Viral fiber art

dengue virus pillowViruses inspire many different types of art, but I was unaware of the number of people who make viruses out of fiber!

On This Week in Virology #266 we heard from Emily who had knitted a dengue virus pillow (photograph at left).

The next week on TWiV #267 we heard from Carolyn who had knitted a picornavirus (photo below).

knit picornavirus

The following week (TWiV #268) we heard from Jessica who has also knitted two different icosahedral structures.

knit icoshadedron

This made me wonder how many people knit viruses, so I searched Ravelry for ‘virus’. Here are some of the interesting creations I found.

Cold virus by Krista:

Cold virus

Dawn’s cold virus (rhinovirus):

rhinovirus

Melini’s phage hat:

phage hat

Two H1N1 influenza viruses:

h1n1 knit

And Susan’s bacteriophage:

knit bacteriophage

There are also bacteria, such as this collection (with some viruses) from Clare:

knit microbes

You can find more by searching for ‘microbe’ at Ravelry (login required), where you’ll also find the patterns to reproduce these wonderful creations. Microbes are clearly inspiring and fascinating to fiber artists!

Do you make fiber viruses? If so let me know and we can include a photograph here.

TWiV 262: Wrong form, right professor

On episode #262 of the science show This Week in Virology, Vincent returns to the University of Wisconsin – Madison to speak with Ann Palmenberg about her career in virology.

You can find TWiV #262 at www.microbe.tv/twiv.

Viruses might provide mucosal immunity

T4 HocThe mucosal membranes that line our respiratory, alimentary, and urogenital tracts and the outer surface of the eyes are portals of entry for microbes. The cells at these surfaces have functions that require that they are exposed to the environment – for example, gaseous exchange in the lung between inspired air and the blood. Mucus, pH extremes, enzymes, and immune cells are some of the antimicrobial defenses that are present at various mucosal surfaces. It now appears that bacteriophages – viruses that infect bacteria – might also be part of the mucosal antimicrobial defense system.

A sampling of the ratio of bacteriophages to bacteria in a variety of mucosal surfaces (sea anemone, hard and soft coral, polychaete, teleost, human gum, and mouse intestine) revealed higher ratios when compared to non-mucosal samples (e.g. neighboring sea water or saliva). A model bacteriophage of E. coli, T4, was used to show that phage specifically attach to mucus: adherence to cultured cells was reduced when mucus was not produced or removed by chemical treatment.

The principal macromolecules in mucus are mucin glycoproteins, which consist of a polypeptide chain linked to hundreds of variable, branched sugar molecules. Mucins are continuously produced at mucosal surfaces which gives rise to a thick protective layer. Phage T4 was found to attach specifically to mucins and not other components of mucus such as protein or DNA. The attachment of phage T4 to mucus-producing cultured cells reduced the number of bacteria that could attach to and kill the cells. This antimicrobial effect was substantially reduced when a strain of phage T4 was used that cannot lyse its bacterial host. Therefore phages bound in mucus protect cells by infecting and lysing bacterial invaders.

Phage T4 attaches to mucins through immunoglobulin-like (Ig-like) proteins present in the viral capsid. First discovered in antibody molecules, the Ig domain has since been found in hundreds of different proteins with various functions and appear to be encoded in ~25% of dsDNA phages. The Ig domain, typically 80 amino acids in length, is often involved in interactions with other proteins or ligands. For example, the Ig domains of antibodies interact with antigens, and the poliovirus receptor interacts with poliovirus via an Ig domain on the receptor molecule. The capsid of phage T4 contains 155 copies of an Ig-like protein called Hoc (colored yellow in the image). Deletion of the phage T4 hoc gene reduced binding of the virus to mucin, showing that adherence to mucin requires Ig-like protein domains.

These results demonstrate that a model bacteriophage, T4, attaches to mucus via an interaction between viral Ig-like capsid proteins and mucins. The ability of phages to attach to mucin clearly helps protect cultured cells from bacterial attachment and killing. Bacteriophages may be part of a previously unrecognized mucosal immune defense system. This suggests a symbiotic relationship between phages and metazoan hosts: the phages provide protection to mucosal surfaces, and in turn are provided hosts (bacteria) in which to reproduce. However, additional experiments are required to prove the authors’ conclusion of a “key role of the world’s most abundant biological entities in the metazoan immune system”. It will be necessary to directly demonstrate that phages attaching to mucins in mucus can protect an animal (e.g. mice) from bacterial invasion. This will not be an easy experiment because the phage and bacteria composition of mucus is likely to be complex and continuously changing as mucus is sloughed from cells and new mucins are produced.

