TWiV 478: A pox on your horse

The TWiV team explains how infectious horsepox virus – likely the ancestor of smallpox vaccines – was recovered from chemically synthesized DNA fragments.

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TWiV 464: Boston baked viruses

At Tufts University Dental School in Boston, Vincent speaks with Katya Heldwein and Sean Whelan about their careers and their work on herpesvirus structure and replication of vesicular stomatitis virus.

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Richard Elliott, virologist

Vincent and Richard ElliottVirologist Richard Elliott passed away on 5 June 2015. I have known Richard since 1979 and I would like to provide some personal recollections of this outstanding virologist. A summary of his work can be found at the MRC-University of Glasgow Centre for Virus Research science blog.

I first met Richard in 1979 when he joined Peter Palese’s laboratory at Mt. Sinai School of Medicine in New York City. We overlapped for only about a year but it was enough to get to know him: he was a hard-working, enthusiastic virologists and a good friend. We shared many beers in New York City. At the end of 1979 I went off to David Baltimore’s laboratory where in 1981 I produced an infectious DNA clone of poliovirus. It proved very difficult to make infectious DNAs of negative strand RNA viruses, and it was Richard who was the first to accomplish this feat in 1996 for a virus with a segmented genome. This work was very important as it showed that infectious DNA clones were not limited to RNA viruses with monopartite genomes.

I remained in contact with Richard over the years but I did not see him in person until the 2010 meeting in Edinburgh of the Society for General Microbiology. The following year he joined me, Connor Bamford, Wendy Barclay, and Ron Fouchier for TWiV #177 recorded in Dublin. Schmallenberg virus had just emerged as a new pathogen of livestock, and he discussed his work on this virus.

I next saw Richard in a 2011 meeting of the Brazilian Society for Virology. When I arrived in Brazil it was quite hot, and I found Richard sitting by the pool, reviewing manuscripts in his bathing suit. I snapped a few photos of him and put them on Facebook. Later that evening he said his laboratory had asked why pornographic photos of him were on the internet – he was shirtless in my pictures (with Grant McFadden in the photo).

Richard Elliott and Grant McFadden

Richard visited New York in the summer of 2014 but we were unable to connect. Early this year Richard had agreed to join TWiV again for an episode from Glasgow. Sadly he became too ill to participate and died on the Friday before I traveled to Scotland. While there I briefly visited the Elliott lab at the University of Glasgow MRC-Centre for Virus Research, nearly a week after his death. I’m happy that I made the lab members smile:

Elliott lab

I can still remember Richard telling me how to spell his name: two ls, two ts. Richard was an excellent virologist, mentor, and friend. I will miss him.

Update: Corrected to reflect the fact that Richard produced the first infectious DNA of a segmented (-) strand RNA virus.

Thirty years of infectious enthusiasm

infectious poliovirus dnaThirty years ago this month I did an experiment that set the course of my career, and provided an important step forward for animal virology. I showed that a cloned DNA copy of the poliovirus RNA genome is infectious in mammalian cells.

When I arrived as a postdoctoral fellow in the laboratory of David Baltimore in 1979, the restrictions placed on cloning complete genomes from pathogenic viruses in bacterial plasmid vectors had just been lifted. Consequently David suggested that I construct a full-length DNA copy of the poliovirus RNA genome, decode the genetic information, and determine if the DNA is infectious. By the fall of 1980 I had produced three different plasmids which contained overlapping DNA copies of poliovirus RNA. For most of the next year I worked on deciphering the complete 7,440-nucleotide sequence of the poliovirus genome. The methods I used – archaic by today’s standards – are worthy of a separate story.

With the viral genome sequence completed, I turned to joining the three plasmids to produce a single DNA copy of poliovirus RNA. I began this task early in 1981 but success eluded me for most of the first half of the year. In the summer I attended a Gordon Research Conference in New Hampshire, during which I presented the poliovirus genome sequence (together with Bert Semler, who had worked on the genome sequence while a postdoctoral fellow in the Wimmer laboratory). The poliovirus sequence was enthusiastically received, and I returned from the June meeting rejuvenated. By July I had assembled a full-length copy of poliovirus DNA in a bacterial plasmid. I called it pVR106, the sixth plasmid I had constructed while in the Baltimore lab.

