A different kind of remote control

nucleusAmong the multitudes of eukaryotic viruses with DNA genomes, some replicate in the cell nucleus, while others avoid the nuclear bureaucracy and remain in the cytoplasm. But biology is not always so rigid: a new giant virus has been found that replicates in the cytoplasm, where it seems to recruit components of the nuclear transcription machinery (link to paper).

Noumeavirus was isolated from a pond near – where else? – Noumea airport in New Caledonia. The 200 nanometer icosahedral particles infect the amoeba Acanthamoeba castellani and have a double-stranded DNA genome of 376,207 base pairs encoding 452 proteins. Sequence comparisons revealed Noumeavirus to be a new member of the family Marseilleviridae, which  includes other previously discovered giant viruses.

Other members of the Marseilleviridae replicate in the cytoplasm of the host cell, so it was assumed that the related Noumeavirus would do the same. However an analysis of the proteins in purified virus particles revealed an absence of components of the transcriptional machinery – which is needed for ths synthesis of mRNA. RNA polymerase, for example, is readily detected in other cytoplasmic viruses such as Mimivirus and poxviruses.

If proteins involved in transcription are not present in the Noumeavirus particle, and the virus does not enter the nucleus, how are viral mRNAs produced?  It appears that early in infection, the required proteins are moved from the nucleus to sites of viral replication in the cytoplasm. When nuclear proteins were labled with green fluorescent protein, within one hour after infection they can be seen moving out of the nucleus into the cytoplasm to sites of viral replication. The nuclear integrity remains intact, as host DNA does not leave the organelle. This recruitment of nuclear proteins is transient:  after 2-4 hours proteins are no longer leaving the nucleus.

This series of events suggests that nuclear proteins needed to initiate viral mRNA synthesis are recruited from the nucleus to sites of viral replication in the cytoplasm. Once viral mRNAs are made, the viral transcriptional machinery can be assembled and the nuclear proteins are no longer needed. The authors call this ‘remote control of the host nucleus.’

Confirmation of this hypothesis will require the demonstration that nuclear proteins involved in viral mRNA synthesis are recruited to early sites of viral replication in the cytoplasm. It will also be essential to identify the mechanism by which these nuclear proteins are extracted. Perhaps one or more virion proteins, such as an abundant 150 amino acid protein of unknown function, is involved.

Other giant viruses, such as Mimivirus, package the viral transcriptional machinery in the virus particle and are independent of the cell nucleus. At the other extreme are viruses that undergo transcription and DNA synthesis entirely in the nucleus (e.g., herpesviruses). Perhaps Noumeavirus is a relic of an evolutionary transition between the two replication strategies.

Image credit

TWiV 439: The purloined envelope

Paul Bieniasz joins the TWiV team to talk about the co-option, millions of years ago, of an endogenous retrovirus envelope protein by hominid ancestors for host defense against viral infection.

 

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TWiV 438: Drs. TWiV go to Washington

On the eve of the March for Science, the TWiV team gathers at ASM Headquarters in Washington, DC with guests Stefano and Susie to talk about the state of science communication.

You can find TWiV #438 at microbe.tv/twiv, or watch above/listen below.

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I have always marched for science

vrrThis Saturday I will be participating in the March for Science in Washginton, DC. It’s all about celebrating science and the role it plays in each of our lives. Frankly, I could have participated in a March for Science one, two, three, or any number of years ago, because these issues have always been important.

Over ten years ago, well into my science career, I recognized the need for scientists to come off the sidelines (to quote March for Science) and start telling the world what it is that we do. It’s why I wrote a virology textbook; started virology blog; produce five science podcasts; teach a virology course and record all the lectures for YouTube, and much more. These activities have been my March for Science.

Whenever I visit a university to give a science talk, I spend the last 15 minutes telling the audience (mainly scientists) why they need to tell the public what we do. I tell them to let the world know that our lives are long and prosperous because of science. I emphasize that every scientist needs to communicate, so that the public sees us all together championing the way science benefits the planet.

The March for Science, held in many different cities, will give the world a view of scientists together defending the roles that science plays in our lives. It’s a large scale view of what I have done myself over the years, and what I have encouraged other scientists to do.