The finding that phages play roles in mucosal immunity would have far-reaching consequences for human health. Some fascinating questions that come to mind include: do phage populations play roles in human diseases? Are they altered in human diseases and can we correct these diseases by restoring phage populations? Are the phage populations altered by antimicrobial therapy that alters bacterial populations? Do phages contribute to development of the immune system by modulating bacterial populations? Might mucus-bound phages stabilize the microbiome?

Update: Michael Schmidt and I discussed these remarkable findings on episode #59 of the science show This Week in Microbiology.

Live from the Society for General Microbiology Conference in Manchester, UK

MicrobeWorld and the Society for General Microbiology (UK) to live stream two events from their Spring Conference 2013 in Manchester, England, March 25-28.

Peter Wildy Prize for Microbiology Education
Monday, March 25, 2013 17:20 GMT (1:20 PM EST | 10:20 AM PST)  

David Bhella, Ph.D., will be accepting the Peter Wildy Prize for Microbiology Education, awarded annually by the Society for General Microbiology for an outstanding contribution to microbiology education. Bhella’s acceptance speech will be live streamed at 17:20 GMT (1:20 PM EST | 10:20 AM PST). Vincent Racaniello was awarded the Wildy Prize in 2012.

This Week in Microbiology
Wednesday, March 27, 2013 15:30 GMT (11:30 AM EST | 8:30 AM PST) 

Join Vincent Racaniello and co-host Laura Piddock, Ph.D., with guests Paul Williams, Ph.D., Kalin Vetsigian, Ph.D., and David Harper, Ph.D., for a live-streaming episode of This Week in Microbiology. The live stream starts at 15:30 PM GMT (11:30 AM EST | 8:30 AM PST) and you can watch it below. If you have any questions for Vincent or his guests during the broadcast you can tweet your question using the #sgmman hash tag or type it into the chat function of the video player.

If you live elsewhere in the world, please use www.everytimezone.com, to calculate when the live streams will start in your area.

 

(If you don’t see the video and it is after the official start time please press the play button or refresh the page.)
 

TWiV 211: Viruses r us

On episode #211 of the science show This Week in Virology, the TWiV four discuss an mRNA-based influenza vaccine, and a phage tubulin that forms a filamentous array in the host cell that is needed for positioning viral DNA.

You can find TWiV #211 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. 

A spike for piercing the cell membrane

bacteriophage t4Some viruses that infect bacteria (bacteriophages) deliver their DNA into the host cell with an amazing injection machine. The tailed bacteriophages (such as T4, illustrated) store their DNA in a capsid attached to a long tail tube that is surrounded by a sheath. At the bottom of the tube is a baseplate with a spike in the center. When the baseplate contacts the host cell, the sheath contracts, driving the spike into the cell membrane. The viral DNA travels down the tube and enters the cell through the opening produced by the spike. The structure of the spike has now been determined, providing insight into how it makes a hole in the cell membrane.spike

Structures of the spike from P2, a well studied virus of E. coli, and the choleraphage phi92 were determined.  The spikes are built from three copies of a single protein (trimers). The trimers are indeed shaped like spikes: they are wider at one end and taper to a rather sharp tip (figure at right). The bulk of the spike is made up of alternating beta-strands which form a corkscrew-like beta-helix. The sharp tip is composed of three beta-hairpins which the authors say “come together like petals in a flower bud”.

iron in spikeAn interesting feature on the interior of the spike tip are three pairs of histidine residues that hold a single iron atom (figure at left). The authors believe that the iron helps the trimers form by keeping the protein chains in register, and also provides increased strength to the tip. The latter would be important as it pierces the cell membrane. This idea could be tested by changing one or more histidines to another amino acid so that iron cannot be held in the tip.

To verify that these structures are those of the spike that is attached to the baseplate, the authors solved the structure of the phage particle by cryo-electron microscopy and image reconstruction. The image clearly shows the spike protruding from the base plate (figure below). The structures of the spike proteins solved by X-ray crystallography could then be computationally fitted in the correct location in the cryo-EM image of the baseplate.baseplate with spike

These structures support the idea that the spike is a rigid needle that pierces the bacterial membrane and forms a channel through which the DNA can pass. An interesting question is how the DNA gets past the spike, which plugs the end of the tail tube. The authors believe that the spike is loosely attached to the tube and might be easily dissociated once it passes through the cell membrane. The spike of phage T4 can be dissociated at low pH, a condition that is found in the periplasm, the space between the inner and outer bacterial membranes.

There are distinct signatures of the spike structure that can be identified in proteins of other contractile injection systems, including diverse bacteriophages. They can also be found in bacterial type VI secretion systems, which are membrane complexes used to transport proteins outside of the cell. Once evolution builds a useful machine, it is often put to many diverse uses.

