, 1970, issue of Nature (1, 2). Remarkably, the Temin & Mizutani paper (submitted with authorship 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 as Mizutani & Temin, but the journal reversed the order) was received at the journal on June 15, probably a record speed from receipt to publication [Watson & Crick’s (38) paper on the structure of DNA took twenty-three days]. Indeed, the proofs of the paper did not arrive in Temin’s office until after the journal itself, allowing a few typographical errors to go uncorrected. The Temin laboratory continued working on the properties of enzymes in RSV virions, dis- covering a DNA-dependent DNA polymerase activity in addition to the RNA-dependent one, now known to be an additional activity of RT itself (39), and a DNA endonuclease activity, most likely the viral integrase (40). However, as discussed below, the work of his laboratory was very quickly overshadowed by that of many other scientists, who, despite being more accomplished biochemists and having had an interest in retrovirus replication, had not thought to do the simple experiment shown in Figure 2. As noted in a Nature editorial three months later, “In the brief time that has elapsed since Temin announced in June that RNA tumor viruses contain an activity which requires RNA and all four nucleoside triphosphates to synthesize DNA, a flood of research work- ers have cast covetous eyes on the pickings to be gleaned from Teminism” (41, p. 998). Temin’s

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Baltimore, 1970

Temin and Mizutani, 1970

Figure 2 The key experiments from Baltimore (top) (1) and Temin & Mizutani (bottom) (2). Reprinted from the original papers, with permission. 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 position as a founder of molecular retrovirology—and indeed, it can be argued, of molecular cancer research—was secure.

DISCOVERY OF REVERSE TRANSCRIPTASE BY DAVID BALTIMORE David Baltimore’s interest in animal virology began in graduate school. He transferred from MIT to the Rockefeller University to study under Richard Franklin. Franklin was a pioneer in investigating genome replication of picornaviruses—small, positive-sense RNA viruses, including poliovirus and mengovirus. These viruses were amenable to study in the era before molecular cloning because they replicate in the cytoplasm and induce shutoff of host protein and RNA synthesis. Their RNA synthesis is also unaffected by actinomycin D, allowing easy experimental shutoff of host RNA synthesis and detection of viral RNA synthesis early after infection. Viral RNA and protein synthesis could be studied by concentrating on cytoplasmic fractions and pulse- labeling with radioactive precursors for RNAs or proteins. Baltimore identiﬁed cytoplasmic RNA

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polymerase activity in virus-infected cells (42), and he extended this work as a postdoctoral fellow in the laboratory of James Darnell at MIT, where he demonstrated that the RNA polymerase activity could be detected in in vitro reactions (43). He also did postdoctoral work with Jerome Hurwitz at the Albert Einstein College of Medicine, where he honed his skills in biochemistry and enzymology. In 1965 Baltimore first established his independent laboratory at the Salk Institute, where he continued his work on poliovirus, also in association with Renato Dulbecco. Because picornaviruses have single-stranded positive-sense (relative to viral mRNA) RNA genomes in virions, genome replication in infected cells would require synthesis of complementary negative- sense RNA (44) and then asymmetric synthesis of progeny positive-sense viral RNA from either negative-sense RNA or a double-stranded RNA intermediate. He and postdoctoral fellow Marc Girard identified the key replication structures, the double-stranded replicative intermediate (RI) and the more complex replicative form (RF) (44, 45). While at the Salk Institute, Baltimore and graduate student Michael Jacobson also studied synthesis of poliovirus proteins, work that continued after his move to MIT in 1968. In a landmark paper, they showed that poliovirus proteins are initially translated as a large polypeptide chain, which is then proteolytically cleaved into the mature viral proteins (46). Synthesis and processing of viral proteins from polyprotein precursors is a common feature of many animal viruses, including retroviruses. At MIT, Baltimore expanded his research interests to another viral system, VSV. This shift was facilitated by his associate (and future wife) Alice Huang, who had worked with this virus as a graduate student. VSV is a rhabdovirus—an enveloped, negative-sense RNA virus (meaning that the genome is complementary to the viral mRNA and cannot be directly translated into protein)—in the same genus as the human pathogen rabies virus. Given the negative-sense nature of the VSV genome (47), it was clear that initial VSV infection would involve steps different from those of picornavirus infection, for which the initial event is translation of the infecting virion RNA into a viral polyprotein. Synthesis of positive-sense VSV mRNA from the incoming virion genomic RNA would be required before expression of viral proteins could take place. In the mid- to late 1960s, several animal viruses had been shown to carry nucleic acid polymerizing enzymes in their virions: poxviruses [e.g., vaccinia virus, with a double-stranded DNA genome and RNA polymerase in the virion (33, 48), and reoviruses, with a double-stranded RNA genome and RNA polymerase in the virion (34, 35)]. Baltimore, Huang, and graduate student Martha Stampfer (36) extended this reasoning and asked whether VSV might contain a virion RNA polymerase that could synthesize complementary mRNA from the viral genome. They confirmed this hypothesis by showing that purified VSV virions have a polymerase activity that incorporates ribonucleotides (an RNA polymerase, now called transcriptase), that RNA synthesis is inhibited by 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 addition of ribonuclease to the reaction (RNA-dependent RNA polymerase), and that the resulting RNA product is complementary to the viral genome. Operationally, the basic experiment was simple: incubating purified virions with ribonucleoside triphosphates (one labeled) and testing for appearance of high molecular weight (acid insoluble) radioactivity. This work was accepted by PNAS in March, 1970, and was published in early June. The discovery of the VSV transcriptase raised the possibility that other RNA-containing viruses may also carry nucleic acid polymerases in their virions. Baltimore next turned his attention to RNA tumor viruses (as they were then designated), which were known to be enveloped single- stranded RNA viruses. He obtained a preparation of Rauscher MLV from a contract supplier to the National Cancer Institute and RSV from Peter Vogt. Although he was aware of Temin’s provirus model, he initially tested whether these viruses might have an RNA polymerase, in light of his experience with VSV and poliovirus as well as a report describing RNA complementary to virion RNA in cells infected with an MLV-MSV complex (49). He employed an approach similar to the one he used for detecting the RNA polymerase in VSV, but the results were negative. However,

