
==== Front
Yale J Biol MedYale J Biol MedThe Yale Journal of Biology and Medicine0044-00861551-4056Yale Journal of Biology and Medicine EssayCancer issueThirty Years into the Genomics Era: Tumor Viruses Led the Way DiMaio Daniel a*Miller George b
a Departments of Genetics and Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut
b Departments of Pediatrics, Epidemiology and Public Health, and Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut* To whom all correspondence should be addressed: To whom all correspondence should be addressed: Daniel DiMaio, MD, PhD, Department of Genetics, Yale University School of Medicine, Sterling Hall of Medicine, I-141, 333 Cedar Street, New Haven, CT 06510; Tel: 203-785-2684; Fax: 203-785-6765; E-mail: daniel.dimaio@yale.edu.10 2007 12 2006 79 3-4 179 185 Copyright ©2006, Yale Journal of Biology and Medicine2006Yale Journal of Biology and MedicineThis is an open access article distributed under the terms of the Creative Commons CC BY-NC license, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited. You may not use the material for commercial purposes.
==== Body
This year marks the 30th anniversary of an important milestone in the history of biology: the dawn of the genomics era with the first report of the complete DNA sequence of a viral genome in 1977.

Viruses are submicroscopic micro-organisms that are dwarfed in size and complexity by all free-living forms of life. Thus, when rudimentary methods for determining short stretches of DNA sequence were developed in the 1970s, a few visionary laboratories realized that viral genomes should be the target of the first large-scale sequencing efforts. The results of this endeavor were eagerly awaited. An entire page of the 1970 edition of Albert Lehninger’s Biochemistry, then the standard text in the field, was devoted to the imaginary DNA sequence of a bacterial virus, the bacteriophage ɸX174.

At the time, the prospect of sequencing the vastly larger human genome seemed impossible. Nevertheless, the sequencing of viral genomes provided seminal insights not only into viral growth, but also into animal and human carcinogenesis. These studies resulted in the discovery of cellular oncogenes and led directly to the identification of viruses as an important cause of human cancer and the idea that non-viral cancer is, in large part, a genetic disease. In addition, the sequence analysis of viral genomes laid the intellectual and technical framework that enabled the eventual sequencing and analysis of cellular genomes, an effort that in turn is unlocking many of the secrets of cancer and providing new opportunities for more effective cancer treatments.

Setting the Stage
The breakthrough that allowed the sequencing of viral genomes (and later, larger cloned genomes) was the use of restriction endonucleases to manipulate and analyze DNA. Bacterial cells synthesize restriction enzymes to defend themselves against invading foreign DNA, and these enzymes were first identified and characterized on the basis of their ability to cleave bacteriophage DNA.

The value of restriction enzymes was established by a series of pioneering studies by Daniel Nathans at Johns Hopkins and Paul Berg at Stanford on the genome of SV40, a small virus that normally infects monkeys. SV40 was discovered in tissue cultures of monkey kidney cells in which poliovirus vaccine was produced, and many vaccinated individuals were inadvertently inoculated with SV40 during early poliovirus vaccination programs.

Enhanced interest in SV40 lay in the fact that it and the closely related mouse polyomavirus caused tumors in rodents. It was hoped that detailed analysis of tumor viruses would shed light on the genesis of common human cancers and suggest new approaches to prevent and treat cancer.

This hope has been realized many times over. SV40 has been implicated in some human cancers, including childhood brain tumors, mesotheliomas, and non-Hodgkins lymphoma, but an etiologic role of SV40 in these tumors has not been established unequivocally [1]. Nevertheless, the early development of molecular biology techniques to analyze SV40 DNA led to the first restriction map and the first localization of a genetic signal, the viral origin of DNA replication, in eukaryotic cells [2-4].

The first recombinant DNA molecule constructed in vitro consisted of SV40 linked to bacteriophage lambda DNA [5].

This work set the stage for much of modern genetics and molecular biology and won Nobel prizes for Nathans and Berg. Studies on SV40 also played a major role in the genetic engineering revolution and the birth of the biotechnology industry.

The First Sequencing Successes
Prior to the use of restriction endonucleases, sequencing studies were largely limited to the handful of bases at the very ends of viral genomes (a striking limitation in the case of circular genomes). Now the entire viral genome was accessible.

The initial large-scale sequencing efforts bore fruit in 1977 with the publication of the complete DNA sequence of ɸX174 by a consortium headed by Frederick Sanger (who already had won a Nobel Prize for developing methods to sequence proteins and later won a second Nobel for DNA sequencing) [6].

