Pandoravirus, bigger and unlike anything seen before

pandoravirusThe discovery of the giant Mimivirus and Megavirus amazed virologists (and also many others). Their virions (750 nanometers) and DNA genomes (1,259,000 base pairs) were the biggest ever discovered, shattering the notions that viruses could not be seen with a light microscope, and that viral genomes were smaller than bacterial genomes. Now two even bigger viruses have been discovered, which are physically and genetically unlike any previously known viruses. They have been called Pandoraviruses.

Both new viruses were isolated by culturing environmental samples in the amoeba Acanthamoeba castellaniPandoravirus salinus was isolated from shallow marine sediment in a river at the coast of central Chile, and Pandoravirus dulcis was obtained from mud at the bottom of a freshwater pond near Melbourne, Australia. The P. salinus genome is at least 2.77 megabases in length (there is some uncertainty in the actual length due to the presence of repeated sequences at the ends of the DNA), while the P. dulcis genome is 2.47 megabases in length. The smaller P. dulcis genome is a subset of the P. salinus genome.

These new genomes are twice as large as those of previously described viruses, and bigger than the genomes of intracellular bacteria such as Tremblaya (138,927 base pairs) and Rickettsia (1,111,523 bp), some free living bacteria, and many free living Archaea.

While the huge sizes of the Pandoravirus virion and genomes are amazing, I find three other features of these viruses even more remarkable. The first is their atypical replication cycle. The virions are taken into amoebae by phagocytic vacuoles, and upon fusing with the vacuole membrane, the virion contents are released into the cytoplasm via a pore on the virion apex. Within 2-4 hours the cell nucleus is reorganized, and by 8-10 hours new particles appear where the nucleus once was. Pandoravirus DNA and virions are synthesized and assembled simultaneously, in contrast to eukaryotic DNA viruses and phages which fill pre-formed capsids with DNA. Virions are released by 10-15 hours as the cells lyse.

A second amazing feature is that most of the P. salinus open reading frames encode brand-new proteins. Of the 2,556 putative protein coding sequences in the P. salinus genome, 93% have no recognizable counterparts among known proteins. Some of the genes found in large DNA viruses are present, such as those encoding DNA polymerase and DNA-dependent RNA polymerase, and several amino acyl-tRNA synthetases, like members of the Megaviridae. Curiously, many of the Pandoravirus coding regions contain intervening sequences, which must be removed by RNA splicing. This process is known to occur only in the cell nucleus, suggesting that some Pandoravirus transcription occurs in that organelle. The lack of gene homology leads to authors to conclude that ‘no microorganism closely related to P. salinus has ever been sequenced’.

I am also impressed by what the authors describe as the ‘alien morphological features’ of the virions. The oval-shaped particles are 1 micron in length and 0.5 microns in diameter, easily visible by light microscopy. They are wrapped in a three-layered envelope with a pore at one end of the particle, and resemble nothing that has ever been seen before (see photograph).

How much bigger can viruses get? I don’t know the answer but I would guess even bigger than Pandoraviruses. The membranous Pandoravirus particle could easily accommodate even larger genomes. How big can a virus get and still be a virus? The answer to that question is easy: it is a virus as long as it requires a cell for replication.

These remarkable findings further emphasize the need for scientists to pursue their curiosity, and not only work on problems of obvious medical relevance. As the authors write,

This work is a reminder that our census of the microbial diversity is far from comprehensive and that some important clues about the fundamental nature of the relationship between the viral and the cellular world might still lie within unexplored environments.

Continuing their playful naming of giant viruses, the authors note that the name Pandoravirus reflects their ‘lack of similarity with previously described microorganisms and the surprises expected from their future study’.

The largest viral genome from a human

Mimivirus LBA111The biggest known viruses are Mimivirus (750 nanometer capsid, 1.2 million base pair DNA) and Megavirus (680 nanometer capsid, 1.3 million base pair DNA). These giant viruses have all been isolated from environmental samples, and many infect amoebae. A new Mimivirus has now been isolated from a human patient with pneumonia.

To search for giant viruses in humans, respiratory samples from 196 Tunisian patients with community-acquired pneumonia were co-cultured with the amoebae Acanthamoeba polyphaga. One sample, a bronchial aspirate, yielded a new Megavirus that was given the catchy name LBA111. The patient from which LBA111 was isolated had been hospitalized with fever, cough, shortness of breath, and bloody sputum. Serum from this patient, but not from those of 50 healthy blood donors, reacted with LBA111 proteins, indicating suggesting that this individual had been infected with the virus.

