Investigating Viruses during the Transformation of Molecular Biology

This Reflections article describes my early work on viral enzymes and the discovery of mRNA capping, how my training in medicine and biochemistry merged as I evolved into a virologist, the development of viruses as vaccine vectors, and how scientific and technological developments during the 1970s and beyond set the stage for the interrogation of nearly every step in the reproductive cycle of vaccinia virus (VACV), a large DNA virus with about 200 genes. The reader may view this article as a work in progress, because I remain actively engaged in research at the National Institutes of Health (NIH) notwithstanding 50 memorable years there.

I ranked Children's number 1 for the intern-matching program because of its academic reputation and association with Harvard Medical School. My wife Toby and I were ecstatic when the acceptance came.
I was 23 and Toby was 20 when we traveled from Brooklyn to Boston in June 1961 and settled in a small apartment off the Fenway, close to the hospital. Toby, a charismatic elementary school teacher, quickly secured a good position. Children's was far more up-to-date and better staffed than Bellevue; on the other hand, interns at that time were on duty every day and every other night, and the life and death responsibilities were exhilarating albeit overwhelming for a new medical graduate. In particular, the rotation in the Jimmy Fund children's cancer center was devastating, as can be imagined from the descriptions in Siddhartha Mukherjee's excellent book The Emperor of All Maladies (2). I intended to stay at Children's for one year and then go elsewhere for basic research training, so after several months I made an appointment to discuss career options with Lewis Thomas, who was Chairman of the Department of Medicine at NYU-Bellevue Medical Center. Wikipedia aptly describes Thomas as an American physician, poet, etymologist, essayist, administrator, educator, policy advisor, and researcher. He is well known for essays in the New England Journal of Medicine, a collection of which was published as The Lives of a Cell: Notes of a Biology Watcher (3). As a student, I had been on medical rounds with Dr. Thomas, but I had not spoken to him previously. Nevertheless, he must have seen some potential in me during our meeting as he offered me a fellowship on a National Institutes of Health (NIH) training grant designed to further educate physicians in basic sciences. I accepted and chose to enter a Ph.D. program rather than seek a postdoctoral position, as the former would allow me to take additional science courses as well as provide a student deferment from the Doctor Draft. While completing my internship, I checked out programs at nearby Harvard and Massachusetts Institute of Technology (MIT) and chose the latter. I have a vivid memory of walking along Longwood Avenue under sunny skies in June 1962 eager to begin a new stage of my life across the river in Cambridge.

From MIT to NIH
The Biochemistry Division at MIT, chaired by John (Jack) Buchanan, attracted me because of its integration within the Biology Department, which at that time was a quiet haven housed on three floors of the 10-year-old Dorrance Building located near the bustling hub of the campus. Additional members of the Biology Department included Eugene Bell, Gene Brown, Jim Darnell (soon to leave for the Albert Einstein College of Medicine), Maurice Fox, Vernon Ingram, Cyrus Levinthal, Salvador Luria, Boris Magasanik, Alex Rich, and Irwin Sizer. Jim Darnell describes this early period before the huge expansion of biology at MIT in a Reflections article (4). Taking into account my medical education and desire to strengthen my background in the core sciences, Jack Buchanan allowed me to substitute mathematics and chemistry for biology elective courses. The first year at MIT provided a good learning experience, although taking final exams in a gymnasium filled with young engineering students seemed anachronistic. At the end of that year, I was ready and eager to begin research, although I still had to choose a topic and a mentor.
I was influenced by the pivotal experiment of Brenner, Jacob, and Meselson (5) identifying unstable RNA as the intermediate between DNA and proteins, which had been published the year before I arrived at MIT. In extending this work to eukaryotes, Jim Darnell was isolating rapidly   (7). B, circulating immature erythrocytes at 12 days after treatment of tadpoles with thyroxin. Animals were injected intraperitoneally with 14 C-labeled amino acids and bled 3 h later. Smears were stained for hemoglobin with o-dianisidine and autoradiographed. Arrows point to heavily labeled cells that are more spherical and contain less hemoglobin than the mature tadpole erythrocytes. From   (8).