The next year, the DNA sequence of the tumor virus SV40 was solved by groups headed by Sherman Weissman at Yale University and Walter Fiers in Belgium [7-8].

ɸX174 and SV40 are relatively simple viruses, each with only about 5,000 DNA base pairs (compared to the 6 billion base pairs in each of our cells), but with the primitive methods available at that time, this sequence analysis required many years and prodigious effort. With today’s technology, the genomes of these small viruses can be knocked off in a busy afternoon.

Viruses Take Center Stage
With the support of the “War on Cancer” declared by the National Cancer Institute in the 1970s, tumor virologists rapidly adopted sequencing as a primary method to analyze viral genomes, and larger DNA tumor viruses soon fell to sequencing efforts: papillomaviruses, the cause of warts and cervical and some other human cancer, with 8,000 base pairs, in 1982; and adenoviruses, which cause respiratory tract disease in humans and tumors in experimental animals, with 36,000 base pairs, in 1984. The following year, 1985, saw the completion of the 172,000 base pair sequence of the DNA of Epstein-Barr virus, a herpesvirus that infects virtually all humans and causes certain infected individuals to develop Hodgkin’s disease, Burkitt’s lymphoma, or nasopharyngeal carcinoma. Since the sequencing was done manually, this heroic feat required eight postdoctoral scientists working full time for several years. The poxviruses, a virus group that includes the dreaded smallpox virus and related viruses that can cause tumors in animals, weighed in at 191,636 base pairs in 1990.

The necessity of sequencing and analyzing ever-larger viral genomes spurred improvements in technology. The first sequences were derived from RNA copied from the viral DNA, but researchers soon moved to sequencing the DNA itself. A key breakthrough was the development of rapid enzymatic DNA sequencing methods by Sanger and colleagues at Cambridge University [9] replacing more cumbersome chemical methods invented by Maxam and Gilbert at Harvard University [10]. The Cambridge group also developed “shot-gun” methods for sequencing the genome of Epstein-Barr virus, in which the sequence of random fragments of viral DNA was determined, and the sequences of overlapping DNA segments were then aligned by computer. This method turned out to be essential for solving the sequences of the much larger bacterial and cellular genomes, and eventually the postdoctoral fellows were replaced by robots for the cloning operations and by automated DNA sequencing machines. Our ability to record and analyze long DNA sequences was also sorely tested, a predicament that helped lead to the development of methods of computational analysis required for the study of bacterial and cellular genomes.

Viral Genomes as Targets and Teachers about Cancer and Other Diseases
The sequences of viral genomes revealed the great promise and limitations of large-scale sequence analysis. For the first time, the entire genetic legacy of an organism was manifest. Genes and regulatory signals were identified, protein structure and function was deduced, and overall genetic organization was laid bare. It seemed possible that a life form could be reduced to a set of simple instructions, thus revealing its most intimate secrets.

Novel human viruses were discovered based on DNA sequencing; genes that viruses captured from cellular DNA were identified; and pathogenic mechanisms and the genetic and evolutionary relationships between different viruses were elucidated. Again, studies of tumor viruses led the way.

Kaposi Sarcoma herpesvirus, which commonly causes cancer in AIDS victims, was discovered solely on the presence of novel viral DNA sequences, related to Epstein-Barr virus in a tumor [11], and hepatitis C virus was defined based on sequence information [12,13]. Hepatitis C virus and its compatriot hepatitis B virus are responsible for the development of hepatocellular carcinoma, the third most common cancer in the world [14,15]. Analysis of oncogenic retroviruses isolated from animal tumors revealed that these viruses carried genes responsible for cancer and that these genes were actually altered versions of cellular oncogenes [16], a discovery that won the Nobel Prize for Michael Bishop and Harold Varmus at the University of California at San Franscisco. Extensive sequencing also resulted in the identification of multiple different types of human papillomaviruses and the discovery that certain high-risk HPV types cause important human cancers [17,18].