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when he tested whether RNA tumor viruses might contain a DNA polymerase (by switching the ribonucleoside triphosphates in the reaction for deoxyribonucleoside triphosphates), the Rauscher MLV preparation showed strong evidence for DNA synthesis, as published in the landmark paper on June 27, 1970 (1) and as shown in Figure 2. In that report [which appeared simultaneously with Temin & Mizutani’s (2)], further characterization of the reaction indicated that the product was DNA, that RNA was the template, and that the enzyme would not make RNA. This result strongly supported Temin’s provirus model. Baltimore initially did not detect DNA polymerase activity in the RSV preparation, but subsequently he was successful, as reported in the initial publication. One difference between Baltimore’s report and Temin & Mizutani’s report was that the latter investigators showed that detection of DNA polymerase activity in RSV virions was greatly enhanced by addition of nonionic detergent to disrupt the viral envelope, whereas Baltimore did not include detergent in his reactions. In retrospect it is likely that Baltimore’s ready detection of DNA polymerase activity in the Rauscher MLV may have reflected partial disruption of the envelopes in the preparation used; subsequent experiments with various MLVs have confirmed that detection of RT activity requires permeabilization of the viral envelope with detergents or other means; a later paper showed a strong dependence of DNA synthesis on detergent for another MLV preparation (50). Baltimore made the first presentation of his results at the 1970 Cold Spring Harbor Sympo- sium on Transcription in early June of 1970. At that time, as mentioned above, he was aware of the results of Temin and Mizutani, who did not attend that conference but submitted a paper to it (51). The independent findings of RNA-dependent DNA polymerase activity in RNA tumor viruses [later dubbed reverse transcriptase (RT) by an editorial writer in Nature (52)] from two laboratories reinforced each other and electrified the fields of virology and cancer biology. In short order other investigators showed that the DNA product of the in vitro RT reaction is complementary to the viral RNA (53), that DNA-dependent DNA polymerase activity is also present in retroviral virions (54, 55), and that exogenous RNA templates [poly(A) or poly(C)] can be copied into their complementary DNAs [i.e., poly(T) or poly(dG), respectively] when added to the in vitro reactions (56). Baltimore and his coworkers (notably postdoctoral fellow Inder Verma) went on to establish that RT requires an oligonucleotide primer to initiate DNA synthesis (57), and that the primer for retroviral reverse transcription is an RNA (58) (see below for the details of retroviral DNA synthesis). The Baltimore group purified the RT enzymes from avian and murine retroviruses, and they showed that RTs from certain RSV mutants that were temperature sensitive for replication were thermolabile in vitro, establishing that retroviruses encode their RTs (59). Finally, they demonstrated that purified RT could use cellular mRNA (globin mRNA) as a template to synthesize complementary DNA (cDNA) (60), an important practical tool funda- 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 mental to much of molecular cell biology and biotechnology. One of us (H.F.) participated in that work.

FOLLOW-UP TO THE DISCOVERY The reaction to the reports of the discovery of RT was instantaneous. There is a story that one well-known scientist in the audience for Baltimore’s Cold Spring Harbor talk left immediately to go back to his laboratory and shortly returned to the meeting to report that he had reproduced the experiment. The original Nature papers were accompanied by a News and Views piece with the breathless headline “Central Dogma Reversed.” In short order, follow-up studies from all over the world reproduced and extended the results, identifying the enzyme in all retroviruses tested, characterizing its capacity to copy both natural RNA and DNA templates, identifying the optimal reaction conditions, and so on, such that another Nature piece, entitled “Apres` Temin, le Deluge”´ (41), appeared three months later, and still another, “Deluge Unabated” (61), a few

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months after that. Over the course of the next few years, a tremendous number of papers on the subject appeared, many in the pages of Nature; the level of familiarity was such that Nature’s editorial writer referred to scientists who studied RT as “Teminists.” The discovery also received considerable attention in the popular press. For example, in 1971, feature articles showcasing Temin and Baltimore appeared in widely circulated magazines, including Der Spiegel in Germany and Newsweek in the United States, with Temin on the cover of the latter. There are several factors that worked together to bring on the deluge. First, the key experiment was simple to reproduce. Any laboratory with access to a stock of virus could do so in a matter of days or less, and most did so. Second, the discovery of RT opened a large new field for investigation, one with the promise to lead to real molecular insight into the fundamental mechanisms of replication of a large group of viruses, and into cancer, a field that was pretty much stalled at the time. Third, the finding came at a key time in cancer research, breathing new life into the National Cancer Institute’s Special Virus Cancer Program, started about ten years previously (62). Fourth, it provided a valuable new tool for molecular biologists desiring to copy RNA into DNA in the laboratory. Fifth, it provided a valuable new tool for virologists as well, both to assay and monitor the growth of retroviruses, most of which have no visible effect on cells in culture, and to detect and identify new viruses, including ones that might be associated with human cancers. Finally, there was the sheer drama of an insightful scientist with a brilliant and revolutionary idea, convinced of its correctness, who labored for ten years publishing papers with data to support it, which, while not incorrect, convinced none of his peers, who either ignored him or labeled him a heretic (and worse) until he found the clincher experiment that changed everyone’s mind overnight. The fact that this experiment was performed and published at the same time by two independent groups only heightened the contrast, leaving no room for doubters. It came as no surprise that Temin and Baltimore, along with Renato Dulbecco, mentor to them both, were awarded the 1975 Nobel Prize in Physiology or Medicine, a remarkably short time after the original discovery.