In early July I attended a lab party at the Baltimore summer home in Woods Hole, MA. There I told David that I had assembled a full-length DNA copy of poliovirus RNA. The next task was to determine if this DNA was infectious. The logic for this experiment is shown in the figure. Poliovirus can initiate infection of cultured cells; poliovirus RNA, extracted from the virion, is also infectious when introduced into such cells by transfection. What would happen if we transfected pVR106 into cells? I asked David if I should first add a promoter sequence to pVR106, to allow synthesis of viral RNA from a DNA template. I remember his words clearly: “Don’t bother. Just transfect the plasmid into cells”. Then he went off to eat lobster.

The next week I sought help from Richard Mulligan, a postdoc in Phil Sharp’s lab and an expert at introducing DNA into cells. He set up two plates of CV-1 cells (African green monkey kidney cells, known to be susceptible to poliovirus) which we used in a transfection experiment done on 29 July 1981. The protocol for the experiment (jpg file) shows that we transfected one plate of cells with 10 micrograms of the plasmid vector, pBR322, and one plate with the same amount of pVR106. Immediately after transfection I removed 1 ml of the medium for virus titration. I also saved 1 ml of medium on each of the next two mornings, and on the third day broke open the cells in a small volume of buffer to determine if there were virus particles inside of the cells.

To determine if virus had been produced by DNA transfection, I did a plaque assay on the culture samples using monolayers of HeLa cells. The positive control for the plaque assay was poliovirus – which gave beautiful plaques (dish #4). No plaques were observed when cell culture medium from the zero time point of cells transfected with pVR106 was used; however infectivity was observed on days 1 and 2. Unfortunately, the negative transfection control was problematic – there were plaques in the lysate of cells transfected with the vector pBR322 (dish #8). I suspected that this sample had been contaminated with virus from one of the other samples, but I could not pinpoint the source.

Just before I began this experiment, David left for a meeting in Europe – the widely attended International Congress of Virology. He had left instructions to call him if the experiment worked. I wasn’t sure if I should tell him yet, having observed plaques on the negative control. So I showed the results to Bob Weinberg – his lab was on the same floor and I often asked him for advice. He looked at the plaques and told me the experiment looked great – pVR106 was most likely infectious. But he told me not to tell David until I had repeated the experiment. “If you tell David, the world will find out”, he said, and that should not happen until the results were confirmed.

So I didn’t call David, and I repeated the experiment on 12 August 1981. This time the results were very clear: cells transfected with pVR106 produced poliovirus, and cells transfected with the vector pBR322 did not. When David returned from Europe, I went to tell him, but Weinberg had reached him first. “Be a hero”, David said to me.

To this day we do not understand why cloned poliovirus DNA is infectious. The most likely explanation is that the plasmid vector contains a sequence that acts as a promoter for RNA synthesis – what we call a cryptic promoter. Some years later Bert Semler inserted a promoter sequence upstream of poliovirus DNA, and found that this DNA is more infectious than pVR106.

Since this work, infectious DNA clones have been produced for many other DNA and RNA viruses. These reagents are double-edged swords: they enable manipulation of the viral genome at will, allowing unprecedented genetic analysis and the use of viruses as vectors for gene therapy. But nearly any virus can now be recovered from the nucleotide sequence – effectively making it impossible to ever truly eradicate a virus from the globe.

I was very lucky to be in the right place at the right time to accomplish this work. It opened doors for me: without an infectious poliovirus DNA clone, I would likely not have obtained a position at Columbia University, where I have been fortunate to work with many talented students, postdoctoral fellows, and technicians who have accomplished outstanding work. One of those students was Eric Moss, who I thank for reminding me of what transpired 30 years ago.