These studies ultimately led to development of prophylactic vaccines designed to prevent virus infection and the resulting human cancers. Vaccines based on the major capsid protein of the high-risk human papillomaviruses will prevent much cervical cancer, and the hepatitis B virus surface antigen vaccine already is causing a reduction in the incidence of hepatocellular cancer [14,19]. These cancer vaccines are produced by using recombinant DNA technology with defined viral DNA segments because of the difficulty of propagating these human tumor viruses in the laboratory. These two vaccines alone have the potential to eliminate greater than 10 percent of the world-wide cancer burden. The prevention of cancers by vaccination is the culmination of a century-long effort to tie infectious agents to cancer and validates this entire enterprise [20]. In addition, ongoing work at Yale and elsewhere demonstrates that viral gene expression is required to maintain the survival and growth of certain cancers, suggesting that anti-viral drugs may be useful in treating these cancers once they develop [21,22,23]. Vaccination against viral non-structural antigens expressed by cancer cells also has emerged as a potential therapeutic approach.

Viruses responsible for acute infections have been subjected to intense sequencing efforts. Complete sequence information enabled the laboratory synthesis of infectious poliovirus, a small virus that has so far eluded sustained efforts for eradication [24]. In a remarkable example of molecular archaeology, sequencing of archived samples of the 1918 pandemic influenza virus, which caused the most deadly acute epidemic in history, allowed the resurrection of this virus [25]. Studies based on this work will have a profound effect on our understanding of these important human pathogens and the genetic basis for future influenza pandemics, but this work also inspired much discussion about the potential misuse of sequence information and scientific knowledge. The very rapid identification of a novel coronavirus as the causative agent of SARS was a stunning achievement based entirely on sequencing [26,27]. Recent sequencing studies also revealed that the environment harbors a vast number of uncharacterized bacteriophage and other viruses [28,29]. Remarkably, the genes contained in these viruses often do not resemble previously known genes, hinting at immense genetic diversity of viruses.

Surprises and Complications
It was anticipated that genomic sequence information would lead to the rapid elucidation of all the details of virus reproduction and myriad interactions between viruses and cells and suggest new rational approaches to combat virus infections and virally induced tumors. But there also were complications. Viruses were more clever, versatile, and enigmatic than we appreciated, and not all their secrets were revealed by simple sequence analysis alone. RNA splicing, first discovered in oncogenic adenoviruses and widespread in cellular genes as well [30,31], results in the synthesis of proteins assembled from segments encoded by DNA scattered about the genome, complicating efforts to deduce protein sequence from DNA sequence.

In other cases, viruses selectively ignored signals that should tell the cellular machinery where genes started and stopped, and RNA editing or ribosomal frameshifting confounded the definition of a gene (e.g., [32,33]). One of the first viral sequences solved revealed the startling situation that the same piece of DNA could encode two proteins with entirely different amino acid sequences [34]. Thus, it became clear that complete understanding of an organism, even one as simple as a virus, cannot be deduced from sequence information alone, but the services of experimental biologists were still needed.

Moving Beyond Viruses
The great value of sequencing was immediately clear to the scientific community and inspired successful attempts to decipher larger cellular genomes.

The first complete DNA sequence of a bacterial genome, that of Mycoplasma genitalium, was reported in 1995 [35], and now hundreds of bacterial genomes have been decoded, providing valuable information about the lifestyle, evolution, and pathogenesis of these organisms. And in 2001, the entire human genome sequence was completed.

In parallel, the genomes of several other pathogens and organisms useful as experimental models, including the malaria parasite, insects, worms, plants, and other mammals, have been sequenced. These sequences uncovered a treasure trove of information that is revolutionizing our understanding of evolution, gene organization and function, cellular function, development, and disease. These studies also revealed the disquieting fact that a substantial fraction of our own genomes is derived from remnants of viruses related to oncogenic retroviruses. It has even been postulated that the transfer of viral genes into primordial RNA-based cells gave rise to the three branches of cellular life on earth: bacteria, archea, and eukaryotes [36].

More Insights into Cancer
DNA sequencing has had a particularly profound effect on our understanding of carcinogenesis and on efforts to control this disease. Sequencing of the cellular versions of the oncogenes first identified in tumor viruses revealed newly arising mutations in many sporadically occurring human cancers [37], helping to establish the notion that much of cancer has a genetic basis.

Many of these oncogenes encode growth factors, growth factor receptors, signal transduction proteins, and transcription factors, which are crucial components of signaling pathways that determine how cells respond to their environment, grow, and die. Based on these and related studies, it is now possible in some cases to predict a patient’s prognosis or response to chemotherapy based on the sequence of cellular proteins involved in cell signaling and growth [38].

The elucidation of the human genome sequence also allowed the construction of microarrays that have been used to assess the transcriptional profile of normal and cancer cells. These studies revealed the existence of subsets of various tumors with markedly different prognosis, suggesting underlying biological differences that can be exploited in developing improved approaches for managing these diseases [39].

