TWiV 291: Ft. Collins abuzz with virologists

Vincent, Rich, and Kathy and their guests Clodagh and Ron recorded episode #291 of the science show This Week in Virology at the 33rd annual meeting of the American Society for Virology at Colorado State University in Ft. Collins, Colorado.

You can find TWiV #291 at www.microbe.tv/twiv.

TWiV 259: Windows into the soul of a cell

On episode #259 of the science show This Week in Virology, Vincent and Rich join Jackie at the University of Texas, Austin to talk about her work on mouse mammary tumor virus.

You can find TWiV #259 at www.microbe.tv/twiv.

From a food blender to real-time fluorescent imaging

single phage infectionAlthough Avery, MacLeod, and McCarty showed in 1944 that nucleic acid was both necessary and sufficient for the transfer of bacterial genetic traits, protein was still suspected to be a critical component of viral heredity. Alfred Hershey and Martha Chase showed that this hypothesis was incorrect with a simple experiment involving the use of a food blender. The Hershey-Chase conclusion has since been upheld numerous times*, the most recent by a modern-day experiment using real-time fluorescence.

Hershey and Chase made preparations of the tailed bacteriophage T2 with the viral proteins labeled with radioactive sulfur, and the nucleic acids labeled with radioactive phosphorus. The virions were added to a bacterial host, and after a short period of time were sheared from the cell surface by agitation in a blender. After this treatment, the radioactive phosphorus, but not the radioactive sulfur, remained associated with bacterial cells. These infected cells went on to produce new virus particles, showing that DNA contained all the information needed to produce a bacteriophage.

In a modern validation of the Hershey-Chase experiment, bacteriophages are mixed with a cyanine dye which binds to the viral DNA (illustrated). Upon infection of the bacterial host, the phage DNA is injected into the cell together with the dye. In time the dye leaves the phage DNA and binds to the host genome. This process can be observed in real-time (as it happens) by fluorescence microscopy.

This technique was used to visualize single bacteriophages infecting an E. coli host cell. It takes about 5 minutes on average for 80% of bacteriophage lambda DNA to exit the capsid, with a range of 1-20 minutes.

These experiments do not simply provide a visual counterpart to the Hershey-Chase conclusion, but reveal additional insights into how viral DNA leaves the capsid. One interesting observation is that the amount of DNA that remains in the capsid apparently is not the sole determinant of how quickly ejection occurs. The amount of DNA ejected from the capsid does appear to regulate the dynamics of the process.

The kitchen blender experiment contrasts vividly with the complexity of real-time fluorescent imaging. Hershey and Chase did not have the technology to visualize phage DNA entering the host cell; they used what was available to them at the time. While improved technology is important for pushing research forward, simple experiments will always make important contributions to our understanding of science.

*The infectivity of cloned viral DNA is one validation of the Hershey-Chase experiment.

Hershey, AD, Chase, M. 1952. Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36:39-56. 

Van Valen, D., Wu, D., Chen, Y-J, Tuson, H, Wiggins, P, Phillips, R. 2012. A single-molecule Hershey-Chase experiment. Current Biol 22:1339-1343. 

TWiV 174: Dog runs and mooing miRs

On episode #174 of the podcast This Week in Virology, Vincent, Alan, and Rich consider whether pet dogs might transmit human noroviruses, and an RNA virus microRNA that might be involved in oncogenesis.

You can find TWiV #174 at www.microbe.tv/twiv.

Renato Dulbecco, 1914-2012

wee plaques 1952For the second time in a week I note the passing of an important virologist. Renato Dulbecco, together with David Baltimore and Howard Temin, received the 1975 Nobel Prize in Physiology or Medicine for discoveries about how tumor viruses interact with the genetic material of the cell. Dulbecco also devised my favorite virological method, the plaque assay, for determining the virus titer, the number of animal viruses in a sample.

