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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Nels joins the TWiV team to talk about his work on genomic accordions in vaccinia virus, hepatitis B virus in a 439 year old mummy, and viral induction of energy synthesis by a long noncoding RNA.
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Show notes at microbe.tv/twiv
I just love it when long standing mysteries in virology are suddenly solved, typically by the use of new technologies. In this story, the long standing mystery was why poxvirus mRNAs have a stretch of poly(A) in their 5′-noncoding regions. The answer is that it allows the ribosome to preferentially translate these viral mRNAs over those of the host (link to paper).
Ribosomes, the sites of protein synthesis, are very large assemblies of RNAs and proteins. A ribosome-associated protein called RACK1 is the major player in this story. This protein was previously found to be important for translation of viral RNAs by internal ribosome entry, a process in which ribosomes bypass the 5′-cap structure on mRNAs.
Cells lacking the gene encoding RACK1 seem to be just fine – they grow normally. This result would suggest that RACK1 is not needed for translation of mRNAs via the 5′-cap structure. But the translation of mRNAs of vaccinia virus, a poxvirus, is blocked in cells lacking RACK1. More specifically, the viral mRNAs produced later in infection cannot be translated.
This effect of RACK1 on vaccinia virus mRNA translation prompted a closer look at the protein in virus infected cells – which revealed that it is phosphorylated by a viral protein kinase. Phosphorylation of RACK1 makes the ribosomes preferentially translate vaccina virus mRNAs – an effect that is completely dependent upon the poly(A) in the 5′-untranslated region! Transfer of this poly(A) sequence to a non-viral mRNA enhanced its translation in cells in which RACK1 is phosphorylated.
The amino acid sequence of RACK1 that is phosphorylated is within a loop that contacts the 18S ribosomal RNA subunit. An examination of RACK1 proteins from different species reveals that in plants, unlike in mammals, this loop contains a stretch of negatively charged amino acids. Because a phosphate is negatively charged, it made perfect sense to see if the plant loop sequence, when transferred to mammalian RACK1, could stimulate translation of poly(A) containing mRNAs. It did!
In other words, phosphorylation by a vaccinia virus protein kinase makes RACK1 plant-like! Which is why the authors called their paper ‘Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase”. Exactly what RACK1 protein is doing in plants is a burning question, as is how RACK1 recognizes mRNAs containing a poly(A) leader.
Phosphorylation of RACK1 by the vaccinia virus kinase explains why viral mRNAs with poly(A) leader sequences are preferentially translated in infected cells (late viral mRNAs have this unusual leader). The presence of poly(A) in the 5′-ends of late vaccinia virus mRNAs is considered to be a consequence of polymerase slippage – but it is clearly not a mistake. At one time, long ago, a polymerase might have slipped for the first time, but the resulting poly(A) in the mRNA conveyed a selective advantage that remained. Today I would no longer call the addition of poly(A) to vaccinia virus mRNAs an error!
Why did it take so long to figure out the role of poly(A) at the 5’-end of vaccinia virus mRNAs? The right technology was not available – the key ones were the ability to knock out specific genes in cells, to knock down mRNA levels with RNA interference, and to identify the sites of RACK1 phosphorylation by mass spectrometry. These are not only recently developed techniques, but they have become widely accessible. But don’t forget the use of long-ago isolated vaccinia virus mutants of the protein kinase that were an essential part of this story.
Rich Condit, a poxvirologist, joined Nels Elde (also a poxvirologist, though he dabbles in other systems) and me to discuss this very cool paper on episode #21 of TWiEVO, This Week in Evolution. Rich had some very good insight on this paper; plus he bristled twice during the episode. You’ll have to listen to find out why.
On episode #329 of the science show This Week in Virology, the TWiV team reviews identification of immune biomarkers in CFS/ME patients, and how a cell nuclease controls the innate immune response to vaccinia virus infection.
You can find TWiV #329 at www.microbe.tv/twiv.
On episode #284 of the science show This Week in Virology, the TWiV team discusses how skin scarification promotes a nonspecific immune response, and whether remaining stocks of smallpox virus should be destroyed.
You can find TWiV #284 at www.microbe.tv/twiv.
On episode #203 of the science show This Week in Virology, Vincent and Rich meet up with Mark Challberg to talk about his scientific career studying viral DNA replication, and his transition to an NIH Program Officer.
You can find TWiV #203 at www.microbe.tv/twiv.
On episode #198 of the science show This Week in Virology, Vincent, Alan, Rich, and Kathy review fatal avian influenza virus in harbor seals, and poxvirus deployment of genomic accordions to counter antiviral defenses.
There once was a virus named pox
Whose genome contained a squeeze-box
When placed under pressure
It expanded its measure
Overcoming the new cellular blocks
You can find TWiV #198 at www.microbe.tv/twiv.
On episode #194 of the science show This Week in Virology, Vincent returns to Madison, Wisconsin and meets with postdocs to discuss their science and their careers.
You can find TWiV #194 at www.microbe.tv/twiv.
One of the most important procedures in virology is measuring the virus titer – the concentration of viruses in a sample. A widely used approach for determining the quantity of infectious virus is the plaque assay. In this technique, the spread of progeny viruses released by individually infected cells is restricted to neighboring cells by a semisolid medium. Consequently, each infectious particle produces a circular zone of infected cells called a plaque. By imagining live, virus-infected cells using a microscope, beautiful movies have been made which show how a plaque develops in real time.
To produce the movies, cells were infected with vaccinia virus, covered with a semi-solid medium, and placed in an incubator. The monolayers were examined periodically until a small plaque became visible. The infected cells were then placed on an inverted microscope fitted with a camera. Images of the plaque were taken every hour for 12-19 hours and assembled into a movie.
The first movie shows plaque formation on monkey cells infected with vaccinia virus. The virus infection begins at a small focus in the center, then spreads radially outwards. As the infection spreads, the cells undergo changes know as cytopathic effect. The large circle of dead cells would appear as a plaque if the monolayer were stained.
The second movie, made at higher magnification, shows spread at the edge of a viral plaque. The vaccinia virus used for this experiment carries the gene encoding enhanced green fluorescent protein (EGFP). Hence the infected cells fluoresce green as viral replication proceeds.
By showing very clearly how a viral plaque develops, these movies will be an invaluable teaching resource for years to come. I am grateful to the authors of this study for providing an up-close view of a technique that animal virologists have been using since 1952.
Doceul, V., Hollinshead, M., van der Linden, L., & Smith, G. (2010). Repulsion of Superinfecting Virions: A Mechanism for Rapid Virus Spread Science DOI: 10.1126/science.1183173