In addition, advances in rapid sequencing and computational analysis allowed comprehensive comparison of the sequences of normal and cancer cell genomes and the identification of crucial shared mutations in independently arising cancers [40]. The genes identified in this manner are likely to play important roles in carcinogenesis and provide new targets for therapy and diagnosis.

Sequencing of DNA isolated from cancers also revealed the frequent occurrence of mutations in genes encoding tumor suppressor proteins, particularly p53, which was first identified as a binding partner of SV40 large T antigen, the viral DNA replication protein [41,42]. Large T antigen also binds the retinoblastoma tumor suppressor protein, a central component of another tumor suppressor pathway often inactivated by mutation in human cancers [43]. Strikingly, three unrelated groups of DNA tumor viruses, SV40, human papillomaviruses, and adenoviruses, all inactivate these same tumor suppressor proteins, implying the existence of a limited number of regulatory nodes essential for viral replication and cellular carcinogenesis.

These important cellular genomics efforts clearly had their origins in studies of viral genomes. Francis Collins, who heads the National Human Genome Research Institute and directed the United States’ effort to sequence the human genome, was trained in the Weissman laboratory at Yale that sequenced SV40. Two of the prime movers of the landmark 1995 M. genitalium sequence were Hamilton Smith and Craig Venter. Smith and his Johns Hopkins’ colleague Nathans supplied essential information and materials for the SV40 sequencing project, and a year before the appearance of the first complete bacterial sequence, Venter published the sequence of smallpox virus DNA.