labeled RNA from HeLa cells and Jon Warner in Alex
Rich's lab was soon to describe polyribosomes from rabbit reticulocytes. Vernon Ingram (Fig. 1), who had made the landmark discovery that sickle cell hemoglobin differed from normal by a single amino acid substitution, suggested that I investigate the regulation of hemoglobin synthesis. It was known that mammals, birds, and amphibians undergo developmental changes in their hemoglobin. Fetal and adult human hemoglobin share two of their four subunits, but the polypeptide changes that occur in birds and amphibians had not yet been determined. I thought that the molecular switch from tadpole to frog hemoglobin might be particularly appropriate for experimentation because metamorphosis could be induced by thyroxin in free-living animals. My thoughts at the time revolved around the possibility that this hormone directly or indirectly regulated transcription of hemoglobin genes.
Vernon was a highly supportive mentor, although his approach to developing an independent investigator consisted of helping outline a project and then evaluating progress after several months. His hands-off style suited me as I had already benefitted from instruction in laboratory technique and close supervision by Robert W. Chambers, whose lab I had worked in while in medical school. Moreover, my responsibilities as a medical student and intern made me confident in attempting new techniques. I found that working with red proteins was a great way to hone biochemical procedures-sloppy loading or packing of a gel filtration column was immediately apparent. The hemoglobins of tadpoles and frogs migrated differently on electrophoresis ( Fig. 2A) and had different subunits as shown by a variety of methods including peptide fingerprinting and N-terminal Edman degradation (6). The circulating erythrocytes of amphibians are nucleated, and the immature ones still synthesize hemoglobin, which could be metabolically labeled in vitro or in vivo with radioactive amino acids and heme precursors. Metamorphosis proceeded synchronously upon immersion of tadpoles in water containing thyroxin, leading to a sharp decline to near zero in synthesis of hemoglobin by red blood cells. After 8 days, hemoglobin synthesis abruptly increased due to entry into the circulatory system of immature erythrocytes that could be radioactively labeled, had a more spherical shape (Fig. 2B), and synthesized the adult frog hemoglobin (7,8). The data suggested a model in which thyroxin regulates the maturation or proliferation of a clonally distinct erythrocyte line.
Research was going well in the spring of 1966, and I was thinking of laboratories working on gene regulation that I might join to gain additional experience when I received a letter informing me that my military deferment would soon end and requesting that I register for the draft. As a draftee with one year of pediatric training, my likely prospect seemed to be an assignment in family medicine on a military base. However, I was now fully dedicated to a career in research and therefore decided to volunteer for the U.S. Public Health Service instead and seek an assignment at the NIH. I was assured during a scouting visit that a position awaited me there upon a satisfactory physical examination and induction into the Public Health Service. With just a few months remaining at MIT, my priority was to complete experiments, and so I deferred writing a thesis, which I later completed during evenings and weekends of my first six months at NIH.

From Frogs to Viruses
I arrived in Bethesda in September 1966 with Toby and our three young children, who reveled in a large house with a backyard after living in the small Cambridge apartment. My position was in the NIAID Laboratory of Biology of Viruses (LBV), which had just merged with the Laboratory of Cell Biology following the departure of Karl Habel and Harry Eagle, the former chiefs of the two separate laboratories. Cell biologists and virologists are indebted to Harry Eagle for defining the nutritional requirements of mammalian cells in culture and will recognize his name on packages of Eagle's medium. I was warmly welcomed into LBV by the new Laboratory Chief Norman Salzman (  later discovered the cellular origin of a retroviral oncogene for which they received a Nobel Prize. I had come to the NIH with the expectation of working with Norman on the isolation and characterization of mammalian chromosomes, although LBV was mainly a virology laboratory. Upon my arrival, however, Norman informed me that the chromosome project was being phased out but that there was an opening to work on vaccinia virus (VACV), because he had recently switched from this virus to the much smaller polyoma virus. Although I had never taken a virology course and had not planned on working with viruses, I was content nonetheless because viruses provide unique models for studying gene expression and of course have important medical significance. Moreover, because of an assignment to write a review of poxviruses as part of my Ph.D. qualifying exam at MIT, I was already fascinated by their unusual life cycle.