 
Browning, C., Shneider, M., Bowman, V., Schwarzer, D., & Leiman, P. (2012). Phage Pierces the Host Cell Membrane with the Iron-Loaded Spike Structure, 20 (2), 326-339 DOI: 10.1016/j.str.2011.12.009

Norton Zinder, 1928-2012

phage ms2Norton Zinder made two important discoveries in the field of virology. While a Ph.D. student with Joshua Lederberg at the University of Wisconsin-Madison he found that viruses of bacteria (bacteriophages) could move genes from one host to another, a process called transduction. Later in his own laboratory at The Rockefeller University he isolated the first bacteriophages that contain RNA as genetic material. These were seminal findings in the growing field of molecular biology.

By the 1950s it was well known that different strains of the bacterium Escherichia coli could exchange genes in a process called recombination. Zinder wanted to know if other bacteria could also exchange genes in a similar manner, and therefore began to study Salmonella typhimurium. The strains that Zinder used for his experiments were lysogens: their chromosomal DNA contained integrated copies of the DNA genomes of bacteriophages. Zinder readily detected genetic exchange in Salmonella, but suspected that the latent phages might play a role. To test this idea, he grew Salmonella in tubes that were connected by a fine filter that allowed passage of viruses but not bacteria between the two cultures. The results showed that a filterable agent, or virus, could mediate the exchange of genetic material between bacterial strains; direct contact between bacteria was not necessary. The authors coined transduction to describe this process. We now understand that transduction occurs because bacteriophages may incorporate bacterial DNA into the viral particle. Transduction remains a common tool to stably introduce a foreign gene into a host cell.

Zinder describes the discovery of RNA-containing bacteriophages, which took place after he had moved to The Rockefeller University, in the Preface to RNA Phages:

In the late fifties, Tim Loeb, a new graduate student at The Rockefeller University, came into my laboratory and asked whether I thought it was possible that there were male-specific bacteriophages for E. coli. I….quickly responded in the affirmative and off he went to a raw sewage plant in New York City. …f2, the second isolate, was chosen for further study. Little did we think at the time that a whole new area of study was in the offing….

The first two bacteriophages that Loeb had isolated from New York City sewage were called f1 and f2. During purification of the phages it was clear that the genome of f1 was DNA. Chemical analyses subsequently demonstrated that the genome of f2 was RNA (later found to be positive-strand RNA). Similar phages were since isolated all over the world, and their study provided much basic information on viral replication, protein biosynthesis, and genome replication. The first genome sequence determined was in 1976 for the related RNA bacteriophage MS2.

Update: Moving eulogy by Jeffrey Ravetch in Eulogy for a brilliant mentor and teacher.

Loeb, T. (1961). A Bacteriophage Containing RNA Proceedings of the National Academy of Sciences, 47 (3), 282-289 DOI: 10.1073/pnas.47.3.282

Robert A. Weisberg, 1937-2011

weisbergRobert A. Weisberg was a Scientist Emeritus at NCI until the time of his death on 1 September 2011. Previously he was Chief of Microbial Genetics at NICHHD, a position he retired from in 2008. He was a pioneer in the study of the bacteriophage lambda. His research lead to seminal contributions about how bacteriophage lambda integrates into the E. coli chromosome. His laboratory produced the first library of cloned genes, using lambda transducing phages. Weisberg’s work was a combination of biochemical and genetic approaches, and he was an expert in both disciplines.

My colleague Max Gottesman was a good friend of Weisberg and writes:

Bob was a wonderful colleague; he shared freely his innovative ideas with others, had no issues with authorship, and welcomed the success of others – in short, a model citizen/scientist. He leaves behind a substantial scientific legacy that taken together established lambda as a model system for the study of recombination and gene control and an important tool for bacterial genetics. And those who knew him personally feel that he enriched their lives, and that an era has ended with his passing.

Weisberg’s recent review, co-authored with Gottesman, Little lambda, who made thee?, is an excellent review of the virus and its contribution to the field of molecular biology.

TWiV 145: The inVinceable TWiV

twortHosts: Alan Dove and Rich Condit

Alan and Rich tackle the discovery of bacteriophages, and treating influenza by calming the cytokine storm.

Click the arrow above to play, or right-click to download TWiV 145 (63 MB .mp3, 87 minutes).

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, by email, or listen on your mobile device with the Microbeworld app.

Links for this episode:

Weekly Science Picks

Alan – AT&T Tech Channel
Rich –
Smallpox and its eradication (pdf)

Listener Pick of the Week

AsifScience Photo Library

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.