REVERSE TRANSCRIPTION AND RETROVIRAL REPLICATION The enzymatic activity of RT in virions made it possible to elucidate the mechanism of viral DNA synthesis (reverse transcription), the key ﬁrst step in retrovirus replication. Younger readers might be surprised to learn that the whole process shown in Figure 3 was worked out (correctly) before molecular cloning and DNA sequencing were available. The model was developed based on a set of observations that accumulated within a few months or years of the initial discovery (63): 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 1. RT can copy both RNA and DNA templates. 2. To do so, like all DNA polymerases, it requires a primer, a complementary nucleic acid sequence base-paired to the template, which provides a free 3-OH end to which nucleotides are added. 3. The natural primer for the synthesis of the DNA strand complementary to the genome RNA (the negative strand) is a molecule of host cell tRNA associated with the genome by base-pairing of its 3-terminal 18 nt to a complementary sequence (PB) near its 5 end. 4. The genome RNA has a short sequence, R, duplicated at the 5 end and near the 3 end adjacent to a poly(A) sequence. A sequence called U5 lies between R and PB, and a sequence called U3 sits adjacent to the 3 R region. 5. RT has another enzyme activity, RNase H, which cleaves RNA only when it exists in an RNA-DNA hybrid. 6. A second primer, a short polypurine tract (PPT) near the 3 end of the genome, is created by RNase H cleavage and used to initiate positive-strand DNA synthesis.

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tRNA 5' AAA 3' RU5 PBS PPT U3 R

DNA synthesis U5 AAA RU5 PBS PPT U3 R

RNase H R U5 Minus-strand AAA strong-stop DNA PBS PPT U3 R

First strand transfer R U5 AAA PBSPPT U3 R

DNA synthesis RNase H PPT U3 R U5

PBS PPT

DNA synthesis RNase H PBS PPT U3R U5

PBS PPT U3 R U5

RNase H

PBS PPT U3R U5 Plus-strand U3 R U5 PBS strong-stop DNA Second strand transfer

U5 PBS

5 P U BS 3 R U 3 U 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

DNA synthesis U3R U5 PPT U3R U5

U3R U5 PBS PPT U3 R U5

LTR LTR

Figure 3 The mechanism of reverse transcription. Adapted from Reference 63, with permission from Cold Spring Harbor Laboratory Press. Abbreviations: LTR, long terminal repeat; PBS, primer binding site; PPT, polypurine tract.

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7. Completion of synthesis of both strands leads to a full-length double-stranded copy of the RNA genome, with some additional sequence (U3-R-U5), known as the long terminal repeat (LTR), at either end. The linear double-stranded DNA is then transferred to the cell nucleus, where the ends of the LTR are covalently joined to cellular DNA at one of a very large number of possible sites. This reaction is carried out by another enzyme, integrase, which is also present in the virion (64). The integrated DNA molecule (only now termed the provirus) serves as the template for synthesis of viral RNA identical to the genome by host RNA pol II, directed by host transcription factors that bind to control sequences present in the upstream LTR (65). The viral RNA is then processed by the normal machinery of the cell and exported to the cytoplasm, where some (both spliced and unspliced) is used as mRNA to direct the synthesis of viral proteins, which then assemble with the unspliced genomic RNA and bud from the cell as virions. Thus, the formation of the LTR neatly solves two problems: how to replicate the ends of a DNA molecule with a primer-requiring enzyme and how to provide transcriptional regulatory sequences for the use of cellular RNA polymerase when those signals lie in DNA sequences upstream of the region to be copied. Once an integrated provirus is formed, there is no mechanism for its excision from cellular DNA (although it can be reduced to a solo LTR by homologous recombination between the two ends), and it remains for the lifetime of the cell, stably inherited as part of its genetic composition. An important consequence of this process is the occasional infection of a germ-line cell, leading to an endogenous provirus (see below).

CONTRIBUTIONS OF REVERSE TRANSCRIPTASE TO VIRAL, CELL, AND MOLECULAR BIOLOGY

Study of Cellular mRNAs Puriﬁed RT [initially from avian myeloblastosis virus (AMV), which can be recovered in high quantities from the blood of leukemic infected chickens] can be used to reverse transcribe cellular mRNA in vitro (60, 66, 67). In the original experiments, globin RNA from rabbit reticulocytes was used, because, before the development of molecular cloning, reticulocytes afforded a system whereby a cellular mRNA could be puriﬁed. Reticulocytes are enucleated precursors to red blood cells that synthesize mainly globin (the protein component of hemoglobin), and globin mRNA from reticulocytes can be further enriched by size selection (10S in sedimentation) (60, 66, 67). It had been relatively recently discovered that mRNAs have poly(A) tracts (68), and in vitro incubation of rabbit reticulocyte 10S mRNA with an oligo(dT) primer, AMV RT, and deoxynucleoside 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 triphosphates led to synthesis of cDNA complementary to the globin mRNA. An immediate application of this technique was to use radioactive globin cDNA to study globin RNA in cells by molecular hybridization (69), an important advance in the era before molecular cloning. This approach was extended to other biological systems in which cellular mRNAs could be enriched (70); once an mRNA was enriched, a radioactive cDNA could be generated to study that RNA in any cell or tissue.