VACV and cowpox virus are well known as the live vaccines used by Edward Jenner to prevent smallpox caused by the closely related variola virus. In fact, 1966 was the year that the World Health Organization mounted its ultimately successful smallpox eradication program with VACV. Poxviruses, of which VACV is the prototype, were the largest known animal viruses at the time (although this honor now falls to pandoravirus). Unlike other viruses with DNA genomes, the poxviruses replicate exclusively in the cytoplasm rather than the nucleus of infected cells (Fig. 4). Early experiments suggesting that poxviruses encode enzymes for replication and reports by Brian McAuslan, Wolfgang (Bill) Joklik, and Norman Salzman, showing that VACV replication is divided into early and late phases, meshed with my interest in the regulation of gene expression. Additionally, there was evidence for rapidly turning-over cytoplasmic poxviral RNA (9, 10), which in 1967 Oda and Joklik (11) characterized as early and late mRNA by hybridization to DNA. The Reflections article by Joklik provides some background for the latter work (12). Although the basis for the hemoglobin switch that I studied at MIT was too complex for molecular studies with the tools available, the tractability of the VACV system, as shown by the research done thus far, gave me hopes that discovering how viral genes are turned on and off would be more feasible. My initial experiments analyzed the time course of gene expression in detail by pulselabeling cells with radioactive amino acids and resolving proteins by gel electrophoresis and autoradiography, methods that I had used at MIT. Those experiments documented a profound inhibition of cellular protein synthesis and the consecutive production of early, intermediate, and late classes of viral proteins (13); my subsequent studies explained these phases by showing that VACV encodes three sets of transcription factors with cognate promoters.
My enthusiasm for studying viruses in general and VACV in particular was further enhanced during attendance at the first Gordon Conference on Animal Cells and Viruses held in June 1967 at the Tilton School in New Hampshire. These annual meetings (emphasizing cell biology and virology in alternate years) became the premier place to meet colleagues and hear the latest advances in the related disciplines. Indeed, many cell biologists got their start in virology because viruses provided relatively simple experimental systems. At the 1967 conference, Joe Kates and Bill Munyon independently announced (and subsequently published, see Refs. 14 and 15) that infectious poxvirus particles have RNA polymerase activity, providing an explanation for the synthesis of mRNA by a cytoplasmic DNA virus. This discovery, suggesting that the encapsulated enzymes are brought into cells by the infecting virus, was particularly exciting because it came at a time when contemporary thinking was influenced by the Hershey-Chase experiment (16), which showed that bacteriophage DNA entered cells but that the detectable proteins were excluded. The presence of RNA polymerase in poxvirus virions motivated subsequent efforts to find polymerase activities in RNA viruses and retroviruses. Indeed, the following year Shatkin and Sipe (17) described RNA polymerase activity in particles of reovirus, a double-stranded RNA virus.
In the meantime, work on poxvirus transcription was invigorated. The findings of Kates and Munyon and other studies strongly implied but did not actually prove a viral origin of the polymerase activity encapsulated in the particle or an essential role of the enzyme in virus replication. A paper (18) ostensibly showing that rifampicin, a specific inhibitor of bacterial but not mammalian DNA-depen- dent RNA polymerase, interferes with VACV gene expression added support for a viral origin. Because a specific inhibitor of the poxvirus RNA polymerase would be a valuable tool, I attempted to confirm and extend the rifampicin story. I discovered, however, that the drug actually has no direct effect on VACV gene expression. In collaboration with Phil Grimley, an electron microscopist at NIH (Fig. 5), and others, we showed that rifampicin was a specific inhibitor of viral membrane assembly (19), which proved valuable in later studies on viral morphogenesis.
