On episode #288 of the science show This Week in Virology,Â the Twivsters discuss how reverse transcriptase encoded in the human genome might produce DNA copies of RNA viruses in infected cells.
You can find TWiV #288 at www.microbe.tv/twiv.
On episode #288 of the science show This Week in Virology,Â the Twivsters discuss how reverse transcriptase encoded in the human genome might produce DNA copies of RNA viruses in infected cells.
You can find TWiV #288 at www.microbe.tv/twiv.
Many years ago a claim was made that cells infected with respiratory syncytial virus contained infectious DNA copies of the viral genome. When this paper was published,Â retroviral reverse transcriptase had been discovered, which explained howÂ DNA copies of retroviral RNA genomes were made in infected cells. Although the respiratory syncytial viral genome is RNA, it does not encode a reverse transcriptase, and how a DNA copy of this genome could be made in infected cells was unknown. The observation was initially met with great fanfare, and was suggested to account for why some RNA virus infections persist, and even to explain autoimmune diseases. However the findings were never duplicated, and the authors fell into scientific obscurity. Now it appears that they might not have been entirely wrong.
It is now quite clear that DNA copies of viral RNA are made in infected cells. A DNA copy of lymphocytic choriomeningitis virus (LCMV) RNA has been detected in mouse cells, and this DNA can beÂ integrated into cellular DNA. LCMV DNA was only detected in cells that produce a retrovirus, implicating reverse transcriptase in this process. DNAs of various RNA viruses, including bornaviruses and filoviruses, have been found integrated into the genome of many animals. An explanation for the genesis of these DNA copies is provided by studies of cells infected with vesicular stomatitis virus.
Vesicular stomatitis virus (pictured) is an enveloped virus with a genome of (-) strand RNA. In infected cells the (-) strand RNA is copied by the viral RNA polymerase to form 5 mRNAs which encode the viral proteins. Infection of various human cell lines resulted in the production of DNA complementary to VSV RNA. The DNAs are single stranded and appear to be produced from the viral mRNAs, not the viral genome.
How might VSV DNA be produced in virus infected cells? About 20% of the human genome consists of a mobile genetic element called LINE-1 (Long Interspersed Nuclear Element). These elements, also called retrotransposons, encode a reverse transcriptase. This enzyme converts LINE-1 mRNA into DNA, which can then integrate elsewhere in the cell genome – hence the name mobile genetic element. LINE-1 encoded reverse transcriptase can also produce DNA copies of cellular mRNAs and other mobile elements that do not encode their own reverse transcriptase. Thanks to LINEs, our genomes are littered with mobile genetic elements.
To determine if LINE-1 could make VSV in infected cells, the authors took advantage of their observation that not all human cells produce VSV DNA after infection. Introduction ofÂ LINE-1 DNA into one of these cell linesÂ enabled it toÂ produce DNA copies of VSV mRNAs. This result shows that LINE-1 can make VSV DNA in infected cells. Whether LINE-1 actually accomplishes this process in unmodified, VSV infected cells remains to be proven.
The authors also show that viral DNA is produced in cells infected with two other RNA-containing viruses, echovirus and respiratory syncytial virus. The latter of course is the virus used to make the claim nearly 40Â years ago that viral DNA is present in infected cells. Whether that viral DNA is infectious, however, is not known. No complete copies of the VSV RNA genome have been observed in infected cells, only copies of viral mRNAs.
It seems likely that in cells infected with RNA viruses, reverse transcriptase encoded by LINE-1 could produce DNA copies of viral RNAs. AfterÂ entering the nucleus these viral DNAs could become integrated into cellular DNA. If a germline cell were infected, the integrated viral DNA would be passed on to subsequent generations, explaining the presence of DNA copies of various RNA viruses in animal genomes.
A key question is whether the production of DNA copies of viral RNAs is an accident, or benefits the virus or host. There is yet no answer to this question. The authors suggest that DNA copies of viral RNA might contribute to innate immune sensing of viral infection. VSV replication is not altered by the presence or absence of viral DNA, but there could be a role for viral DNA during infection of a host animal.