Reverse Transcriptase and Molecular Cloning The development of recombinant DNA cloning revolutionized cell and molecular biology, and RT played a key role in this revolution. RT provided a means to generate double-stranded DNA from an RNA, the ﬁrst step in molecular cloning of speciﬁc cellular mRNAs. RNA preparations enriched for a particular mRNA can be reverse transcribed in vitro into double-stranded DNA, typically using an oligo(dT) primer to pair with the poly(A) tail on the mRNA. The resulting DNAs

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can then be ligated into a plasmid to give cDNA clones, and individual cDNA clones can be tested by DNA sequencing or other techniques to identify those corresponding to the gene of interest. These cDNA clones can then be used for multiple purposes. First, they can be radioactively labeled and used as hybridization probes to characterize specific mRNAs (and their unspliced precursors) in cells. Second, cellular cDNAs are also of central importance for gene cloning. An mRNA- specific radioactive cDNA can be used as a hybridization probe to screen recombinant (e.g., λ phage) libraries of cellular DNA for genomic clones containing sequences of the cDNA. Iteration of this approach (using labeled sequences from the resulting genomic clones to rescreen the library) allows stepwise cloning of an entire gene, including exons, introns, and upstream and downstream nontranscribed sequences. Third, cDNA clones comprising the entire protein-coding sequence of an mRNA can be inserted into expression vectors for production of the pure target protein in bacteria or eukaryotic cells. These expressed cDNAs have a range of applications; they can be used to produce large quantities of pure proteins (e.g., enzymes, including RT itself ), as immunogens in the generation of specific antibodies, or to produce medically important bioproducts such as hormones or growth factors.

Reverse Transcriptase in the Discovery of Oncogenes Retroviral RT also played a major role in discovery of oncogenes—cellular genes associated with cancer. The presence of RT in virions allowed the preparation of molecular probes for virus- related sequences, which were highly useful both to monitor virus replication and to search for related viruses and genes in cells in the days before molecular cloning. A useful technical fact is that in RT reactions (either copying native viral RNA in virions or copying mRNAs with purified RT), actinomycin D does not interfere with DNA synthesis from an RNA template, but it does block DNA synthesis from a DNA template (explaining the early block to RSV replication discussed above). In the absence of actinomycin D, the ultimate product is double-stranded DNA, whereas in its presence, the product is a single-stranded DNA complementary to the template RNA (71). Probes made in this way were used to discover the cellular origin of viral oncogenes (72) in the days before molecular cloning. The seminal experiments involved studies of RSV. Prior studies identified transformation- defective (td ) variants of RSV that arose spontaneously by deletion of particular RNA sequences (73). The loss of these sequences resulted in the loss of the capacity of the td variants to morpho- logically transform cells in culture or cause rapid cancers in infected chickens. This gave rise to the concept of viral oncogenes—sequences responsible for the capacity of the virus to rapidly cause 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 cancers. For RSV, the transforming oncogene was named v-src. Michael Bishop, Harold Varmus, and their colleagues generated radioactive cDNA complementary to RSV via the endogenous RT reaction in RSV virions. They then hybridized the radioactive RSV cDNA with an excess of RNA from td RSV and removed the hybridized cDNA (74). The remaining unhybridized radioactive cDNA was therefore specific for the v-src sequences. When v-src cDNA was hybridized with genomic DNA from uninfected chicken cells, strong hybridization was observed. Hybridization to DNA from other eukaryotic species also occurred, with the extent of hybridization related to the evolutionary distance from chickens (72). These results indicated that the v-src gene of RSV was originally captured from a normal cellular gene—a proto-oncogene termed c-src. The concept that viral oncogenes of acute transforming retroviruses such as RSV are derived from normal cellular proto-oncogenes was expanded and further developed by vigorous experi- mentation on various viruses by many research groups during the 1980s. These studies led to a second deluge—this time of cellular proto-oncogenes, including many that have risen to promi- nence in cell and developmental biology as well as cancer biology, such as H-ras, K-ras, c-myc, c-abl,

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c-raf, c-fos,andc-jun (75). The importance of proto-oncogenes was further highlighted by discoveries that nonviral cancers frequently have genetic alterations in proto-oncogenes that activate them and drive oncogenesis—for example, mutations in H-ras or K-ras (76), gene ampliﬁcation of c-myc (77), and chromosomal translocation/activation involving c-abl (78). Knowledge of these mutations has provided great insights into the molecular and biochemical abnormalities in cancer cells, and in some cases the mutated proto-oncogenes have been successfully targeted to yield therapeutic drugs for speciﬁc cancers (79, 80). The discovery of cellular proto-oncogenes was recognized by award of the Nobel Prize in Medicine or Physiology to Bishop and Varmus in 1989.