Although a specific chemical inhibitor would have been extremely useful for investigating poxvirus transcription, in any case a direct biochemical approach such as that taken in 1969 by Roeder and Rutter (97) in partially purifying eukaryotic DNA-dependent RNA polymerases was needed. Although it took six more years to determine the subunit structure of mammalian RNA polymerase II (20), we were motivated to attempt purification and characterization of the viral enzyme. Up to this time, my experience purifying proteins was limited to hemoglobin. Nevertheless, as a descendent of Harry Eagle's lab, LBV was a great place to undertake a project requiring large amounts of virus: the support staff carried over from Harry Eagle's time made cell culture medium from scratch, bled horses at a nearby farm, and filter-sterilized the serum, allowing us to routinely grow large volumes of HeLa cells, for which we used custom-made 6-liter vented bottles set on magnetic stirrers.
Virus particles are designed to withstand environmental stresses, and this is particularly true for poxviruses, which are resistant to high temperatures and desiccation. Therefore, my first task was to devise a way to extract soluble proteins from the virus core. Non-ionic detergents removed only membrane-embedded surface proteins, whereas strong detergents such as SDS inactivated enzyme activity. Through experimentation, I discovered that appropriate concentrations of deoxycholate, NaCl, and dithiothreitol released the enzymes including the RNA polymerase while leaving the abundant structural proteins insoluble. The next step was to devise quantitative assays for the RNA polymerase and other enzymes and proteins needed for mRNA synthesis that were predicted to be present in the virus, as the genome had not yet been sequenced and was essentially a black box. Enzyme purifications were carried out with 60 -90 mg of starting virus, making high recovery of activity at each step important. Looking back, it seems remarkable that we succeeded in purifying numerous enzymes from virus cores, including the two-subunit poly(A) polymerase (Fig. 6) (21), DNA-and RNA-dependent nucleoside triphosphatases    (27), the eight-subunit DNA-dependent RNA polymerase (28), as well as transcription (29) and termination (30) factors to near homogeneity, and in some cases before proteins with comparable functions were isolated from mammalian cells. These studies were carried out with a succession of talented postdoctoral fellows; some with MDs had their first research experience in my laboratory.

Uncovering an Unusual mRNA Terminal Structure
In 1974, Perry and Kelley (31) took advantage of new methods of rigorously purifying mRNA based on the poly(A) tail to demonstrate that polyadenylated RNA from mouse L cells contains on average 2.2 methyl groups per 1,000 nucleotides. Their data suggested that both the base and the ribose moieties were methylated, although the modified nucleotides were not identified. They further speculated that the methyl groups were in a non-coding portion of the mRNA, possibly at the 5Ј end. Because VACV mRNA is also polyadenylated, my immediate thought was to determine whether VACV mRNA is also modified by methylation. At this particular time Aaron Shatkin was visiting NIH, and he told me that Kin-ichiro Miura and colleagues in Japan had found that the ends of encapsulated double-stranded RNAs of cytoplasmic polyhedrosis virus (CPV) were blocked and that methylation was suspected, which they subsequently published (32). In turn, I cited Bob Perry's recent paper and my thought that viral mRNAs might be methylated. Although VACV is a double-stranded DNA virus, whereas CPV and reovirus-Shatkin's focus-are double-stranded RNA viruses, the infectious particle contains endogenous transcriptase activity in each case. We agreed to test the idea that viral mRNAs are methylated in my laboratory by incubating the four ribonucleoside triphosphates and radioactive S-adenosylmethionine with purified VACV, and in Aaron's laboratory by performing the experiment with reovirus. The next week, I called Aaron and left a message that VACV mRNA is methylated (Fig. 7A), and a few hours later, he called back to say that reovirus mRNA is also methylated. After further work, we separately but simultaneously submitted these exciting results for publication (33,34). The next step was to investigate the nature and location of the methylated nucleotides, which I determined with a postdoc Cha Mer Wei, and which was independently determined by Aaron Shatkin working with Yasuhiro Furuichi-originally from Miura's group-along with other colleagues. The ability to synthesize the viral RNAs in vitro specifically labeled with [␣-, ␤-, or ␥-32 P]ribonucleoside triphosphates was an important factor in deriv-ing the cap structure and its mode of formation in both of our labs. Without divulging the structure in advance, Aaron and I simultaneously submitted our findings for publication (35,36) and mailed copies of the accepted manuscripts to each other. It was gratifying to find that we had independently arrived at the same novel structure (Fig. 7B), which was subsequently termed a cap, with a terminal 7-methylguanosine connected to the penultimate 2Ј-O-methylated nucleotide via a novel 5Ј to 5Ј triphosphate linkage. The only difference between the VACV and reovirus caps are that the former caps can contain either a guanine or an adenine base (m 7 G(5Ј)ppp (5Ј)A m -or m 7 G(5Ј)ppp(5Ј)G m -), whereas the latter only contain guanine (m 7 G(5Ј)ppp(5Ј)G m -).
