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John M. Coffin1 and Hung Fan2

1
Department of Molecular Biology and Microbiology, Tufts University, Boston,
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

Massachusetts 02111; email: [email protected]

2
Department 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
This article’s doi: 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
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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
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

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
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
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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

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1968 Baltimore moves to the Massachusetts Institute of Technology

1968 Discovery of virion RNA polymerase in reovirus

Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

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

Summary Figure
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
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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
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

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 bacterio-
phage 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
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 Delbrück 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
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 lab-
oratory, 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, consis-
tent 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 hy-
pothesis, 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 molec-
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ular hybridization. He found that a small fraction of 3 H-labeled virion RNA (prepared by growing
virus in the presence of 3 H-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 un-
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

infected 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 stud-
ies, 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
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
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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-
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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
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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 VI03CH02-Coffin ARI 10 September 2016 9:14

Baltimore, 1970
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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.

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

36 Coffin · Fan
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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)
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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
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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
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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 VI03CH02-Coffin ARI 10 September 2016 9:14

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 investi-
gators 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
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detergent in his reactions. In retrospect it is likely that Baltimore’s ready detection of DNA poly-
merase 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
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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 comple-
mentary 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 sen-
sitive 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-
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 “Après Temin,
le Déluge” (41), appeared three months later, and still another, “Deluge Unabated” (61), a few

38 Coffin · Fan
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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.
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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
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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):
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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 VI03CH02-Coffin ARI 10 September 2016 9:14

tRNA
5' AAA 3'
R U5 PBS PPT U3 R

DNA
synthesis
U5
AAA
R U5 PBS PPT U3 R

RNase H
R U5
Minus-strand AAA
strong-stop DNA PBS PPT U3 R
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First strand
transfer
R U5
AAA
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

PBS PPT U3 R

DNA synthesis
RNase H
PPT U3 R U5

PBS PPT

DNA synthesis
RNase H
PBS PPT U3 R U5

PBS PPT U3 R U5

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

U3

DNA synthesis
U3 R U5 PPT U3 R U5

U3 R 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
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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
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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
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
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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
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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
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 ge-
nomic 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, and c-jun (75). The importance of proto-oncogenes was further highlighted by discover-
ies 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

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The availability of radioactive retroviral cDNAs as hybridization probes in the era before molec-
ular 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
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into host chromosomal DNA. Once integrated, the proviral DNA is inherited by progeny result-
ing 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 un-
infected 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 retrovi-
ral 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 iden-
tification of endogenous retroviruses based on sequence homologies with known retroviruses.
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 endoge-
nous 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 en-
dogenous 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 re-
verse 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 cen-
tral 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 compo-
nents of telomerase are telomerase reverse transcriptase (TERT) (88) and telomerase RNA (89).
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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.
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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 3 H 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)
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
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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.
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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,
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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 VI03CH02-Coffin ARI 10 September 2016 9:14

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
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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
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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 po-
liovirus, rhabdoviruses, and retroviruses, which became his predominant focus. In retrovirology,
his laboratory made important discoveries in the mechanisms of viral replication, including elu-
cidation 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 lym-
phoma 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
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, includ-
ing 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 Leukämie bei Hühnern. Zentralbl. Bakteriol. Parasitenkd.
Access provided by Universidad Nacional Autonoma de Mexico on 10/17/23. For personal use only.

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
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

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. Cancer 12:256–63
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
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.