It is unfortunate that the March for Science had to be triggered by an administration that shows disdain for science and facts. I bet we could have organized a similar march years ago. But the march is happening now, and whether or not we are branded as left or right doesn’t matter – science doesn’t care about your political party. Neil de Grasse Tyson put it perfectly: “The good thing about science is that it’s true whether or not you believe in it”.

More important than the March for Science is what happens afterwards. Does it build a “global movement to defend the vital role science plays in our health, safety, economies, and governments” as envisioned by its organizers, or is it back to business as usual for most scientists?

I don’t know the answer, but I do know that there are many scientists who do engage the public, and their work will continue. The work that me and my co-hosts do to bring science and scientists to everyone will go on, as it has before the March.

That’s why I will be wearing a This Week in Virology t-shirt to the March in Washington DC. It’s the way I’ve been communicating science, making a difference by reaching as many people as I can. That’s the spirit of the March for Science, which will go beyond one Saturday in April.

TWiV 437: Kathy’s new spindle virus

The TWiVsters reveal new giant viruses that argue against a fourth domain of life, and discovery of viruses in the oceanic basement.

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Viruses under the sea

CORKsViruses infect every living organism on the planet, but not every habitat has been explored for their presence. The igneous ocean crust had not yet been examined for viruses, but seek and ye shall find: there are plenty of viruses under the seas.

The oceanic basement is an enormous ecosystem that lies at the bottom of the seas, beneath a thick layer of sediment. It is composed of igneous rock through which percolates 20 million cubic kilometers of water. Previous study of cores from this region has revelealed the presence of prokaryotes, but no one had looked for viruses.

Facilitating the study of the oceanic basement are seafloor observatories that have been placed into existing boreholes. Two have been placed in 3.5 million year old rock in the northeastern Pacific Ocean. They penetrate hundreds of meters through sediment and into the basement (illustrated; image credit) and are fitted with plumbing that allows sampling of uncontaminated fluids from different depths in the basement rock.

Analysis of fluids recovered from these sites revealed both prokaryotes (8,000 per ml) and virus particles (90,000 per ml). Ribosomal RNA sequence analysis showed that bacteria dominated these communities, with some Archaea but virtually no eukaryotes.

Examination of the fluids by electron microscopy showed virus particles of different kinds: tailed and untailed icosahedral particles, untailed globular particles, and rod and spindle shaped. My favorite is the lemon shaped particle, for its form and implied taste.

To provide information on the viral genomes in the oceanic basement, sequences were determined from total cellular DNA (material retained on a 0.2 micron filter) extracted from the samples. Most viral sequences likely had archaeal hosts. Some prophage sequences were identified – viral genomes integrated into host DNA – which allowed more certain identification of the infected cell.

Most of the identified archaeal and bacterial virus sequences came from the families Myoviridae and Siphoviridae (think tailed, icosahedral viruses). One complete circular DNA genome identified is 55,906 nucleotides in length with 81 open reading frames. Twenty of these encode proteins with recognizable functions, such as capsid proteins, a primase and a DNA polymerase. No genes encoding tRNAs, such as those found in giant viruses, were identified.

Some sequences were similar to those of giant viruses like mimiviruses and phycodnaviruses. These viruses are known to only infect eukaryotes. Eukaryotic genomes were rare in the basement metagenome collections (1% of the community in one location).

I am not surprised that viruses have been found in ocean’s basement. Still, I’m amazed when I think about how far down they are, in warm water (65 degrees C) in 3.5 million year old rock.

To paraphrase Samuel E. Wright (Under The Sea):

The viruses are always greener
In somebody else’s lake
You dream about going up there
But that is a big mistake
Just look at the viruses around you
Right here under the ocean floor
Such wonderful viruses surround you
What more is you lookin’ for?

 

TWiV 436: Virology above Cayuga’s waters

At Cornell University in Ithaca, New York, Vincent speaks with Susan, Colin, and Gary about the work of their laboratories on parvoviruses, influenza viruses, and coronaviruses that infect dogs, cats, horses and other mammals.

You can find TWiV #436 at microbe.tv/twiv, or listen below.

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Forget the fourth domain of life

three domains of lifeWhen giant viruses were discovered – with genomes much larger than any previously seen – some suggested that they had descended from a fourth domain of life (the current three are bacteria, archaea, and eukaryotes). Part of the reason for such a claim was the finding of homologs of bacterial and eukaryotic genes, including molecules involved in translation. Analysis of new giant viruses encoding even more components of the translation machinery has thrown cold water on the fourth domain hypothesis.