After determining the nature of viral caps and describing the relevant analytical methods, it was straightforward for us and others to determine the 5Ј-RNA structures of additional mRNAs including HeLa cell polyadenylated RNA (37), histone mRNA (38), and viral mRNAs of adenovirus (39) and herpesvirus (40). The RNAs made in vivo contain the same basic cap structure but with additional methylations, such as m 7 G(5Ј)ppp(5Ј)N m pN m with the four common 2Ј-O-methylribonucleosides (G m , A m , U m , C m ) and a novel dimethylated nucleoside N 6 ,O 2 Ј-dimethyladenosine (m 6 A m ) in the penultimate position, as well as internal N 6 -methyladenosine residues in the sequence m 6 ApC and Apm 6 ApC (41). The importance of these additional methylations is just becoming apparent (42,43).
The small number of encoded mRNAs made reovirus better suited than VACV for studying the role of caps in translation and mRNA stability, which was carried out in Aaron's lab (44,45). On the other hand, VACV had a technical advantage over reovirus in that we already knew how to solubilize the enzymes in the virus core and quickly determined that the soluble enzymes could cap and meth- ylate exogenous RNA. For this reason, my lab tackled the enzymology: we purified a heterodimeric guanylyltransferase/guanine-7-methyltransferase that transferred GMP from GTP to the di-or triphosphate end of substrate RNAs and methylated the added GMP at the N-7 position (23). A second enzyme that methylated the penultimate nucleotide at the 2Ј-O position was also purified (24). We further demonstrated that the guanylyltransferase was also an RNA triphosphatase (25) and established that cap formation occurs in the following steps (where AdoMet is S-adenosylmethionine and AdoHcy is S-adenosyl-L-homocysteine) Methylation of the guanosine in step iii importantly prevents the reversible reaction of step ii. Stewart Shuman and Jerry Hurwitz (46) further demonstrated that the transfer of GMP from GTP to the diphosphate end of RNA is a two-step reaction with a VACV enzyme-GMP intermediate. Curiously, VACV also encodes decapping enzymes that regulate mRNA stability (47,48).
Following the purification of the VACV capping enzymes, we partially purified the corresponding enzymes from eukaryotic cells and established that capping occurred by the same mechanism, although the individual activities were associated with separate enzymes (49 -54).
The years 1974 -1978, during which the structure and mechanism of formation of the cap were established, rank among the most intense and enjoyable of my scientific career in part because I was still working nearly full time at the bench.