www.annualreviews.org • The Discovery of Reverse Transcriptase 47

 VI03CH02-Coffin ARI 10 September 2016 9:14

26. Temin HM. 1963. The effects of actinomycin D on growth of Rous sarcoma virus in vitro. Virology
20:577–82
27. Temin HM. 1964. The participation of DNA in Rous sarcoma virus production. Virology 23:486–94
28. Boeke JD, Stoye JS. 1997. Retrotransposons, endogenous retroviruses, and the evolution of retroele-
ments. In Retroviruses, ed. JM Coffin, SH Hughes, HE Varmus, pp. 343–435. Cold Spring Harbor, NY:
Cold Spring Harbor Lab. Press
29. Temin HM. 1969. Control of cell multiplication in uninfected chicken cells and chicken cells converted
by avian sarcoma viruses. J. Cell. Physiol. 74:9–16
30. Svoboda J, Machala O, Deozanek T. 1968. Rescue of Rous sarcoma virus in mixed cultures of virogenic
mammalian and chicken cells, treated and untreated with Sendai virus and detected by focus assay.
J. Gen. Virol. 2:461–64
31. Boettiger D, Temin HM. 1970. Light inactivation of focus formation by chicken embryo fibroblasts
Access provided by Universidad Nacional Autonoma de Mexico on 10/17/23. For personal use only.

infected with avian sarcoma virus in the presence of 5-bromodeoxyuridine. Nature 228:622–24
32. Temin HM. 1970. The DNA provirus of RNA tumor viruses. In Oncology 1970: Being the Proceedings of the
Tenth International Cancer Congress, ed. RL Clark, RW Cumley, JE McCay, MM Copeland, pp. 776–80.
Chicago: Yearb. Med. Publ.
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

33. Kates JR, McAuslan BR. 1967. Poxvirus DNA-dependent RNA polymerase. PNAS 58:134–41
34. Borsa J, Graham AF. 1968. Reovirus: RNA polymerase activity in purified virions. Biochem. Biophys. Res.
Commun. 33:895–901
35. Shatkin AJ, Sipe JD. 1968. RNA polymerase activity in purified reoviruses. PNAS 61:1462–69
36. Baltimore D, Huang AS, Stampfer M. 1970. Ribonucleic acid synthesis of vesicular stomatitis virus. II.
An RNA polymerase in the virion. PNAS 66:572–76
37. Cancer: DNA from an RNA template. 1970. Nature 226:1003
38. Watson JD, Crick FH. 1953. Molecular structure of nucleic acids; a structure for deoxyribose nucleic
acid. Nature 171:737–38
39. Mizutani S, Boettiger D, Temin HM. 1970. A DNA-dependent DNA polymerase and a DNA endonu-
clease in virions of Rous sarcoma virus. Nature 228:424–27
40. Mizutani S, Temin HM, Kodama M, Wells RT. 1971. DNA ligase and exonuclease activities in virions
of Rous sarcoma virus. Nat. New Biol. 230:232–35
41. Après Temin, le déluge. 1970. Nature 227:998
42. Baltimore D, Eggers HJ, Franklin RM, Tamm I. 1963. Poliovirus-induced RNA polymerase and the
effects of virus-specific inhibitors on its production. PNAS 49:843–49
43. Baltimore D. 1964. In vitro synthesis of viral RNA by the poliovirus RNA polymerase. PNAS 51:450–56
44. Baltimore D, Becker Y, Darnell JE. 1964. Virus-specific double-stranded RNA in poliovirus-infected
cells. Science 143:1034–36
45. Baltimore D. 1968. Structure of the poliovirus replicative intermediate RNA. J. Mol. Biol. 32:359–68
46. Jacobson MF, Baltimore D. 1968. Polypeptide cleavages in the formation of poliovirus proteins. PNAS
61:77–84
47. Schaffer FL, Hackett AJ, Soergel ME. 1968. Vesicular stomatitis virus RNA: complementarity between
infected cell RNA and RNAs from infectious and autointerfering viral fractions. Biochem. Biophys. Res.
Commun. 31:685–92
48. Munyon W, Paoletti E, Grace JT Jr. 1967. RNA polymerase activity in purified infectious vaccinia virus.
PNAS 58:2280–87
49. Biswal N, Benyesh-Melnick M. 1969. Complementary nuclear RNAs of murine sarcoma-leukemia virus
complex in transformed cells. PNAS 64:1372–79
50. Baltimore D. 1970. RNA-dependent DNA synthesis by virions of mouse leukemia virus. Cold Spring
Harb. Symp. Quant. Biol. 35:843–46
51. Mizutani S, Temin H. 1970. An RNA-dependent DNA polymerase in virions of Rous sarcoma virus.
Cold Spring Harb. Symp. Quant. Biol. 35:847–49
52. Cancer viruses: more of the same. 1970. Nat. New Biol. 227:887–88
53. Spiegelman S, Burny A, Das MR, Keydar J, Schlom J, et al. 1970. Characterization of the products of
DNA-directed DNA polymerases in oncogenic RNA viruses. Nature 227:563–67