Racaniello, V., & Baltimore, D. (1981). Cloned poliovirus complementary DNA is infectious in mammalian cells Science, 214 (4523), 916-919 DOI: 10.1126/science.6272391

Viruses and journalism: Off-the-shelf chemicals

artificial_poliovirusI have had many opportunities to speak with journalists of different kinds during the more than 30 years that I have studied viruses. I wrote previously about my negative experience with CNN. I’d like to relate a much more positive encounter with newspaper reporters.

As a postdoctoral fellow in David Baltimore’s laboratory I was fortunate to have found that a cloned DNA copy of the RNA genome of poliovirus is infectious in mammalian cells. It was the first such finding for an animal virus and made it possible to create and genetically modify viruses.

These findings attracted a good amount of press coverage. On November 15, 1981, the New York Times published an article by Harold Schmeck entitled “Polio Virus Made With Artificial Genetic Material” (click image above for a larger view). I was a bit taken aback by the headline: up to that point in my career I’d had few encounters with journalists. What did he mean by ‘artificial genetic material’? I’d placed the viral genome in a bacterial plasmid, which I didn’t view as artificial nucleic acid. The article began:

Artificially fabricated genetic material has been used for the first time to produce an active, infectious polio virus in the laboratory…The genetic material was of a kind never used by the virus in nature. Furthermore, the starting material was fabricated from off-the-shelf chemicals.

It’s true that poliovirus nucleic acid is never found in a bacterial plasmid in nature. But what did he mean by ‘off-the-shelf chemicals’? Then I realized: we had explained to Mr. Schmeck that poliovirus RNA was converted to a DNA copy using the enzyme reverse transcriptase. The enzyme was provided with the four nucleoside triphosphates – ATP, TTP, CTP, and GTP – which were used to produce the DNA copy. Those were the ‘off-the-shelf chemicals’: not exactly the kind of thing you might find in the local supermarket, but I got the analogy. In the end I liked the article; it was factually correct and presented in an engaging manner.

On the following Sunday, in ‘News of the Week in Review’, the Times included a short piece on our results in the ‘Ideas and Trends’ section entitled ‘Reinventing a Polio Virus’ (click to view). It began:

Off-the-shelf chemicals are seldom the stuff of which genetic material is made. But scientists at Massachusetts Institute of Technology used just that to manufacture the genetic core of a polio virus. Then, even the scientists were surprised. When tested on cell samples, the artificially contrived virus appeared indistinguishable from the real thing; both initiated polio infection.

I suppose the off-the-shelf analogy helped make it clearer to readers that we had done something different. In 1981 there was no Science Times section and readers did not have the amount of science journalism that we have today.

After Mr. Schmeck’s article was published, he told us that it was originally scheduled for the front page of the newspaper. But November 15, 1981 was the day the first space shuttle was launched, and as a result our story was pushed to the inner pages. Not bad, though, for my first encounter with journalists.

Later in 1981 I was interviewed for a BBC radio program about my work on poliovirus. I’ve located a recording of that interview and will post it here tomorrow.

Racaniello, V., & Baltimore, D. (1981). Cloned poliovirus complementary DNA is infectious in mammalian cells Science, 214 (4523), 916-919 DOI: 10.1126/science.6272391

Infectious DNA clones

img_0420The development of recombinant DNA methods by Cohen and Boyer in 1973, together with the discovery of reverse transcriptase by Temin and Baltimore in 1970, made it possible to introduce a mutation at any location in a viral genome. The essential reagent is an infectious DNA clone, a double-stranded DNA copy of the viral genome carried in a bacterial plasmid. These DNAs (or RNAs produced from them) can be introduced into cells by transfection1 to produce infectious virus.

Infectious clones of viral genomes were initially produced in the late 1970s and early 1980s. The first was made in 1978 by inserting a DNA copy of the RNA genome of the bacteriophage QB, made with reverse transcriptase, into a plasmid vector. Infectious virus was produced when the cloned viral DNA was inserted into E. coli. In 1980,  infectious cloned retroviral DNA was produced by inserting the integrated viral DNA from the cellular genome into a plasmid vector. The next year, a DNA copy of the RNA genome of poliovirus was produced by reverse transcription and inserted into a plasmid vector. When the cloned copy of the viral genome was introduced into mammalian cells, infectious virus was produced.