Virologist John Holland passed away on 11 October 2013. I asked former members of his laboratory for their thoughts on his career and what he meant to them.
For more than 35 years, John Holland was a major figure and leading contributor in the study of RNA viruses. Â His early pioneering work on poliovirus was the first demonstration that the block to poliovirus growth in non-primate cells was due to the absence of specific receptors on non-susceptible cells. Â This critical discovery has been a guiding beacon for many subsequent studies on virus-cell interactions. Â Holland was also a central figure in applying molecular biology approaches to the study of viruses. Â His work on picornavirus protein processing and replication was innovative and characterized by a “simple but elegant” experimental approach.
For the last 20 – 25 years of his scientific career, Holland focused his studies on vesicular stomatitis virus (an RNA-containing rhabdovirus) as a model system for viral persistence and for the study of evolution of viral genomic RNAs. Â His work on persistent infections lead to important discoveries about genome interactions between infecting viruses and the defective-interfering particles that they generate. Â Some very far-reaching conclusions about the nature of virus-virus and virus-cell interactions during viral infections have emerged from Holland’s research efforts. Â Perhaps his most influential findings resulted from experiments devoted to quantitation of the rates of mutation during the replication of viral genomic RNAs. Â These studies, many of which were carried out with his long-term collaborator Esteban Domingo, led to the now widely-accepted concept of virus quasispecies. Indeed, using novel, quantitative experimental approaches, Holland and his co-workers greatly advanced our understanding of the molecular mechanisms that have generated genetic diversity among RNA viruses and, ultimately, among living organisms in general.
As one indication of Holland’s significant contributions to our basic understanding of viruses, one need only look at the more than 150 original research papers and review articles that he published. The publications are in the most respected and prestigious scientific journals. Â Moreover, a number of these papers provided seminal biological tenets and have gone on to become classics in the field of molecular virology. Â Holland also trained a significant number of Ph.D. students, postdoctoral fellows, and undergraduate researchers who now have academic positions in major universities and research institutions in the United States and Europe or positions in big pharma and biotechnology. Many of these individuals still carry out research in virology.
I spent six and a half years in Johnâ€™s lab from 1987 to 1993. This period was a truly wonderful time in my life, in large part due to how John ran his lab and interacted with me and other lab members. John promoted a great sense of scientific freedom and actively encouraged creative thought. Â He also was tremendously supportive when the pathway forward was fraught with difficulties and challenges and I found his immense knowledge and insights to be very helpful in rapidly determining how to proceed. John was a supremely competent and confident scientist and yet he maintained a great sense of humility about his own highly significant contributions, in part I believe due to his interest and global understanding of the many other branches of science, which extended to quantum mechanics and â€œstring theoryâ€. On a personal and worldly level John was also tremendously supportive and understanding; his advice in these areas nearly always hit the mark. When I first arrived at UCSD John accompanied me while I was arranging to rent an apartment and buy a car. As a newcomer to the USA it was a great comfort to have the presence of this physically imposing, experienced and knowledgeable guy at my side while I made the deals. My admiration and respect for John continued to grow during my tenure at UCSD; I saw eye-to-eye with John in nearly all things. When I left the Lab I stayed in regular contact with John by E-mail and visited John and his wife Dottie from time to time when they moved to Taos, NM and then Mesquite, NV. John was a very special and influential person in my life. Â I feel hugely privileged to have known him as a mentor and a friend.
From my sabbatical stays with him, I remember a few anecdotes and I include also some thoughts.
A visitor comes into John’s lab at UCSD and sees a guy with dirty hands trying to fix an oil pump on the floor. Do you know where can I find Professor Holland? I am Professor Holland. What can I do for you?
When I was on sabbatical with him, I prepared a manuscript for J. Virol. on work done in Madrid. John liked it, reviewed it prior to submission and corrected a few English mistakes. I sent the manuscript and one of the referees comments stated: Â “English must be improved”. John’s comment: “You see Esteban, this is how things work. This incompetent reviewer added this comment because he / she knows you are Spanish”.