The ability to decode bacterial and cellular genomes is a watershed achievement in biology, one that is transforming our concepts of microbial pathogenesis, cellular function, evolution, and human disease, particularly cancer. This remarkable accomplishment is the logical extension of pioneering studies begun a quarter of a century earlier on viruses, the simplest organisms at the border between chemistry and life.
==== Refs
Shah KV   Virus 40 and human disease J Infect Dis 2004 190 2061 2064 15551202 
Danna KJ  Nathans D   Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae Proc Natl Acad Sci USA 1971 68 2913 2917 4332003 
Danna KJ  Nathans D   Bidirectional replication of simian virus-40 DNA Proc Natl Acad Sci USA 1972 69 3097 3102 4343955 
Danna KJ  Sack GH  Nathans D   Studies of simian virus-40 DNA. Cleavage map of SV40 genome J Mol Biol 1973 78 363 376 4355833 
Jackson DA  Symons RH  Berg P   Biochemical method for inserting new genetic information into DNA of Simian Virus 40: Circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli Proc Natl Acad Sci USA 1972 69 2904 2909 4342968 
Sanger F  Air GM  Barrell BG  Brown NL  Coulson AR  Fiddes CA    Nucleotide sequence of bacteriophage phi X174 DNA Nature 1977 265 687 695 870828 
Fiers W  Contreras R  Haegemann G  Rogiers R  Van de Voorde A  Van Heuverswyn H    Complete nucleotide sequence of SV40 DNA Nature 1978 273 113 120 205802 
Reddy VB  Thimmappaya B  Dhar R  Subramanian N  Zain BS  Pan J    The genome of simian virus 40 Science 1978 200 494 502 205947 
Sanger F  Nicklen S  Coulson AR   DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci USA 1977 74 5463 5467 271968 
Maxam AM  Gilbert W   A new method for sequencing DNA Proc Natl Acad Sci USA 1977 74 560 564 265521 
Chang Y  Cesarman E  Pessin MS  Lee F  Culpepper J  Knowles DM  Moore PS   Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma Science 1994 266 1865 1869 7997879 
Choo QL  Kuo G  Weiner AJ  Overby LR  Bradley DW  Houghton M   Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome Science 1989 244 359 362 2523562 
Miller RH  Purcell RH   Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups Proc Natl Acad Sci USA 1990 87 2057 2061 2156259 
Beasley RP   The major etiology of hepatocellular carcinoma Cancer 1988 61 1942 1956 2834034 
DiBisceglie AM   Hepatitis C and hepatocellular carcinoma Hepatology 1997 26 34S 38S 9305661 
Stehelin D  Varmus HE  Bishop JM  Vogt PK   DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA Nature 1976 260 170 173 176594 
Bosch FX  Lorincz A  Munoz N  Meijer CJ  Shah KV   The causal relation between human papillomavirus and cervical cancer J Clin Pathol 2002 55 244 265 11919208 
Durst M  Gissmann L  Ikenberg H  zur Hausen H   A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions Proc Natl Acad Sci USA 1983 80 3812 3815 6304740 
Koutsky LA  Ault KA  Wheeler CM  Brown DR  Barr E  Alvarez B    A controlled trial of a human papillomavirus type 16 vaccine N Engl J Med 2002 347 1645 1651 12444178 
Klein G  DiMaio D   Garcea R  DiMaio D   Principles of Human Tumor Virology The Papillomaviruses 2007 New York Springer 19 29 
Hwang ES  Riese DJ  Settleman J  Nilson LA  Honig J  Flynn S  DiMaio D   Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene J Virol 1993 67 3720 3729 8389903 
Kennedy G  Komano J  Sugden B   Epstein-Barr virus provides a survival factor to Burkitt's lymphomas Proc Natl Acad Sci USA 2003 100 14269 14274 14603034 
von Knebel Doeberitz M  Oltersdorf T  Schwarz E  Gissmann L   Correlation of modified human papilloma virus early gene expression with altered growth properties in C4-1 cervical carcinoma cells Cancer Res 1988 48 3780 3786 2837324 
Cello J  Paul AV  Wimmer E   Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template Science 2002 297 1016 1018 12114528 
Tumpey TM  Basler CF  Aguilar V  Zeng H  Solorzano A  Swayne DE    Characterization of the reconstructed 1918 Spanish influenza pandemic virus Science 2005 310 77 80 16210530 
Drosten C  Gunther S  Preiser W  van der Werf S  Brodt HR  Becker S    Identification of a novel coronavirus in patients with severe acute respiratory syndrome N Engl J Med 2003 348 1967 1976 12690091 
Ksiazek TG  Erdman D  Goldsmith CS  Zaki SR  Peret T  Emergy S    A novel coronavirus associated with severe acute respiratory syndrome N Engl J Med 2003 348 1953 1966 12690092 
Angly FE  Felts B  Breitbart M  Salamon P  Edwards RA  Carlson C    The marine viromes of four oceanic regions PLoS Biology 2006 4 2121 2131 
Venter JC  Remington K  Heidelberg JF  Halpern AL  Rusch D  Eisen JA    Environmental genome shotgun sequencing of the Sargasso Sea Science 2004 304 66 74 15001713 
Berget SM  Moore C  Sharp PA   Spliced segments at the 5' terminus of adenovirus 2 late mRNA Proc Natl Acad Sci USA 1977 74 3171 3175 269380 
Chow LT  Gelinas RE  Broker TR  Roberts RJ   An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA Cell 1977 12 1 8 902310 
Jacks T  Varmus HE   Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting Science 1985 230 1237 1242 2416054 
Sanchez A  Trappier SG  Mahy BW  Peters CJ  Nichol ST   The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing Proc Natl Acad Sci USA 1996 93 3602 3607 8622982 
Barrell BG  Air GM  Hutchison CA   Overlapping genes in bacteriophage phiX174 Nature 1976 264 34 41 1004533 
Fraser CM  Gocayne JD  White O  Adams MD  Clayton RA  Fleischmann RD    The minimal gene complement of Mycoplasma genitalium Science 1995 270 397 403 7569993 
Koonin EV  Senkevich TG  Dolja VV   The ancient Virus World and evolution of cells Biol Direct 2006 1 29 16984643 
Tabin CJ  Bradley SM  Bargmann CI  Weinberg RA  Papageorge AG  Scolnick EM    Mechanism of activation of a human oncogene Nature 1982 300 143 149 6290897 
Lynch TJ  Bell DW  Sordella R  Gurubhagavatula S  Okimoto RA  Brannigan BW    Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib N Engl J Med 204 350 2129 2139 15118073 
Golub TR  Slonim DK  Tamayo P  Huard C  Gaasenbeek M  Mesirov JP    Molecular classification of cancer: class discovery and class prediction by gene expression monitoring Science 1999 286 531 537 10521349 
Sjoblom T  Jones S  Wood LD  Parsons DW  Lin J  Barber TD    The consensus coding sequences of human breast and colorectal cancers Science 2006 314 268 274 16959974 
Lane DP  Crawford LV   T antigen is bound to a host protein in SV40-transformed cells Nature 1979 278 261 263 218111 
Linzer DI  Levine AJ   Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells Cell 1979 17 43 52 222475 
DeCaprio JA  Ludlow JW  Figge J  Shrew JY  Huang CM  Lee WH    Large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene Cell 1988 54 275 283 2839300