Reverse Transcriptase and Endogenous Retroviruses The availability of radioactive retroviral cDNAs as hybridization probes in the era before molecular cloning provided important insights into endogenous retroviruses. Endogenous retroviruses result from rare retroviral infection of germ-line cells, leading to integration of the viral DNA into host chromosomal DNA. Once integrated, the proviral DNA is inherited by progeny resulting from that germ cell, and the DNA is passed in a Mendelian fashion to further generations; these vertically transmitted proviral DNAs are referred to as endogenous retroviruses. Prior to the discovery of RT, there already was evidence of retroviral genetic information in the genomes of uninfected individuals. For instance, some but not all uninfected chickens were known to ex- press proteins antigenically and functionally related to viral proteins of RSV and related viruses, and in genetic crosses, the expression of the viral proteins had a pattern of Mendelian inher- itance (81). With the discovery of RT, radioactive retroviral cDNA probes could be used for sensitive detection and analysis of endogenous retroviruses related to the infectious (exogenous) retrovirus used to generate the probe. When a cDNA probe for MLV was hybridized with uninfected mouse cell DNA, the kinetics of hybridization indicated that there were multiple (∼50) copies of endogenous MLV-related proviruses (82). The endogenous MLVs in the mouse genome were subsequently confirmed by Southern blot hybridizations employing radioactive MLV cDNA probes (83). These endogenous retroviruses complicated molecular cloning of exogenous retroviral proviruses from infected cells. Screening of λ phage libraries from infected cell DNA with most retroviral cDNA probes identified clones containing endogenous retroviral DNA; these clones outnumbered those containing the desired exogenous provirus. With time (and the advent of molecular cloning), investigators developed molecular techniques that allowed classification and mapping of endogenous retroviruses (84). Ultimately, whole-genome sequencing allowed identification of endogenous retroviruses based on sequence homologies with known retroviruses. 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 This work has revealed the startling fact that most eukaryotes carry large numbers of endogenous retroviruses in their genomes. For instance, ∼8% of the human genome is made up of endogenous retroviral DNA—more than the DNA that encodes proteins (85). The human endogenous retroviruses reflect invasion into the human genome (or those of ancestral species) of multiple different retroviruses over long evolutionary scales (tens of millions of years). The human genome does not harbor any known endogenous proviruses that are infectious, but the genomes of other animals, such as mice and chickens, do. In addition to endogenous retroviruses, other genetic elements with some similarities to endogenous retroviruses are present in the genomes of many eukaryotes. They are referred to as non-LTR retrotransposons, because active ones can undergo reinsertion into the genome by reverse transcription of RNA intermediates, but they lack the characteristic LTRs of retroviruses. Long interspersed nuclear elements (LINEs) encode the RT necessary for their retrotransposition, whereas short interspersed nuclear elements (SINEs) depend on LINE RT for retrotransposition (86). Together, LINEs and SINEs account for 40–50% of human DNA (85).

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Reverse Transcriptase and Telomerase When retroviral RT was discovered, it appeared to be catalyzing a reaction counter to the central dogma (DNA → RNA → protein), although Francis Crick, who had enunciated the central dogma, wrote a commentary on how RT’s activity is not really incompatible with it (18). Ini- tially, RT was considered to be a viral enzyme unique to retroviruses and pararetroviruses (e.g., hepadnaviruses), although Temin proposed that it was derived from a cellular system—which he called the protovirus—used to transfer information within an organism (87). Although pro- toviruses were never found, reverse transcription was subsequently found to be fundamental to cellular telomerase. Telomerase is a multiprotein complex responsible for maintaining the ends of chromosomes (telomeres) by generating tandem copies of hexanucleotide repeats. Key components of telomerase are telomerase reverse transcriptase (TERT) (88) and telomerase RNA (89). Telomerase generates the telomere repeats by cyclically extending the lagging strand of telomere DNA 6 nt at a time, with TERT using telomerase RNA as the template. Thus, cells use RT in a basic process; it is possible that cellular RT was originally derived from a viral RT, or vice versa.

REVERSE TRANSCRIPTASE IN RETROVIRUS ASSAY AND DISCOVERY Although Temin & Rubin’s (23) focus assay and similar assays for transforming strains of murine and feline viruses (90) enabled quantitative analysis of oncogene-containing viruses such as RSV and MSV, most retroviruses lack oncogenes, and although they replicate well, most have no discernible effect on infected cells. Before 1960, assays for such viruses relied on their effects in animals, on electron microscopy, or on use of speciﬁc antibodies. All of these procedures left much to be desired; they were complex, too expensive, lacked adequate sensitivity, and required special- ized reagents. RT provided a simple, sensitive, and rapid assay that could detect any retrovirus. It could be used to quantify the amount of a known virus (e.g., for growth curves in cell culture) and to detect new viruses or classify newly discovered viruses as retroviruses. The endogenous RT assay that measured nucleotide incorporation in disrupted virions as directed by the genome RNA and the natural tRNA primer was found in short order to be relatively insensitive, compared with assays for RT that used incorporation of a single labeled deoxynucleotide into DNA with an exogenously added simple homopolymer template and a short complementary oligonucleotide primer. A commonly used combination is 3H TTP with a poly(rA):oligo(dT) template (53, 55), which is used to this day for retrovirus quantiﬁcation. A more recent—and much more sensitive— update of the exogenous RT assay is the polymerase chain reaction (PCR)-enhanced RT (PERT) 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 assay, based on reverse transcribing a natural (usually bacteriophage) RNA template followed by PCR to detect and quantify the cDNA product (91). Although numerous retroviruses had been found to cause cancer in experimental and domestic animals, as of 1970, no such association had been shown for human cancer, and RT promised to provide a far more sensitive means to detect human retroviruses than any other assay in use at the time. It was only a few months after the discovery when reports began to appear of RT, both intracellular and in particles, in various human cancers (92–94). Unfortunately, these reports were soon proven incorrect, a result of the fact that normal cellular enzymes, particularly mitochondrial (γ) DNA polymerases, can also use RNA as a template (95). Nonetheless, over the next ten years, numerous new retroviruses were reported in human cancers, most of them detected with RT assays. As reviewed by Weiss and colleagues (96–98), all such reports were incorrect, due to cell polymerase–RNA complexes masquerading as viruses as well as to real viruses present in the cultures either from laboratory contamination or expression of an endogenous retrovirus. Indeed, such reports continued for several more decades, most recently with the discovery of xenotropic