Most remarkably, John worked on his bench until retirement. He would supply cells to all of us (students, postdocts, visiting professors) for us to do the experiments. He maintained continuous conversation with us on scientific issues (from the lab or elsewhere) while working on the bench. These conversations were a great inspiration but he would allow absolute freedom to do the experiments each of us wanted to do. He was an example of honesty, authenticity and vision to anticipate towards where virology was heading to. It is said that he was offered by Joshua Lederberg to head a new institute on emerging infections, but he declined in order to remain active in the lab (if true, this would be very much in line with John’s personality, but I have no confirmation of such offer; he never told me anything about it).
He was very critical of population geneticists who objected to quasispecies. He would often say things such as: “You see Esteban, these guys treat viruses as if they were frogs. Just ignore them. Data are data”.
His review published inÂ ScienceÂ in 1982 (vol. 215, pages 1577- 1585) was a great inspiration to me and the beginning of a permanent contact until he passed away. One of his students (I believe David Steinhauer) told me that the Q beta work that allowed a calculation of a mutation rate in Charles Weissmann’s laboratory Â (in which I participated) made John understand what was going on in the dynamics of DI [defective-interfering particle] generation and competition with standard VSV, and this is the reason why he honestly recognized the Q beta work so enthusiastically in the Science review.
When Bert sent the message a few weeks ago telling us that he had passed away I couldnâ€™t stop thinking about him and my days in his lab at UCSD. He had a big influence on all of us that passed through his lab.
I came to his lab in May 1985, to do post-doctoral training after completing my Ph.D. at the University of Buenos Aires where I worked with Foot and Mouth Disease Virus. For all of us virologists in the late 70’s-early â€˜80s, John Holland was a big name in the field, and to receive his letter welcoming me to pursue a post-doctoral fellowship in his lab was very exciting to me. But I did not know how different he (and his lab) was from what I imagined: He and everything in his lab was so relaxed and informal!
If I have to describe John, I would say that in addition to be very intelligent and creative, he was modest and extremely generous of his time and his lab resources. He strongly believed in being independent and a free thinker: he would not tell you what to do. I remember that when I arrived he said, â€œHere is the lab, take any open bench, talk to the people and think about what you would like to do and we could talk in a few weeksâ€. He always implied that you were able to do great work; he expressed confidence, and respect for the work we did and always listened with a very open mind. I believe that the sense of support that we experienced allowed us to grow. He was an excellent model for the ideal scientist.
It is no secret what an amazing a scientist John was, his scientific legacyÂ is there for everybody to see. He was the smartest person I ever met, andÂ knowledgeable to the point of ridiculous. We had a sort of running jokeÂ trying to come up with a question about anything, from history to quantumÂ physics, that he did not have an answer for. Didn’t find it.
He was an even better person. Many of us think of John as a mix of mentor,Â friend and father (I’m stealing David Clark’s words). My most vividÂ memories are usually related to John worrying about the possibility ofÂ somebody being unhappy, or finding excuses for somebody’s human weaknesses.
There was a janitor, D., who would tell everybody the story of his ongoingÂ divorce and how his wife wanted to take his two kids away from him. One dayÂ he disappeared, which was normal due to high turnout for his job. MonthsÂ later John comes to the lab and says “Have you heard about D.? He is inÂ jail. The police came to his home and it was full of computers and otherÂ stuff stolen from the University. The divorce drove him crazy!”
AccordingÂ to John, the devil himself is a really nice guy.
The worst of my time with him, hands down, was when I had to leave.Â However, on the positive side, that gave me a good enough excuse to finallyÂ give him a hug (he was very shy). He was, is, and will always be my hero.Â He made me a better person and is helping me even after his death. NotÂ because I believe in any beyond, but because of his lessons in humanity.