Mimivirus LBA111 is not likely to be a contaminant introduced from the laboratory in which it was isolated, because its genome sequence is original. The LBA111 genome is double-stranded DNA 1,230,522 base pairs in length, mostly closely related to the genome of Megavirus chilensis, a giant virus isolated off the coast of Chile. In addition to other differences, the LBA111 genome encodes two  tRNAs (histidine and cysteine) not present in the genome of M. chilensis.

While these findings indicate an association of Mimivirus LBA111 with human pneumonia, they do not prove that this virus is the causative agent of human disease. However, the possibility that Mimivirus causes human disease makes sense. Mimiviruses are present in soil and water where they multiply in amoebae. A known agent of human respiratory disease, Legionella pneumophila, also colonizes amoebae. Antibodies to Mimivirus, as well as Mimivirus DNA, have been found in patients with pneumoniaMimivirus should therefore be added to the list of agents that should be considered in patients with pneumonia.

TWiV 206: Viral turducken

On episode #206 of the science show This Week in Virology, Vincent, Alan, Dickson, and Kathy discuss how the innate immune response to viral infection influences the production of pluripotent stem cells, and the diverse mobilome of giant viruses.

You can find TWiV #206 at www.microbe.tv/twiv.

This year in virology

XMRVFor some time I have thought about reviewing this year’s topics on virology blog in 2001, not only to get a sense of what I thought was significant, but more importantly, to highlight areas that need more coverage. I went through all the articles I wrote in 2011, put them in subject categories, and listed them by number of articles. The results are both obvious and surprising.

I wrote most frequently about the retrovirus XMRV and its possible role in chronic fatigue syndrome and prostate cancer. This extensive coverage was warranted because we had an opportunity to learn how disease etiology is established, followed by development of therapeutics. By the end of the year we learned that XMRV does not cause human disease, but the journey to that point was highly instructive.

The next most frequently visited topic on virology blog was influenza. Writing often about this virus makes sense because it is a common human infection that occurs every year, and controlling it is a continuing goal of virology research.

There were five  posts noting the death of virologists, colleagues, or someone I thought made a substantial impact on my career.

I wrote more about poliovirus than any other virus except XMRV and influenza. Eradication of poliomyelitis continues to be difficult and faces periodic setbacks.

I only wrote three articles about topics in basic virology.

Like many others, I find the biggest viruses and their virophages compelling.

The past year saw the release of Contagion, a movie about a virus outbreak. Look for an analysis on TWiV in 2012.

The state of science education and science funding is becoming more of a concern. It is not a topic I write about often – I prefer to focus on the science of virology – but for future scientists it is extremely important.

The other posts covered a variety of topics and viruses, including HIV, human papilloma viruses, hepatitis C virus, and smallpox virus.

What have I learned from looking back? The best covered viruses – XMRV, influenza, and poliovirus – deserve the attention. I am surprised that there were so few articles on important viruses such as HIV, HCV, rotaviruses, and herpesviruses. That shortcoming will have to change. I did not write enough about basic virology. One could argue that teaching a virology course is enough – but I think that concise, informative articles on basic virology are very useful. I’ll try to do more of that in 2012. There is one topic I’d like to write less about, but over which I have little control – the passing of scientists.

Thank you for coming here to learn about virology.

Megavirus, the biggest known virus

megavirusThe mantle of world’s biggest virus has passed from Mimivirus to Megavirus. But in this case, size doesn’t matter. It’s the genes that these viruses share and do not share that make this story important.

The discovery of Mimivirus in a French cooling tower amazed virologists. At 750 nanometers in diameter, it dwarfed all other known viruses and shattered the notion of viruses as ‘filterable agents’. That definition came from using filters that exclude any particle larger than 200 nanometers. The 1.2 million base pair Mimivirus DNA genome was found to encode 979 proteins – more than any other virus. This treasure chest included some proteins never found in any viral genome, such as amino-acyl tRNA synthetases, the enzymes that attach amino acids to transfer RNAs in preparation for protein synthesis. Many other cellular genes were also encoded in the Mimivirus genome, as well as proteins that had never been seen before. This discovery led to the idea that Mimivirus evolved from an ancestral cellular organism by losing genes. The alternative idea is that viruses are ‘pickpockets’ – they began as small pieces of DNA that escaped from cells, acquired a capsid, and then slowly stole genes from other organisms.

The problem with the idea that Mimivirus is a gene loser is that it was the only really big virus of its kind – until Megavirus was discovered in the ocean off the coast of Chile. At 680 nm in diameter, Megavirus is slightly smaller than Mimivirus, but the DNA genome is larger – 1,259,197 base pairs versus 1,182,000. The Megavirus genome encodes 1,120 putative proteins compared with 979 for Mimivirus. What is significant about Megavirus is that its genome resembles that of Mimivirus, including the presence of cellular genes such as those encoding amino-acyl tRNA synthetases. Also important is the fact that 258 of the Megavirus proteins have no counterparts in Mimivirus, including three amino-acyl tRNA synthetases.

