Ancient proteins block modern viruses

T7-like virusCould ancient host proteins contribute to the replication of a modern virus? The answer is, not very well (link to paper).

Viruses are obligate intracellular parasites, which means that they have to get inside of a host cell to produce more viruses. The genomes of all viruses, even the biggest ones, do not encode anywhere near the number of proteins that are needed to replicate. The cell provides thousands of proteins that are involved in energy production, membrane synthesis, protein synthesis, transport, and so much more.

The difficulty in studying ancient proteins is that none of them exist. But we can make good guesses about what very old proteins might look like, by examining modern proteins, seeing how they vary among organisms, and calculating how they might look like billions of years ago. The field of predicting what ancient proteins might look like is quite active.

Investigators have predicted what ancient versions of a cell protein called thioredoxin might have looked like. They have synthesized such ‘ancient’ thioredoxins and shown that they are stable and active. Thioredoxins are found in nearly all organisms, where they act as antioxidants.

Ancient thioredoxins that have been synthesized include those from the last common ancestors of bacteria; of archaea; and of archaea and eukaryotes (all around 4 billion years old); the last common anestor of cyanobacterial, deinococcus, and thermus groups (about 2.5 billion years old);  the last common ancestor of gamma-proteobacteria; of eukaryotes; and of fungi and animals (around 1.5 billion years old).

These ancient thioredoxins work in a modern E. coli. This bacterium has two thioredoxin genes, and if they are both deleted, growth occurs, but very slowly. If genes encoding ancient thioredoxins are introduced into these mutated bacteria, they can compensate for the growth deficiency. The older thioredoxins (4 billion years) compensate less well than ones that are closer in time (1.5 billion years).

It’s amazing that an ancient protein can work in a modern E. coli. But could ancient thioredoxins support viral growth?

Thioredoxin from E. coli is an essential part of the DNA polymerase complex of the bacteriophage T7 (pictured – image credit). This virus does not form plaques on E. coli lacking the two thioredoxin genes. The only ancient thioredoxin gene that allows phage T7 plaque formation is from the last common ancestor of cyanobacterial, deinococcus, and thermus groups, which is about 2.5 billion years old and has 57% amino acid identity with the E. coli enzyme. But the effienciency of plaque formation was very poor – about 100 million times worse than on regular  E. coli. None of the older thioredoxins worked.

Why would an ancient thioredoxin work for E. coli but not for bacteriophage T7? Over billions of years, thioredoxin evolves but it must still be able to carry out its function for E. coli. The viruses that infected bacteria 4 billion years ago were very different from contemporary viruses, and so the ancient thioredoxin does not work for modern viruses. Today’s thioredoxin could change so that it would not support T7 replication – as long as the enzyme still works for E. coli.

The authors of this work view it as a proof of principle: that virus growth is not supported by an ancient version of a modern protein required for virus replication. They would like to apply this approach to produce plants that are resistant to viruses, which have serious effects on global agricultural productivity.

I think the work is amazing not only because an ancient protein can be made, but it supports growth of the host and not that of a virus. It might therefore be possible to reconstruct the host-virus arms race, starting from ancient proteins. In this race, the gene encoding an essential cell protein can evolve so that it no longer supports virus replication. Next, the viral genome changes to adapt to the altered cell protein. And so the game goes back and forth.

The authors have shown that they can select mutant bacteriophage T7 isolates that replicate in the present of an ancient thioredoxin. This result suggests that it might be possible to reconstruct host-virus arms races beginning with an ancestral host protein. If we can make an ancient protein, could we also make an ancient virus? Why not?

TWiV 450: Ben tenOever and RNA out

Ben tenOever joins the TWiVoli to discuss the evolution of RNA interference and his lab’s finding that RNAse III nucleases, needed for the maturation of cellular RNAs, are an ancient antiviral RNA recognition platform in all domains of life.

 

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A virus with a green thumb

PoxvirusI 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.

Image credit.

 

TWiV 449: The sound of non-silencing

The TWiV Council explores the finding that facial appearance affects science communication, and evidence that RNA interference confers antiviral immunity in mammalian cells.

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The Traditional Lecture is Not Dead. I Would Know – I’m A Professor

Virology 2017Wired Magazine recently published an article with a headline distinctly opposite of mine, which claims that the traditional lecture is dead. I disagree, and here is why.

The thesis of the article, by Rhett Allain, is that modern technologies have made the traditional lecture obsolete. The traditional lecture is one during which a teacher stands at the front of the room and ‘disseminates knowledge to students’. Allain claims – rightly so – that animated videos like The Mechanical Universe are far more engaging. He suggests that, to teach physics, just show the students episodes from this show. If they have questions, just pause the show.

He claims that showing these videos – or equivalents for other subjects – beats most lectures. Lectures in which teachers drone on and on.

Well guess what – I teach a virology course at Columbia University, and at the end of the year, most of the students say its one of the best, or the best class they have taken in their college years. I don’t show The Viral Universe or any other videos during my class. I talk to the students about my knowledge of viruses, gained from researching them for over 30 years.