John was a terrific scientist and mentor. He taught us by example: he worked in the lab every day with joy, focus, and a sense of purpose. We had our scientific discussions with him at his lab bench or ours â€“ not in his office. John also set an example of balancing science and other things in life; his outside pursuits included oil painting, gliding, hiking, and baking delicious French bread. For me his mentorship has been very important in the last couple years when I have been a professor with more administrative responsibilities – he was always willing to provide wise advice. John and I had an active email correspondence, and the week before he passed away we corresponded about some recent defective interfering particle research. Likewise he corresponded with many former Holland lab members, making each of us feel like we had a special bond with him and with each other.
Iâ€™m not sure who taped it up there, but during my time in John Hollandâ€™s lab there was a rather inconspicuous elementary school-like drawing of a palm tree with the caption â€œFantasy Islandâ€ that hung as a permanent fixture above the sink at the center of the lab. In thinking back, it seems like an appropriate symbol for my memories of a special time of exciting science and great collegiality in Johnâ€™s lab.
When I approached John in my first year of graduate school to ask if I could do my final rotation in his lab, he agreed, and pointed to the collection of lab reprints that were stacked in files on one of the office walls; â€œGrab five or six papers from there, go home, or go to the beach or something, and come back in a week and tell me what you want to doâ€. This was different from my previous rotations, and a week later I came back with a few rough ideas in my head, but no definitive project outlined. I was a bit apprehensive, but I had thoroughly digested the recent lab publications, and had a pretty good idea of the general themes and directions of the group. After a few minutes of discussion alongside Johnâ€™s bench (not in his office), we informally outlined a set of experiments and goals that probably didnâ€™t deviate much from what he would have â€œgivenâ€ me as a rotation project right from the start – if John was a â€œnormalâ€ PI. The beauty of the process was that he made me think that I had come up with the idea for this project on my own! This encapsulates the secret to John Hollandâ€™s style of mentorship; it was a fluid blend of independent thought on our part, and guidance on his part that was sometimes firm, but more often so subtle that it bordered on the undetectable. During my graduate career John never had a single formal lab meeting, and he never micromanaged. In fact, by todayâ€™s admin-infested, documentation-oriented standards, John didnâ€™t manage us at all. However, he fostered an environment of personal responsibility, collegiality, and support, that would have made formal lab meetings redundant. Maybe it worked because John was in the lab every day, but we all knew the ins-and-outs of other projects, and when anyone obtained a new result or developed a gel, we would congregate around the lightbox and offer feedback and suggestions on the spot. Soon after joining the lab, I learned that there should never be any embarrassment or shame in an â€œunexpected resultâ€ or â€œugly gelâ€, because we all got them from time-to-time and you couldnâ€™t get away with trying to hide it! Though such results usually led to a standard series of bad jokes and gentle ribbing, this was inevitably followed by the kind of support and constructive commentary that would have distinguished any formal lab meeting. Though unconventional, those of us fortunate enough to have been a part of Johnâ€™s group know that the supportive environment, and the freedom to design experiments and pursue projects, allowed us to grow as independent thinkers and scientists, and offered the best training we could hope for.
The test of time reveals that everyone I overlapped with in Johnâ€™s lab actually had career ambitions as a scientist, and we all now hold respectable jobs in academia and industry. However, at the time this was probably less apparent, and the lab often resembled a clubhouse for overgrown kids, with a generous degree of joking around, and people vacating periodically during the day for softball games and other outdoor pursuits in the inviting San Diego climate. True to his nature, John left us to our own devices; somehow having faith that we were also self-motivated, determined scientists who would let the experiments dictate our schedules, and in fact the lab routinely became a beehive of activity well into the evening and was always well populated over the weekends. Late afternoons were the highlight of each day as John was always in the lab, either passaging his viruses in the glass milk bottles that we used for cell culture, or synthesizing oligos for sequencing studies. Though he was inherently shy, John was always in his element during this time, when he was one of the gang participating in the banter that drifted aimlessly from science, to subjects such as politics and sports, and back again – jokes and cackles of laughter punctuating the proceedings all the way. However, in retrospect, my favorite time for interacting with John came during relatively quiet times on Saturday and Sunday mornings, when he would come in to the lab to passage his lines, and sometimes it was just the two of us. It was then that John would often discuss his experiences in science and in life, informally and interspersed with stories and anecdotes. These occasions constituted the best mentoring experiences I ever received, and bonded a friendship that lasted until his passing. The amazing thing is that John somehow seems to have created this kind of personal time for each of us that were privileged enough to have worked with him, and we have all led richer, more meaningful lives because of it.