Klosneuvirus, with a 1.57 million base pair DNA genome, was discovered in a wastewater treatment plant in Austria, and three related viruses – Indivirus, Hokovirus, and Catovirus – were found in environmental samples.  Sequence analyses suggests that these viruses should be classified in a subfamily of the Mimiviridae.

The Klosneuviruses encode far more components of the translational machinery than do mimiviruses – 25 tRNAs, 19 aminoacyl tRNA synthetases, 11 initiation and elongation proteins, a chain release factor, and tRNA modifying enzymes.

Phylogenomic analyses demonstrate that the aminoacyl tRNA synthetase and translation factor genes are likely derived from protists. This finding is not compatible with the hypothesis that these viruses are derived from a fourth domain of life. It is more likely that smaller ancestors of giant viruses acquired these genes from known eukaryotes.

Why these components of the translational system have been maintained in these giant virus genomes is an excellent question. They might confer some advantage to the viruses, for example when host translation is shut off as a viral defense. Having components of the translational apparatus might allow viral protein synthesis to proceed.

Note that genes encoding ribosomal RNAs or proteins have not been found in any virus. In fact no virus encodes a complete protein synthesis machinery. Maybe they have yet to be discovered? Or perhaps these energetically costly activities are best left to the cell?

 

TWiV 435: Two virus particles walk into a cell

The TWiVome discuss the blood virome of 8,420 humans, and thoroughly geek out on a paper about the number of parental viruses in a plaque.

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The purity of plaques

dose-response-plaque-assayThe plaque assay – my favorite assay in the world – is a time-honored procedure to determine the number of viruses in a sample, and to establish clonal virus stocks. The  linear relationship between the number of infectious particles and the plaque count (illustrated; image credit) shows that one infectious particle is sufficient to initiate infection. Despite the one-hit kinetics of plaque formation, could more than one virus contribute to a plaque?

To answer this question, ten genetically marked polioviruses were mixed and subjected to plaque assay. Of 123 plaques, 6 (4.9%) contained more than one virus. Similar results were found when polioviruses with phenotypic markers were studied.

Examination of poliovirus stocks by electron microscopy revealed both single particles and aggregates of 2 to 10 particles. Increasing particle aggregation by treatment of viruses with low pH increased co-infection frequency, indicating that aggregation of particles leads to multiply infected cells.

When these experiments were repeated with mutagenized polioviruses, the co-infection frequency increased – probably because recombination and complementation between two defective genomes leads to rescue of the defects.

Do these findings indicate that poliovirus plaque formation does not follow one-hit kinetics? The results do not prove that, in unmutagenized virus stocks, more than one poliovirus is needed to form a plaque. They only show that a small percentage of plaques contain more than one poliovirus. The presence of more than one poliovirus in 5-7% of plaques is likely a consequence of virion aggregation. It would be informative to prepare poliovirus stocks with no aggregates, and determine if co-infected plaques are still observed.

Some viruses of plants and fungi follow two-hit kinetics: two virus particles, with two different genomes, are needed for infection (illustrated). Assuming that 4-7% of poliovirus plaques are initiated by multiple viruses, the resulting plots deviate only slightly from a straight line, and do not resemble the curves of two-hit kinetics.

What are the implications of these findings for the use of plaque assays to produce clonal virus stocks? Even though the frequence of multiply infected plaques is low, the possibility of producing a mixed population is still possible, if only one plaque purification is done. In our laboratory we have always repeated the plaque purification three times, which should ensure that no multiply infected plaques are isolated.

Update 3/31/17: I would like to see similar experiments done with other viruses, to see how often multiple viruses can be found in a plaque. Examples included hepatitis A virus, which is released from cells in membranous vesicles containing multiple virus particles; and enveloped viruses, which might aggregate more frequently than naked viruses.

I looked back at the 1953 publication in which Dulbecco and Vogt first described the plaque assay for poliovirus, and demonstrated one hit kinetics. The dose-response curve clearly shows one-hit kinetics with little deviation of the individual data points from a straight line.

plaque dose response

Linear relationship between the number of plaques and the virus concentration. Image credit.