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murine leukemia virus–related virus (XMRV), an MLV-like virus thought to be associated with prostate cancer and chronic fatigue syndrome (99, 100), which turned out to be a recombinant between two endogenous MLVs acquired during passage of a human xenograft in nude mice (101). A related area that immediately received a great deal of attention following the discovery of RT was attempts to find drugs that inhibited RT for potential treatment of human cancer. Although well supported by the Special Virus Cancer Program, such studies were misguided for two reasons. First, no human retrovirus had yet been found. Second, even if one had been, it was already well known from the observation that RSV could transform cells in the complete absence of virus replication (30, 102) that ongoing virus replication was not necessary for maintenance of the transformed state, so even if a retrovirus-induced cancer were known to be present, treatment by blocking retroviral infection would not be successful. RT inhibitors were to become medically important much later, for their critical role in the development of effective therapies to treat HIV infection. It wasn’t until 1980 that the first (and, so far, the last) human cancer–causing retrovirus, HTLV-1 (the cause of adult T cell leukemia), was discovered by the Gallo laboratory (103), with the identification of its associated RT activity playing the key role in establishing it as a retrovirus, but with biochemical properties distinct from other known, laboratory strain viruses. Within six months of the report of HTLV-1, the first case reports of another disease, which came to be known as acquired immunodeficiency syndrome (AIDS), started appearing (104, 105). About two and a half years later, the virus now known as HIV was isolated from an AIDS patient in France, again with its RT as the definitive piece of evidence for its retroviral identity (106). The emergence of the AIDS pandemic in 1981 was profoundly affected by the fact that HIV is a retrovirus. Fortunately, a great deal of knowledge that had been gained from research on oncogenic retroviruses, beginning with the discovery of RT in 1970, could be quickly applied. The first successful drug to treat AIDS patients was azidothymidine (AZT), an RT inhibitor, which was first used in 1987. AZT is a chain-terminating thymidine analog (107) that was first synthesized as an antibacterial drug but failed in that capacity and was languishing in the chemical archives of Burroughs-Wellcome. It was found to inhibit HIV replication with high efficiency and was shown to be phosphorylated in cells and readily incorporated by HIV RT but not by cellular DNA polymerase (108). In a clinical trial, its dramatic short-term effects in delaying the death of AIDS patients (109) led to its rapid approval for use, although the virus rebounded after a few months due to the appearance of mutations in RT that render it AZT resistant (110). The resistance problem has been solved by the development of additional antiviral drugs—including drugs that, 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 like AZT, are termed nucleoside RT inhibitors (NRTIs); other RT inhibitors (non-NRTIs, or NNRTIs); and drugs targeting other viral enzymes such as protease or integrase. The current armamentarium of anti-HIV drugs, used in combinations of at least three at a time, has largely converted HIV infection/AIDS from an almost certain death sentence to a manageable chronic disease. In management of HIV-infected individuals, RT also plays a critical role in monitoring the status of virus replication because it is used in quantitative RT-PCR reactions to measure levels of HIV RNA in the blood—so-called viral load measurements. Current assays can detect less than 1 HIV RNA copy per milliliter of blood (111).

CODA: LIFE AFTER REVERSE TRANSCRIPTASE The discovery of RT came while both Baltimore and Temin were early in their careers: Baltimore, a pretenure associate professor, was thirty-two and had been an independent investigator for ﬁve years; Temin, a professor, was thirty-ﬁve and had been an independent investigator for ten years.