I was sorry to hear of John Hollandâ€™s death. Â John meant a lot to me. I was one of the first post-docâ€™s in his lab after he moved to Irvine, California. He was a very good mentor , and taught me the intricacies of cell culture Â and virology, since I arrived in his lab with no previous experience of cell culture. It was a fun time in the lab, we were exploring the differences in tRNA profiles in cancers and normal cells, and working on mengovirus restriction, projects I continued with later in my own laboratory. Â John created a very lively, questioning atmosphere. Â He basically posed a problem or question and then said “go to it”. Â He was a very modest person, but yet was willing to buck authority, particularly university administration. He and Dottie made us very welcome, and I felt really a part of this new institution (Irvine had only been open a year). There were always very lively discussions, not only of science but of the world at large. Â This was a small group, and we really worked as a team, Clayton Buck, Morrie Granger, and one or two students. Â John could be very hot tempered at times, but when his temper subsided there was no grudge. Â We had quite a number of Â undergraduate students around the lab, and I remember our conversations about avoiding the coffee when they were around, since this was the height of the LSD epidemic Â that was occurring in Southern Orange County (Laguna beach in the 1960â€™s ). I was attracted to Johnâ€™s lab because of his previous work with poliovirus. Amazing to think of how we worked in those days, using glass pipettes, mouth pipetting solutions including virus, glass prescription bottles instead of expensive plastic flasks, and most work done in the open bench. These were simpler bygone days.
Scott Vande Pol
John’s passing touched me, and has prompted some thoughts I am happy to share with you. Â HIs output as a scientist was pretty impressive (defining viral receptors, viral protein translation, Â the mechanism of viral persistence, the nature of RNA virus populations and evolution), but I think his life as a scientific mentor was certainly as great. Â Â As I noted to Bert, by modern theories of proper scientific mentorship (weekly documented meetings with progress reports and specific aims achieved on schedule) John was a terrible mentor because he did none of those things. Â But what a great mentor he was! Â He treated his students as his colleagues and equals. Â This man, who was smarter than me, more experienced, and more knowledgable, wanted to know MY opinion, because he was genuinely interested in what I thought. Â And then we would talk. Â This created a space into which we all had to grow, and it was a completely natural outcome of his humility. Â Our formal lab meetings were nonexistent, but in some ways continuous, because he was almost always in the lab. Â That does not mean it was all sweetness, because John had real scientific standards and would let you know when you did not meet them (but without crushing your spirit). Â Â I remember when Mark Spector from Racker’s lab came to give a seminar at UCSD, and as we were walking back I asked John what he thought about the seminar and he replied that the seminar was entirely untrue (actually, stronger words to that effect). Â He did not spare fools, and, he had a real temper that on rare occasions could be pretty ghastly. Â You did not want to be on the receiving end of that. Â But mostly he laughed a lot, and his lab was a place where games, tricks, and joy prevailed. Â I remember that a sure-fire trick was to put a message on the blackboard that some super-prominent scientist has called him leaving a return number, which was actually “dial-a-prayer” in Seattle or New York. Â John would always fall for this, but then laugh and laugh and laugh every time. Â It was a happy place.