48 Coffin · Fan
 VI03CH02-Coffin ARI 10 September 2016 9:14

54. Riman J, Beaudreau GS. 1970. Viral DNA-dependent DNA polymerase and the properties of thymidine
labelled material in virions of an oncogenic RNA virus. Nature 228:427–30
55. Spiegelman S, Burny A, Das MR, Keydar J, Schlom J, et al. 1970. DNA-directed DNA polymerase
activity in oncogenic RNA viruses. Nature 227:1029–31
56. Spiegelman S, Burny A, Das MR, Keydar J, Schlom J, et al. 1970. Synthetic DNA-RNA hybrids and
RNA-RNA duplexes as templates for the polymerases of the oncogenic RNA viruses. Nature 228:430–32
57. Baltimore D, Smoler D. 1971. Primer requirement and template specificity of the DNA polymerase of
RNA tumor viruses. PNAS 68:1507–11
58. Verma IM, Meuth NL, Bromfeld E, Manly KF, Baltimore D. 1971. Covalently linked RNA-DNA
molecule as initial product of RNA tumour virus DNA polymerase. Nat. New Biol. 233:131–34
59. Verma IM, Mason WS, Drost SD, Baltimore D. 1974. DNA polymerase activity from two temperature-
sensitive mutants of Rous sarcoma virus is thermolabile. Nature 251:27–31
Access provided by Universidad Nacional Autonoma de Mexico on 10/17/23. For personal use only.

60. Verma IM, Temple GF, Fan H, Baltimore D. 1972. In vitro synthesis of DNA complementary to rabbit
reticulocyte 10S RNA. Nat. New Biol. 235:163–67
61. Deluge unabated. 1970. Nature 228:410
62. Wade N. 1971. Special Virus Cancer Program: travails of a biological moonshot. Science 174:1306–11
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