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Both went on to make further important discoveries and contributions to their fields, and both became leaders and international spokesmen and statesmen. Temin continued to operate a small research laboratory in the same location for the rest of his life, despite attractive job offers from other prestigious institutions. His research continued to focus on retroviruses, switching from RSV to reticuloendotheliosis virus for obscure technical reasons. His laboratory made further important contributions to the field, in areas including the determination of the structure of the provirus, the early development of retroviral vectors, the discovery of mechanisms of cell killing, and the first measurements of retroviral mutation and recombination rates. His laboratory was the first to recognize the phenomenon of G-to-A hypermutation, later found to be an important antiviral defense mechanism. Beyond his scientific accomplishments, Temin took full advantage of the platform afforded him by his newfound fame to advocate for social and medical causes. Despite working on cancer- causing viruses for his whole career, he felt very strongly about the role of smoking as a cause of cancer. In one well-known example, in his acceptance speech for the Nobel Prize, he both thanked the assembled royalty and other dignitaries and asked them to please put out their cigarettes and cigars. He also took his religion very seriously, and once took advantage of an invitation to the Soviet Union to meet with oppressed Jewish scientists, distribute banned scientific literature to them, and gather information so he could publicize their plight after his return to the United States. Although he never took on any official administrative or leadership role, he often served as an advisor to the National Institutes of Health and other agencies, and was much valued for his contributions. Ironically, Temin was diagnosed with metastatic lung adenocarcinoma and died in early 1994, at the age of fifty-nine. An avid walker, he is memorialized at UW by the naming of a beautiful and widely used path along Lake Mendota on campus after him. Baltimore continued his research in animal virology, initially furthering his interests in poliovirus, rhabdoviruses, and retroviruses, which became his predominant focus. In retrovirology, his laboratory made important discoveries in the mechanisms of viral replication, including elucidation of the mechanism of positive-strand DNA synthesis and LTR formation (112) and the mechanisms of host-virus restriction. He investigated Abelson MLV, which induces B cell lymphoma in mice, discovered its v-abl oncogene (113), and found that it is a tyrosine-specific protein kinase (114)—one of the first discoveries of this important enzyme activity. His laboratory also was able use Ab-MLV to transform primary B cells in culture and follow them as they differentiated into antibody-producing cells. This was a powerful system to study B lymphoid differentiation, and it led him into immunology. His immunology discoveries included molecular cloning of 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 RAG1 and RAG2, genes critical for VDJ recombination in immunoglobulin formation. Another area of interest was the transcription factor NF-κB, which is important in gene expression in immune cells. Baltimore also was instrumental in developing retroviral vectors, originating the first retroviral packaging cell line Psi-2. He has had a long-standing interest in using viruses to control or combat human disease, an interest that continues today. Throughout his career he has been a mentor to numerous postdoctoral fellows and graduate students, many of whom have gone on to establish their own scientific careers (including five members of the National Academy of Sciences). Baltimore has also played leadership roles in academia. At MIT he was the founding director of the Whitehead Institute (1982–1990), and he served as president of the Rockefeller University (1990–1991) and Caltech (1997–2004), where he continues his research program as President Emeritus and Millikan Professor. He has served on numerous national policy committees, including appointment as the first head of the National Institutes of Health AIDS Vaccine Research Committee (1997).

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DISCLOSURE STATEMENT The authors are not aware of any afﬁliations, memberships, funding, or ﬁnancial holdings that might be perceived as affecting the objectivity of this review.

LITERATURE CITED 1. Baltimore D. 1970. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 226:1209–11 2. Temin HM, Mizutani S. 1970. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226:1211–13 3. Ellerman V, Bang O. 1908. Experimentelle Leukamie¨ bei Huhnern.¨ Zentralbl. Bakteriol. Parasitenkd. Infectionskr. Hyg. Abt. Orig. 46:595–609 4. Rous P. 1911. A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J. Exp. Med. 13:397–411 5. Varmus HE. 1990. Nobel lecture. Retroviruses and oncogenes. I. Biosci. Rep. 10:413–30 6. Bishop JM. 1990. Nobel lecture. Retroviruses and oncogenes. II. Biosci. Rep. 10:473–91 7. Gross L. 1957. Filterable agent causing leukemia following inoculation into newborn mice. Tex. Rep. Biol. Med. 15:603–16; discussion 16–26 8. Moloney JB. 1966. A virus-induced rhabdomyosarcoma of mice. Natl. Cancer Inst. Monogr. 22:139–42 9. Bittner JJ. 1936. Some possible effects of nursing on the mammary gland tumor incidence in mice. Science 84:162 10. Jarrett WF, Martin WB, Crighton GW, Dalton RG, Stewart MF. 1964. Transmission experiments with leukemia (lymphosarcoma). Nature 202:566–67 11. Fauquet CM, Mayo MA. 2001. The 7th ICTV report. Arch. Virol. 146:189–94 12. Sharp DG, Beard JW. 1957. Electron micrography of Rous sarcoma virus preparations. Ann. N.Y. Acad. Sci. 68:454–58 13. Bather R. 1958. Relationship between infectivity and the ribonucleic acid content of partially puriﬁed Rous sarcoma virus preparations. Br.J.Cancer12:256–63 14. Shope RE, Hurst EW. 1933. Infectious papillomatosis of rabbits: with a note on the histopathology. J. Exp. Med. 58:607–24 15. Duesberg PH. 1968. Physical properties of Rous sarcoma virus RNA. PNAS 60:1511–18 16. Billeter MA, Parsons JT, Cofﬁn JM. 1974. The nucleotide sequence complexity of avian tumor virus RNA. PNAS 71:3560–64 17. Stent G. 2007. Phage and the Origins of Molecular Biology. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press 18. Crick F. 1970. Central dogma of molecular biology. Nature 227:561–63 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 19. Temin HM. 1976. Howard M. Temin—biographical. In Les Prix Nobel en 1975, ed. W Odelberg. Stockholm: Nobel Found. http://www.nobelprize.org/nobel_prizes/medicine/laureates/1975/ temin-bio.html 20. Dulbecco R, Vogt M. 1954. Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99:167–82 21. Dulbecco R. 1964. Transformation of cells in vitro by DNA-containing viruses. JAMA 190:721–26 22. Groupe V, Dunkel VC, Manaker RA. 1957. Improved pock counting method for the titration of Rous sarcoma virus in embryonated eggs. J. Bacteriol. 74:409–10 23. Temin HM, Rubin H. 1958. Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology 6:669–88 24. Temin HM. 1960. The control of cellular morphology in embryonic cells infected with Rous sarcoma virus in vitro. Virology 10:182–97 25. Rubin H. 1970. Replication and persistence of the RNA oncogenic viruses. In Oncology 1970: Being the Proceedings of the Tenth International Cancer Congress, ed. RL Clark, RW Cumley, JE McCay, MM Copeland, pp. 763–68. Chicago: Yearb. Med. Publ.