In John’s lab we did not work for John, we worked for ourselves with John. Â When I came to the lab, he said: Â “You can do anything you want for your thesis as long as it involves RNA viruses.” Â That was it. Â It was up to me to decide what I wanted to do, even for a rotation project. Â So a student’s project in John’s lab belonged entirely to the student and John never told me what to do. Â Some students wallowed, hell, I wallowed months away too. Â But it developed in his students the ability to make things happen, because John was not going to do it for you. Â Really, a BIT of structure might have helped, but that was not John’s way. Â Â If you wanted advice about a specific idea, he was always there to give it, but your fate was entirely in your own hands. Â In retrospect, it seems Â to have worked well, although in today’s pressured environment few mentors seem to take that approach.
Without trying overtly, John attracted students who opened themselves to him, and part of him then seeped in. We cannot help being inspired by this man because we were changed by him. Â I bet you are going to hear comments such as these a lot.
John was the consummate academic scientist, and being aÂ student or postdoc in his lab was a unique experience. Even in the midst ofÂ teaching major introductory biology classes at UCSD, he worked in the labÂ everyÂ day and was always available for informal discussions at his bench or atÂ theÂ chalk board. During the later stages of his career, John maintained aÂ small-to-mediumÂ sized research group so that he could continue to interact personally withÂ hisÂ mentees every day. He also was also devoted to providing undergraduatesÂ withÂ the opportunity to experience a basic research environment. In the era ofÂ disposable plasticware and reagent kits, he continued to use glassÂ pipettes andÂ cell culture bottles so that students could be employed in his lab and getÂ aÂ taste of research. The best ones progressed to experimental work and manyÂ ofÂ his most talented undergraduates went on to graduate, medical or veterinaryÂ school. Unlike many senior faculty, John always found time to meet withÂ undergraduates to explain difficult concepts from biology classes or toÂ provideÂ career advice. Throughout his career, he influenced hundreds of studentsÂ through his caring teaching and mentoring.
Photo credit: Kathy Spindler
Since electron micrographs first revealed the bullet-shaped morphology of vesicular stomatitis virus (a virus related to rabies virus), understanding the architecture has been elusive. It was known that the RNA genome is wrapped in a helical structure by the viral nucleocapsid (N) protein, but how this structure was encased by the viral matrix (M) protein and the envelope was not clear. These questions have been elegantly answered by a new model of the VSV virion determined by cryo-electron microscopy.
The RNA genome of VSV is coated with many copies of the N protein to form aÂ nucleocapsid with helical symmetry. The nucleocapsid is in turn surrounded by the M protein and then the viral membrane. The reconstructed image (right) not only shows the helical nature of the N protein – RNA assembly, but reveals that the M protein also forms an outer helix. The inner and outer helices are aligned by interactions between N and M protein subunits. Consequently the virion is more rigid than enveloped viruses of other families, such as influenza viruses.
Both the inner (purple) and outer (magenta) lipid bilayers of the virion are visible in the reconstructed image. Close inspection of the inner leaflet (left) reveals that part of the envelope contacts each M protein subunit. In this area is a thin line that may represent part of the G glycoprotein that passes through the membrane. Virions attach to cell receptors via the G glycoprotein, which is embedded in the viral membrane. The interaction of G protein with M, known to be important for budding of viral particles, can now be visualized.
The VSV nucleocapsid contains 37.5 N protein subunits per helical turn, except near the bullet-shaped tip. The structure reveals that the angle of the N protein changes markedly to allow insertion of fewer subunits per turn. Assembly of the nucleocapsid probably begins at the tip of the bullet, as the 5′-end of the viral RNA genome becomes bound with N proteins. Six coils of larger diameter are formed, and then the angle of the N proteins change so that each ring contains 37.5 subunits.
These revealing new data are made even more beautiful in this movie, which shows the virion trunk rotated first around the helical axis, then around the horizontal axis. Parts of the membrane and the M and N protein helices have been removed to reveal the virion interior. The viral RNA is not visible as it is smaller than the resolution of the reconstructed image.
Ge, P., Tsao, J., Schein, S., Green, T., Luo, M., & Zhou, Z. (2010). Cryo-EM Model of the Bullet-Shaped Vesicular Stomatitis Virus Science, 327 (5966), 689-693 DOI: 10.1126/science.1181766