The Poxvirus Genome Revealed
Three technical advances in the 1970s-restriction endonuclease mapping, DNA cloning, and DNA sequencing-revolutionized the way research is carried out with all biological systems including viruses. The construction of recombinant SV40 DNA was reported in 1972 (55). Further recombination experiments were delayed by safety fears, leading to the 1975 Asilomar Conference, which was followed by a voluntary moratorium on cloning DNA. Under the guidelines issued by the NIH in 1977, cloning of viral DNA was allowed only under biosafety level 4 conditions, effectively precluding nearly all such research. After further deliberation, the NIH drafted revised guidelines in 1978 that permitted cloning segments of viral DNA at biosafety level 2 using attenuated phage lambda vectors. During this same period, restriction endonucleases were first used to map the cleavage sites of the small genome of SV40 and subsequently the large VACV genome (56). Riccardo Wittek had come to my laboratory from Zurich to collaborate on cloning, and together with other colleagues, we prepared and cut VACV and lambda DNA in advance so that the ligations could be legally performed on January 2, 1979, the day the new recombinant DNA rules became effective. In November of that year, we submitted the first of a series of papers describing the cloning of VACV DNA and mapping viral transcripts (57,58). Using the recently described Maxam-Gilbert sequencing method (59), Bahige Baroudy and S. Venkatesan in my group obtained the first glimpses of poxvirus genes and regulatory sequences and determined that the two DNA strands are joined to each other by a novel incompletely base-paired hairpin at each end of the genome followed internally by repeat sequences (60,61). We subsequently showed that this hairpin structure is formed by cleavage of a concatemeric intermediate by a virus-encoded Holliday junction endonuclease (62). Improved methods of DNA sequencing with chain termination inhibitors (63) allowed entire poxvirus genomes to be sequenced (64,65), revealing that these genomes are organized with most of the ϳ100 conserved genes in the central region and a similar number of variable ones involved in host interactions nearer to the two ends. Although seeing the complete genome sequences was   exciting and clearly accelerated research in the field, I confess to feeling a loss of the mystery regarding the gene content.

Genetic Engineering
In addition to enabling cloning experiments, the revised NIH Recombinant DNA Guidelines made it permissible to insert foreign DNA into the VACV genome. As we were gaining an understanding of how poxvirus mRNA synthesis is regulated, it seemed possible to incorporate foreign genes and use poxvirus promoters to express them. At this propitious time, two bright young investigators from the UK-Michael Mackett and Geoffrey Smith-came to my laboratory. Jointly, they developed VACV into a novel expression vector (66). Dennis Panicali and Enzo Paoletti (a former postdoc) working at the New York State Department of Health also succeeded in a related effort, although they initially tried to use a poxvirus-incompatible herpesvirus promoter to obtain expression (67). Because VACV had been extensively used as the smallpox vaccine, this vector suggested the exciting prospect of making recombinant vaccines for other pathogens. Over the next two years, we showed that hepatitis B virus antigen and influenza virus hemagglutinin could be expressed from infectious VACV, and that chimpanzees and hamsters, respectively, could be protected against disease (68 -70). Enzo and co-workers (71, 72) also described the potential use of recombinant VACV as a vaccine vector. The description of a general method for production and selection of recombinant VACV and the distribution of transfer plasmids (73)(74)(75) led to widespread use of VACV expression vectors for immunology and infectious disease research. Moreover, as all chordopoxviruses have a similar arrangement of genes, interchangeable promoters, and conserved RNA polymerase and transcription factors, a similar strategy could be used for other poxviruses. Currently, several poxvirus veterinary vaccines have been licensed, and human vaccines and therapeutics are in clinical trials for cancer and infectious diseases including HIV (76 -78). The idea of using virus vectors for vaccines, first established with VACV, has been taken up by others, and now many viruses including adenoviruses, herpesviruses, and vesicular stomatitis virus to name a few are being developed for similar purposes.
Although my laboratory has always been devoted to basic research, I saw the vaccine potential of the expression vectors as a kind of repayment on my medical education and the NIH training grant to educate physicians in basic sciences that started my research career. Therefore, I have maintained collaborations with other research groups as well as industry to share my expertise in the development of vaccines.

From Biochemistry to Virology
It may be evident from these recollections that during the 1960s and 1970s I viewed poxviruses mainly as exceptional systems for biochemical analyses. However, with the sequencing of poxvirus genomes, I became more and more intrigued with the life cycle of these complex viruses. How do the ϳ200 genes orchestrate the complex events needed for synthesis and spread of progeny virions? It was evident that the ability to mutate individual genes would be key to understanding their function. Although genes that are not essential for replication in cell culture could be readily deleted, conditional lethal mutants are required to study essential genes. Richard Condit, in particular, pro- vided a great service to the community by freely distributing his lab's panel of temperature-sensitive VACV mutants (79). Despite their tremendous value, the mutants did not cover the entire genome and were sometimes difficult to work with because of their narrow temperature range, and in some cases, the phenotypes were inconsistent with the known functions of gene products. In the mid-1980s, Tom Fuerst with others in my lab developed an inducible system in which the Escherichia coli Lac repressor and lac operator stringently regulate the expression of foreign genes inserted into the VACV genome (80). Although originally developed as a vector for overexpression, we adapted the repressor system for the regulation of VACV genes (81) and in this way made numerous inducible viruses that behave as null mutants in the absence of inducer (Fig. 8). Complementing cell lines have also been used to make deletion mutants, particularly for early genes (82)(83)(84). With general methods for making conditional mutants and available technologies, we turned our attention to interrogating the steps in the poxvirus replication cycle including entry (85,86), regulation of gene expression (87), genome replication (88), cytoplasmic disulfide bond formation (89), and virion assembly (90,91). Although much work remains to be done, at least some role has been assigned to all 100 essential genes (92).