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Annual Review of Virology Contents Volume 3, 2016

History The Language of Life Ann C. Palmenberg pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1 The Discovery of Reverse Transcriptase John M. Cofﬁn and Hung Fan ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp29 Ecology and Evolution The Strange, Expanding World of Animal Hepaciviruses Alex S. Hartlage, John M. Cullen, and Amit Kapoor ppppppppppppppppppppppppppppppppppppppp53 Bats as Viral Reservoirs David T.S. Hayman pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp77 The Genus Tospovirus: Emerging Bunyaviruses that Threaten Food Security J.E. Oliver and A.E. Whitﬁeld ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp101 Climate Change and the Arboviruses: Lessons from the Evolution of the Dengue and Yellow Fever Viruses Walter J. Tabachnick ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp125 Epidemiology and Management of the 2013–16 West African Ebola Outbreak M.L. Boisen, J.N. Hartnett, A. Goba, M.A. Vandi, D.S. Grant, J.S. Schieffelin, R.F. Garry, and L.M. Branco pppppppppppppppppppppppppppppppppppppppppp147

Access provided by Tulane University on 11/25/20. For personal use only. Genomic Analysis of Viral Outbreaks Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org Shirlee Wohl, Stephen F. Schaffner, and Pardis C. Sabeti ppppppppppppppppppppppppppppppp173 Viruses as Winners in the Game of Life Ana Georgina Cobián Güemes, Merry Youle, Vito Adrian Cantú, Ben Felts, James Nulton, and Forest Rohwer pppppppppppppppppppppppppppppppppppppppppppppppppppppp197 Attachment and Cell Entry Integrins as Herpesvirus Receptors and Mediators of the Host Signalosome Gabriella Campadelli-Fiume, Donna Collins-McMillen, Tatiana Gianni, and Andrew D. Yurochko ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp215 Structure, Function, and Evolution of Coronavirus Spike Proteins Fang Li pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp237

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Genome Replication, Regulation of Gene Expression, and Biosynthesis Properties and Functions of the Dengue Virus Capsid Protein Laura A. Byk and Andrea V. Gamarnik ppppppppppppppppppppppppppppppppppppppppppppppppp263 A Cap-to-Tail Guide to mRNA Translation Strategies in Virus-Infected Cells Eric Jan, Ian Mohr, and Derek Walsh pppppppppppppppppppppppppppppppppppppppppppppppppppp283 Unraveling the Mysterious Interactions Between Hepatitis C Virus RNA and Liver-Speciﬁc MicroRNA-122 Peter Sarnow and Selena M. Sagan ppppppppppppppppppppppppppppppppppppppppppppppppppppppp309 Human Cytomegalovirus Latency: Approaching the Gordian Knot Felicia Goodrum pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp333 Epstein-Barr Virus: The Path from Latent to Productive Infection Ya-Fang Chiu and Bill Sugden pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp359 Assembly and Egress More than Meets the Eye: Hidden Structures in the Proteome Hal Wasserman and Erica Ollmann Saphire pppppppppppppppppppppppppppppppppppppppppppppp373 Nuclear Exodus: Herpesviruses Lead the Way Janna M. Bigalke and Ekaterina E. Heldwein ppppppppppppppppppppppppppppppppppppppppppp387 Moving On Out: Transport and Packaging of Inﬂuenza Viral RNA into Virions Seema S. Lakdawala, Ervin Fodor, and Kanta Subbarao pppppppppppppppppppppppppppppppp411 The Structural Biology of Hepatitis B Virus: Form and Function Balasubramanian Venkatakrishnan and Adam Zlotnick ppppppppppppppppppppppppppppppppp429 Single-Cell Studies of Phage λ: Hidden Treasures Under Occam’s Rug Ido Golding pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp453 Transformation and Oncogenesis Transgenic Mouse Models of Tumor Virus Action

Access provided by Tulane University on 11/25/20. For personal use only. pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org Paul F. Lambert 473 Pathogenesis Tombusvirus-Host Interactions: Co-Opted Evolutionarily Conserved Host Factors Take Center Court Peter D. Nagy ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp491 Polyomavirus Persistence Michael J. Imperiale and Mengxi Jiang pppppppppppppppppppppppppppppppppppppppppppppppppp517 Viruses and the Diversity of Cell Death Pranav Danthi pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp533 Modeling Viral Spread Frederik Graw and Alan S. Perelson pppppppppppppppppppppppppppppppppppppppppppppppppppppp555

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Immunity Bugs Are Not to Be Silenced: Small RNA Pathways and Antiviral Responses in Insects Vanesa Mongelli and Maria-Carla Saleh ppppppppppppppppppppppppppppppppppppppppppppppppp573 Rotavirus Strategies Against the Innate Antiviral System Susana L´opez, Liliana S´anchez-Tacuba, Joaquin Moreno, and Carlos F. Arias pppppppp591 Errata An online log of corrections to Annual Review of Virology articles may be found at http://www.annualreviews.org/errata/virology 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

Contents xi