Since these early findings, infectious DNAs of members of nearly every virus family have been reported. Some have been more difficult to produce than others. For example, to recover infectious influenza virus from cloned DNA, an expression system is used in which cloned DNA copies of the eight RNA segments are flanked by two promoters. Upon introduction of the eight plasmids into cultured cells, two types of RNAs are produced: mRNAs for the synthesis of viral proteins, and viral RNAs for replication and incorporation into virions. The production of infectious DNAs of  (-) strand RNA viruses was counterintuitive. The (-) strand genomic RNA of these viruses is not infectious because it cannot be translated or copied into mRNA in the cell. In the first attempts, full-length (-) strands, produced by in vitro transcription of cloned DNA, were introduced into cells that produce the proteins required for mRNA synthesis. However, no infectious virus was recovered. The solution, found first with rabies virus, was to transfect full-length (+) strand RNA into cells that produce the viral nucleocapsid protein, phosphoprotein, and polymerase. In these cells, the (+) strand RNA is copied into (-) strand RNAs which then initiate an infectious cycle.

The double-stranded RNA genome of reoviruses is not infectious because it cannot be translated. To produce an infectious clone, DNA copies of the genome segments are placed in plasmids under the control of a T7 RNA polymerase promoter. When all 10 plasmids are introduced into cells that synthesize T7 RNA polymerase, viral mRNAs are produced which initiate an infectious cycle.

The complete genomes of many DNA viruses, including polyomaviruses, papillomaviruses, and adenoviruses, are sufficiently small to be carried in plasmid vectors. However, conventional plasmid vectors cannot accommodate the larger DNA genomes of herpesviruses and poxviruses; therefore cosmids and bacterial artificial chromosomes vectors, which can accept larger inserts, have been used. Such vectors have also been used to carry DNA copies of the largest RNA genomes, those of members of the Nidovirales. Poxvirus DNA is not infectious, because cellular DNA-dependent RNA polymerase cannot recognize the viral promoters. Viral DNA-dependent RNA polymerase and transcription proteins must therefore be provided.

The infectious viral DNA clone is a double-edged sword.  It enables manipulation of the viral genome at will, allowing unprecedented genetic analysis and the use of viruses as vectors for gene therapy. But nearly any virus can now be recovered from the nucleotide sequence – effectively making it impossible to ever truly eradicate a virus from the globe.

1The introduction of DNA or RNA into cells with the object of obtaining infectious virus is called transfection (transformation-infection). This phrase was originally coined to describe production of bacteriophage lambda after transformation of cells with viral DNA. Transfection is now incorrectly used to describe the introduction of any DNA into cells. This usage has come about to avoid confusing DNA-mediated transformation with the process of oncogenic transformation.

Taniguchi T, Palmieri M, & Weissmann C (1978). A Qbeta DNA-containing hybrid plasmid giving rise to Qbeta phage formation in the bacterial host [proceedings] Annales de microbiologie, 129 B (4), 535-6 PMID: 754572

Lowy DR, Rands E, Chattopadhyay SK, Garon CF, & Hager GL (1980). Molecular cloning of infectious integrated murine leukemia virus DNA from infected mouse cells. Proceedings of the National Academy of Sciences of the United States of America, 77 (1), 614-8 PMID: 6244569

Racaniello, V., & Baltimore, D. (1981). Cloned poliovirus complementary DNA is infectious in mammalian cells Science, 214 (4523), 916-919 DOI: 10.1126/science.6272391

Schnell MJ, Mebatsion T, & Conzelmann KK (1994). Infectious rabies viruses from cloned cDNA. The EMBO journal, 13 (18), 4195-203 PMID: 7925265

KOBAYASHI, T., ANTAR, A., BOEHME, K., DANTHI, P., EBY, E., GUGLIELMI, K., HOLM, G., JOHNSON, E., MAGINNIS, M., & NAIK, S. (2007). A Plasmid-Based Reverse Genetics System for Animal Double-Stranded RNA Viruses Cell Host & Microbe, 1 (2), 147-157 DOI: 10.1016/j.chom.2007.03.003