Since the early 1920s bacteriologists had used the plaque assay to quantify the number of infectious bacteriophages (viruses that infect bacteria). Dulbecco noted in 1952 that “research on the growth characteristics and genetic properties of animal viruses has stood greatly in need of improved quantitative techniques, such as those used in the related field of bacteriophage studies.” One limiting factor was the development of suitable animal cell cultures that could be used to determine viral titer. By the 1950s the techniques for reliably producing and propagating human cell cultures were developed, and in 1951 the first immortal human cell line, HeLa, was isolated. Dulbecco took advantage of these advances and showed in 1952 that western equine encephalitis virus formed plaques on monolayers of chicken embryo fibroblasts (figure). Dulbecco also made the important observation that one virus particle is sufficient to produce one plaque. He drew this conclusion from his observation of a linear dependence of the number of plaques on virus concentration. This seminal advance made possible the application of genetic techniques to the study of animal viruses.

Dulbecco’s work on tumor viruses was focused on polyomaviruses – small DNA-containing viruses such as murine polyomavirus and SV40. He found that cells from the natural host of the virus – mice for polyomavirus and monkeys for SV40 – were killed as the viruses replicated and produced new viral progeny. However, these viruses did not replicate in or kill cells from other animals. For example, when hamster cells were infected with murine polyomavirus, no viral replication took place, the cells survived, and a few rare cell were transformed  – their growth properties in culture were altered and they induced tumors when injected into hamsters. Dulbecco later found that the polyomaviral DNA is a circular, double-stranded molecule; and that in non-permissive cells (in which the virus does not replicate) the viral DNA became integrated into the host cell chromosome. He also suspected that a viral protein called T (for tumor) antigen was a key to cell transformation.

Today we understand why polyomaviruses transform cells in which they do not replicate: infection does not kill these cells, and the rare transformed cells contain only viral DNA encoding T antigen. This protein is needed for viral replication in permissive cells because it drives cell proliferation, activating cellular DNA replication systems that are required for producing more viral DNA. In a non-permissive cell, T antigen drives the cell to divide endlessly, immortalizing it and allowing the accumulation of mutations in the cell genome that make the cells tumorigenic.

While the details of how DNA tumor viruses transform cells were being elucidated, other investigators were attempting to understand how another class of viruses – with RNA genomes – had similar effects on cells. In 1951 a young scientist named Howard Temin joined Dulbecco’s laboratory to study how Rous sarcoma virus (RSV) caused tumors. This virus had been discovered by Peyton Rous in 1911, but would only cause tumors in chickens, limiting progress. In Dulbecco’s laboratory, Temin found that RSV induced transformation of cultured chicken embryo fibroblasts – the same types of cells that were being used to develop the plaque assay for animal viruses. Temin took this transformation assay to his own laboratory, where he reasoned that a DNA copy of the RSV viral genome must be integrated into the chromosome of transformed cells. This led him to discover the enzyme reverse transcriptase in RSV particles, which produces a DNA copy of the viral RNA.

By embracing a new technology for the study of animal viruses – cell culture – Dulbecco set the study of both DNA and RNA tumor viruses on a path that would lead to understanding viral transformation, an achievement recognized by the 1975 Nobel Prize.

Dulbecco, R. (1952). Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus Proceedings of the National Academy of Sciences, 38 (8), 747-752 DOI: 10.1073/pnas.38.8.747

TWiV 80: How much X could a woodchuck chuck?

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Michael Bouchard

Vincent, Alan, and Rich speak with Michael Bouchard about hepatitis B virus discovery, replication, and pathogenesis.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #80 (58 MB .mp3, 80 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email.

Links for this episode:

Weekly Science Picks

Rich PBS Frontline: The Vaccine War
Alan
Readability
Vincent Starswarm by Jerry Pournelle

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

Virology lecture #19: Transformation and oncogenesis

Get the Flash Player to see this player.


Download: .wmv (352 MB) | .mp4 (89 MB)

Visit the virology W3310 home page for a complete list of course resources.

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