63. Telesnitsky A, Goff SP. 1997. Reverse transcriptase and the generation of retroviral DNA. In Retroviruses,
ed. JM Coffin, SH Hughes, HE Varmus, pp. 121–60. Cold Spring Harbor, NY: Cold Spring Harbor
Lab. Press
64. Brown PO. 1997. Integration. In Retroviruses, ed. JM Coffin, SH Hughes, HE Varmus, pp. 161–203.
Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press
65. Rabson AB, Graves BJ. 1997. Synthesis and processing of viral RNA. In Retroviruses, ed. JM Coffin, SH
Hughes, HE Varmus, pp. 205–61. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press
66. Kacian DL, Spiegelman S, Bank A, Terada M, Metafora S, et al. 1972. In vitro synthesis of DNA
components of human genes for globins. Nat. New Biol. 235:167–69
67. Ross J, Aviv H, Scolnick E, Leder P. 1972. In vitro synthesis of DNA complementary to purified rabbit
globin mRNA (RNA-dependent DNA polymerase/reticulocyte/hemoglobin/density gradient centrifu-
gation/oligo(dT) primer). PNAS 69:264–68
68. Edmonds M, Vaughan MH Jr., Nakazato H. 1971. Polyadenylic acid sequences in the heterogeneous
nuclear RNA and rapidly-labeled polyribosomal RNA of HeLa cells: possible evidence for a precursor
relationship. PNAS 68:1336–40
69. Ross J, Ikawa Y, Leder P. 1972. Globin messenger-RNA induction during erythroid differentiation of
cultured leukemia cells. PNAS 69:3620–23
70. Berns AJ, Bloemendal H, Kaufman SJ, Verma IM. 1973. Synthesis of DNA complementary to 14S calf
lens crystallin messenger RNA by reverse transcriptase. Biochem. Biophys. Res. Commun. 52:1013–19
71. McDonnell JP, Garapin AC, Levinson WE, Quintrell N, Fanshier L, Bishop JM. 1970. DNA poly-
merases of Rous sarcoma virus: delineation of two reactions with actinomycin. Nature 228:433–35
72. Stehelin D, Varmus HE, Bishop JM, Vogt PK. 1976. DNA related to the transforming gene(s) of avian
sarcoma viruses is present in normal avian DNA. Nature 260:170–73
73. Duesberg PH, Vogt PK. 1970. Differences between the ribonucleic acids of transforming and nontrans-
forming avian tumor viruses. PNAS 67:1673–80
74. Stehelin D, Guntaka RV, Varmus HE, Bishop JM. 1976. Purification of DNA complementary to nu-
cleotide sequences required for neoplastic transformation of fibroblasts by avian sarcoma viruses. J. Mol.
Biol. 101:349–65
75. Rosenberg N, Jolicoeur P. 1997. Retroviral pathogenesis. In Retroviruses, ed. JM Coffin, SH Hughes,
HE Varmus, pp. 475–585. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press
76. Tabin CJ, Bradley SM, Bargmann CI, Weinberg RA, Papageorge AG, et al. 1982. Mechanism of acti-
vation of a human oncogene. Nature 300:143–49
77. Alitalo K, Schwab M, Lin CC, Varmus HE, Bishop JM. 1983. Homogeneously staining chromosomal
regions contain amplified copies of an abundantly expressed cellular oncogene (c-myc) in malignant
neuroendocrine cells from a human colon carcinoma. PNAS 80:1707–11
78. de Klein A, van Kessel AG, Grosveld G, Bartram CR, Hagemeijer A, et al. 1982. A cellular oncogene is
translocated to the Philadelphia chromosome in chronic myelocytic leukaemia. Nature 300:765–67

www.annualreviews.org • The Discovery of Reverse Transcriptase 49

 VI03CH02-Coffin ARI 10 September 2016 9:14

79. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, et al. 2001. Activity of a specific inhibitor
of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic
leukemia with the Philadelphia chromosome. N. Engl. J. Med. 344:1038–42
80. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, et al. 2001. Use of chemotherapy plus a
monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N. Engl. J.
Med. 344:783–92
81. Payne LN, Chubb RC. 1968. Studies on the nature and genetic control of an antigen in normal chick
embryos which reacts in the COFAL test. J. Gen. Virol. 3:379–91
82. Gelb LD, Aaronson SA, Martin MA. 1971. Heterogeneity of murine leukemia virus in vitro DNA;
detection of viral DNA in mammalian cells. Science 172:1353–55
83. Steffen D, Weinberg RA. 1978. The integrated genome of murine leukemia virus. Cell 15:1003–10
84. Frankel WN, Stoye JP, Taylor BA, Coffin JM. 1990. A linkage map of endogenous murine leukemia
Access provided by Universidad Nacional Autonoma de Mexico on 10/17/23. For personal use only.

proviruses. Genetics 124:221–36

85. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, et al. 2001. Initial sequencing and analysis of
the human genome. Nature 409:860–921
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