Much of our current work emphasizes the other 50 -100 VACV proteins that are involved in host interactions. In 1990, my group provided the first description of a viral protein that interferes with host immunity (93). The role of the latter VACV-encoded complement inhibitor was deduced by homology and verified experimentally. Since then, proteins targeting additional host defense pathways have been identified in a similar way or in an ad hoc manner for many viruses. However, we found that large-scale screens in which expression of individual cellular genes is reduced or prevented by RNAi were successful in identifying host proteins that VACV uses for various steps in replication but not for anti-viral genes, probably because poxviruses evolved adequate defenses during their co-evolution with cells (94). In ongoing work, we have shown that this roadblock to discovery could be overcome by screening mutant viruses that have had one or more defense genes disrupted and consequently have lost the ability to replicate in certain non-permissive cells (95). Furthermore, with CRISPR/Cas9 technology, we have been able to inactivate cellular genes to confirm virus-host interactions (96). Without doubt, variations of the CRISPR system as well as other technologies that cannot yet be envisioned will be applied to the study of viruses and their hosts.

Concluding Remarks
I have tried to illustrate with my own career the way in which new ideas and technologies have influenced molecular biology in general and virology in particular. I could not imagine in 1966 when Norman Salzman suggested that I work on VACV that this would consume 50ϩ years of my scientific life. However, the study of this complex virus has been a continuous learning experience as I have had to become knowledgeable in many areas of molecular and cell biology to investigate the varied steps in the virus life cycle.
Before completing this article, I would like to express my appreciation of and admiration for the many enthusiastic and talented postdocs, staff scientists, students, and support personnel that over the years have made my career both productive and enjoyable. I wish that I could have named each individually and cited their contribution. The best that I can do is to include a group photo taken at the poxvirus workshop in 2010 ( Fig. 9) and congratulate those who have established laboratories and continue to advance the field.
I want to add a few lines about the poxvirus workshop mentioned above because it has served an important purpose in bringing together the community of investigators interested in poxviruses. The workshop, which has been held biennially since 1977, was initially attended by 50 -60 researchers at the Cold Spring Harbor Laboratories but has expanded to more than 200 attendees and is now held internationally. Nevertheless, the grassroots format remains essentially unchanged with no invited speakers; instead the selection of presentations is based entirely on the quality of the submitted abstracts. At each meeting, the previous organizers gather together (Fig. 10) to recruit one of the attendees to serve as a future chairman and select the new meeting location, which will be in Taiwan next.
I also want to take this opportunity to applaud the NIAID intramural program for continuing to be an extraordinary place for basic and translational research on viruses. In particular, the wise policy of evaluating research support by past performance allowed me great flexibility in choosing new topics to investigate. In 1984, I became Chief of a reorganized Laboratory of Viral Diseases (LVD), and a group photo probably taken the following year shows the NIAID laboratory chiefs at that time, Tony Fauci-the long time Director of the institute, and Ken Sellthe Director of the intramural program then (Fig. 11). Over time I have been fortunate in recruiting outstanding and congenial investigators including Mark Challberg (now in the NIAID extramural program), Jon Yewdell, Jack Bennink, Ed Berger, Alison McBride, Tom Kristie, and Ted Pierson to lead independent LVD sections investigating herpesviruses, influenza virus, HIV, papilloma virus, and flaviviruses.