Not every virology lecturer has my experience in the field. Many of them learn virology from a book. I agree that lectures given by those individuals are dead.

I do record each lecture and post them at YouTube, so that the students in the class, or anyone in the world for that matter, can watch them. It’s a new technology that Allain likes. I like allowing the students to time-shift their learning: some never come to class. But it’s still me making the videos and sharing my knowledge via a traditional lecture format.

Allain is also fond of the flipped classroom – assign a video for students to watch before class, and then use class time to discuss it. I love this idea. But I still think that for my introductory virology class, it’s better for me to talk to them. To walk around the room, without notes, look them in the eye, and muster all my passion and love for the field and send it their way. And don’t think that doesn’t matter – many of my students tell me that my passion for the subject is what makes them interested in viruses.

Research says that most students learn better by doing. I do pause a few times during each lecture to have students complete an online quiz – something Allain also likes. It gives me time to see if what I’m saying is sinking in, and to clarify complex material.

When I lecture, students come to me afterwards with questions, and some even walk with me to the subway, to talk about viruses. How can a video provide that experience?

Allain’s response might be that if anyone wants to teach a virology course, just play my lectures and discuss them in class. I’m all for that. But every spring semester, I’ll be in front of the class, talking about viruses, and making new videos. There is no substitute for a expert who is passionate about their subject. I realize that every physics course can’t be taught by Einstein, but he can teach at least one, and the students at his university will love it.

Not every passionate researcher will make a great lecturer, and for them,  videos and flipped classrooms are a great way to teach. But for those passionate researchers who can teach – why not put them in front of a class and inspire the next generation? I do it every year.

TWiV 448: Mavis the Structure Maven

From ASV 2017 in Madison, Wisconsin, the complete TWiV team speaks with Mavis Agbandje-McKenna about her career and her work solving virus structures by x-ray crystallography and cryo-electron microscopy.

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TWiV 447: Un-impacting an elephant

The glorious TWiVerati un-impact their email backlog, anwering questions about viruses, viruses, viruses, viruses, viruses, and more. You should listen – our fans ask great questions!

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TWiV 446: Old sins die hard

The TWiV hosts review an analysis of gender parity trends at virology conferences, and the origin and unusual pathogenesis of the 1918 pandemic H1N1 influenza virus.

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The first human virus discovered

PlaqueOn the wall of a Columbia University Medical Center building just across the street from my laboratory is a plaque commemorating two participants in the discovery of a mosquito vector for yellow fever virus.

The plaque reads:

Aristides Agramonte, Jesse William Lazear, Graduates of the Columbia University College of Physicians and Surgeons, class of 1892. Acting Assistant Surgeons, U.S. Army. Members of the USA Yellow Fever Commission with Drs. Walter Reed and James Carroll. Through devotion and self-sacrifice they helped to eradicate a pestilence of man.

Yellow fever, known in tropical countries since the 15th century, was responsible for devastating epidemics associated with high rates of mortality. The disease can be mild, with symptoms that include fever and nausea, but more severe cases are accompanied by major organ failure. The name of the illness is derived from yellowing of the skin (jaundice) caused by destruction of the liver. For most of its history, little was known about how yellow fever was spread, although it was clear that the disease was not transferred directly from person to person.

Cuban physician Carlos Juan Finlay proposed in 1880 that a bloodsucking insect, probably a mosquito, was involved in yellow fever transmission. The United States Army Yellow Fever Commission was formed in 1899 to study the disease, in part because of its high incidence among soldiers occupying Cuba. Also known as the Reed Commission, it comprised four infectious disease specialists: U.S. Army Colonel Walter Reed (who was the chair); Columbia graduates Lazear and Agramonte, and James Carroll. Lazear confirmed Finlay’s hypothesis in 1900 when he acquired yellow fever after being experimentally  bitten by mosquitos who had fed on sick patients. Days later, he died of the disease.

The results of the Reed Commission’s study proved conclusively that mosquitoes are the vectors for this disease. Aggressive mosquito control in Cuba led to a drastic decline in cases by 1902.

The nature of the yellow fever agent was established in 1901, when Reed and Carroll injected filtered serum from the blood of a yellow fever patient into three healthy individuals. Two of the volunteers developed yellow fever, causing Reed and Carroll to conclude that a “filterable agent,” which we now know as yellow fever virus, was the cause of the disease.

Sometimes you don’t have to wander far to find some virology history.

Update 6/16/17: The statement on the plaque that Agramonte and Lazear “helped to eradicate a pestilence of man” is of course incorrect, as yellow fever has never been eradicated. Recent large outbreaks of yellow fever in Brazil and Angola are examples of the continuing threat the virus poses, despite the availability of a vaccine since 1938.

TWiV 445: A nido virology meeting

From Nido2017 in Kansas City, Vincent  meets up with three virologists to talk about their careers and their work on nidoviruses.

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