86. Singer MF. 1982. SINEs and LINEs: highly repeated short and long interspersed sequences in mam-
malian genomes. Cell 28:433–34
87. Temin HM. 1971. The protovirus hypothesis: speculations on the significance of RNA-directed DNA
synthesis for normal development and for carcinogenesis. J. Natl. Cancer Inst. 46:3–7
88. Lingner J, Hughes TR, Shevchenko A, Mann M, Lundblad V, Cech TR. 1997. Reverse transcriptase
motifs in the catalytic subunit of telomerase. Science 276:561–67
89. Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, et al. 1995. The RNA component of human
telomerase. Science 269:1236–41
90. Hartley JW, Rowe WP. 1966. Production of altered cell foci in tissue culture by defective Moloney
sarcoma virus particles. PNAS 55:780–86
91. Boni J, Pyra H, Schupbach J. 1996. Sensitive detection and quantification of particle-associated reverse
transcriptase in plasma of HIV-1-infected individuals by the product-enhanced reverse transcriptase
(PERT) assay. J. Med. Virol. 49:23–28
92. Gallo RC, Sarin PS, Allen PT, Newton WA, Priori ES, et al. 1971. Reverse transcriptase in type C virus
particles of human origin. Nat. New Biol. 232:140–42
93. Schlom J, Spiegelman S. 1971. Simultaneous detection of reverse transcriptase and high molecular weight
RNA unique to oncogenic RNA viruses. Science 174:840–43
94. Schlom J, Spiegelman S, Moore D. 1971. RNA-dependent DNA polymerase activity in virus-like particles
isolated from human milk. Nature 231:97–100
95. Kasiviswanathan R, Copeland WC. 2011. Ribonucleotide discrimination and reverse transcription by
the human mitochondrial DNA polymerase. J. Biol. Chem. 286:31490–500
96. Voisset C, Weiss RA, Griffiths DJ. 2008. Human RNA “rumor” viruses: the search for novel human
retroviruses in chronic disease. Microbiol. Mol. Biol. Rev. 72:157–96
97. Weiss RA. 1982. The search for human RNA tumor viruses. In RNA Tumor Viruses, ed. RA Weiss, N
Teich, HE Varmus, JM Coffin, pp. 1205–81. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press
98. Bishop JM, Varmus HE. 1982. Functions and origins of retroviral transforming genes. In RNA Tumor
Viruses, ed. RA Weiss, N Teich, HE Varmus, JM Coffin, pp. 999–1108. Cold Spring Harbor, NY: Cold
Spring Harbor Lab. Press
99. Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, et al. 2009. Detection of an infectious
retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 326:585–89
100. Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, et al. 2006. Identification of a novel Gam-
maretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLOS Pathog.
2:e25
101. Paprotka T, Delviks-Frankenberry KA, Cingoz O, Martinez A, Kung HJ, et al. 2011. Recombinant
origin of the retrovirus XMRV. Science 333:97–101
102. Hanafusa H, Hanafusa T, Rubin H. 1963. The defectiveness of Rous sarcoma virus. PNAS 49:572–80

50 Coffin · Fan
 VI03CH02-Coffin ARI 10 September 2016 9:14

103. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. 1980. Detection and isolation
of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell
lymphoma. PNAS 77:7415–19
104. CDC (Cent. Dis. Control). 1981. Kaposi’s sarcoma and Pneumocystis pneumonia among homosexual
men—New York City and California. Morb. Mortal. Wkly. Rep. 30:305–8
105. CDC (Cent. Dis. Control). 1981. Pneumocystis pneumonia—Los Angeles. Morb. Mortal. Wkly. Rep.
30:250–52
106. Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, et al. 1983. Isolation of a T-
lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science
220:868–71
107. Horwitz JP, Chua J, Noel M. 1964. Nucleosides. V. The monomesylates of 1-(2 -deoxy-β-
D-lyxofuranosyl) thymines. J. Org. Chem. 29:2076–79
Access provided by Universidad Nacional Autonoma de Mexico on 10/17/23. For personal use only.

108. Furman PA, Fyfe JA, St. Clair MH, Weinhold K, Rideout JL, et al. 1986. Phosphorylation of 3 -azido-
3 -deoxythymidine and selective interaction of the 5 -triphosphate with human immunodeficiency virus
reverse transcriptase. PNAS 83:8333–37
109. Fischl MA, Richman DD, Grieco MH, Gottlieb MS, Volberding PA, et al. 1987. The efficacy of azi-
Annu. Rev. Virol. 2016.3:29-51. Downloaded from www.annualreviews.org

dothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex. A double-blind,
placebo-controlled trial. N. Engl. J. Med. 317:185–91
110. Larder BA, Darby G, Richman DD. 1989. HIV with reduced sensitivity to zidovudine (AZT) isolated
during prolonged therapy. Science 243:1731–34
111. Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, et al. 2003. New real-time reverse
transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1
RNA in plasma. J. Clin. Microbiol. 41:4531–36
112. Gilboa E, Mitra SW, Goff S, Baltimore D. 1979. A detailed model of reverse transcription and tests of
crucial aspects. Cell 18:93–100
113. Witte ON, Rosenberg N, Paskind M, Shields A, Baltimore D. 1978. Identification of an Abelson murine
leukemia virus-encoded protein present in transformed fibroblast and lymphoid cells. PNAS 75:2488–92
114. Witte ON, Sun L, Rosenberg N, Baltimore D. 1980. A trans-acting protein kinase identified in cells
transformed by Abelson murine leukemia virus. Cold Spring Harb. Symp. Quant. Biol. 44:855–57

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 VI03-FrontMatter ARI 10 September 2016 8:14

Annual Review of
Virology

Contents Volume 3, 2016

History
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The Language of Life

Ann C. Palmenberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
The Discovery of Reverse Transcriptase
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John M. Coffin and Hung Fan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p29

Ecology and Evolution
The Strange, Expanding World of Animal Hepaciviruses
Alex S. Hartlage, John M. Cullen, and Amit Kapoor p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p53
Bats as Viral Reservoirs
David T.S. Hayman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p77
The Genus Tospovirus: Emerging Bunyaviruses that Threaten Food Security
J.E. Oliver and A.E. Whitfield p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 101
Climate Change and the Arboviruses: Lessons from the Evolution of the
Dengue and Yellow Fever Viruses
Walter J. Tabachnick p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 125
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 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 147
Genomic Analysis of Viral Outbreaks
Shirlee Wohl, Stephen F. Schaffner, and Pardis C. Sabeti p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 173
Viruses as Winners in the Game of Life
Ana Georgina Cobián Güemes, Merry Youle, Vito Adrian Cantú, Ben Felts,
James Nulton, and Forest Rohwer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 197
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 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 215
Structure, Function, and Evolution of Coronavirus Spike Proteins
Fang Li p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 237

ix
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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 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 263
A Cap-to-Tail Guide to mRNA Translation Strategies in Virus-Infected Cells
Eric Jan, Ian Mohr, and Derek Walsh p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 283
Unraveling the Mysterious Interactions Between Hepatitis C Virus RNA
and Liver-Specific MicroRNA-122
Peter Sarnow and Selena M. Sagan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 309
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Human Cytomegalovirus Latency: Approaching the Gordian Knot

Felicia Goodrum p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 333
Epstein-Barr Virus: The Path from Latent to Productive Infection
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Ya-Fang Chiu and Bill Sugden p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 359

Assembly and Egress
More than Meets the Eye: Hidden Structures in the Proteome
Hal Wasserman and Erica Ollmann Saphire p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 373
Nuclear Exodus: Herpesviruses Lead the Way
Janna M. Bigalke and Ekaterina E. Heldwein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 387
Moving On Out: Transport and Packaging of Influenza Viral RNA into Virions
Seema S. Lakdawala, Ervin Fodor, and Kanta Subbarao p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 411
The Structural Biology of Hepatitis B Virus: Form and Function
Balasubramanian Venkatakrishnan and Adam Zlotnick p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 429
Single-Cell Studies of Phage λ: Hidden Treasures Under Occam’s Rug
Ido Golding p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 453
Transformation and Oncogenesis
Transgenic Mouse Models of Tumor Virus Action
Paul F. Lambert p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 473
Pathogenesis
Tombusvirus-Host Interactions: Co-Opted Evolutionarily Conserved
Host Factors Take Center Court
Peter D. Nagy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 491
Polyomavirus Persistence
Michael J. Imperiale and Mengxi Jiang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 517
Viruses and the Diversity of Cell Death
Pranav Danthi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 533
Modeling Viral Spread
Frederik Graw and Alan S. Perelson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 555

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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 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 573
Rotavirus Strategies Against the Innate Antiviral System
Susana López, Liliana Sánchez-Tacuba, Joaquin Moreno, and Carlos F. Arias p p p p p p p p 591
Errata
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