When Mimivirus was first discovered, it was not clear if it was an anomaly, or if it would provide information on the emergence of viruses. Other giant viruses were subsequently discovered, but they were either nearly identical to Mimivirus (Mamavirus) or little genome sequence was available (Terra, Courdo, Moumou). The evolutionary distance of Megavirus and Mimivirus is sufficient to allow identification of similar features and the selection forces that lead to their emergence. A comparison of their DNA sequences reveals a set of genes in common between the two viruses, such as those that function during protein synthesis. These observations suggest that Megavirus and Mimivirus arose from a common cellular ancestor – one that could carry out protein synthesis – and lost the genes that were no longer needed. The alternative scenario, that these giant viruses started out small and acquired additional genes, seems unlikely. Seven different gene acquisition events would have been needed to acquire just the genes encoding amino-acyl tRNA synthetases.

More details about how Megavirus and Mimivirus emerged from a cellular ancestor will require the isolation of other giant DNA viruses. Which leads to the inevitable question – what is the biggest virus on earth? Have we reached the limit of viral genome size? Probably not, but I doubt that we will find viruses with much larger genomes that encode almost all the genes needed for independent replication. Such viruses likely evolved from primitive cells a very long time ago, and only the evolutionary descendants remain on Earth today.

If you are wondering how these giant viruses are named, the authors provide revealing background:

We believe it is useful and desirable that the name of a newly isolated microorganism convey some of its most distinctive properties. After the initial naming of Mimivirus (for “microbe mimicking”), already not a very good name because the prefix “mimi” does not convey a helpful scientific notion, newly isolated related viruses are receiving increasingly random/funny names such as “Mamavirus,” “Moumouvirus,” “Courdovirus,” and “Terra”. …we believe the current trend is counterproductive and should give way to more informative names….a distinctive feature of the above giant viruses (or of their close ancestors) is to possess genome in excess of a “megabase”. Hence, the term “Megavirus,” and the proposed family/genus “Megaviridae” that will be proposed… “Chilensis” then refers to the location where this virus was first isolated.

The authors did not incorporate the species of the viral host in the name of the virus. They justify this break with tradition because the natural host of  Megavirus chilensis is not known – it was isolated from the ocean by co-culture with acanthamoeba, a procedure the authors will continue to use to identify giant DNA viruses.

 Arslan D, Legendre M, Seltzer V, Abergel C, & Claverie JM (2011). Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae. Proceedings of the National Academy of Sciences of the United States of America, 108 (42), 17486-91 PMID: 21987820

TWiV 139: Honey, I shrunk the virus

mimivirusHosts: Vincent Racaniello, Alan Dove, and Dickson Despommier

Vincent, Alan, and Dickson discuss the reduction in genome size of Mimivirus upon passage in amoeba, and analysis of the microbiome of honeybees.

Click the arrow above to play, or right-click to download TWiV #139 (96 MB .mp3, 80 minutes).

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Weekly Science Picks

Alan – Life Before the Dinosaurs by ABC
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Lance – A History of the World since 9/11 by Dominic Streatfeild

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Virophage, the virus eater

mavirusA second virophage has been identified. The name does not signify a virus that infects another virus – it means virus eater.

The story of virophages begins with the giant mimivirus, originally isolated from a cooling tower in the United Kingdom. It is the largest known virus, with a capsid 750 nanometers in diameter and a double-stranded DNA genome 1.2 million base pairs in length. If these statistics are not sufficiently impressive, consider that shortly after its discovery, an even larger related virus was discovered and called mamavirus. These huge viruses replicate in amoeba such as Acanthamoeba; in this host they form large, cytoplasmic ‘factories’ where the DNA replicates and new virions are assembled. While examining mamavirus infected Acanthamoeba polyphaga, investigators noted small icosahedral virions, 50 nm in diameter, within factories and in the cell cytoplasm. They called this smaller virus Sputnik. This new virus does not replicate in amoebae unless the cell is also infected with mimivirus or mamavirus. Surprisingly, infection with Sputnik reduces the yields of mamavirus, and also decreases the extent of amoebal killing by the larger virus.

The second virophage is called Mavirus (for Maverick virus – because the viral DNA is similar to the eponymous DNA transposon). Mavirus was identified in the marine phagotropic flagellate Cafeteria roenbergensis infected with – you guessed it – a giant virus, CroV.  Like Sputnik, Mavirus cannot replicate in C. roenbergensis without CroV, and it also reduces the yield of CroV particles produced. The figure shows a viral factory (VF) in C. roenbergensis surrounded by large CroV particles (white arrowhead) and the smaller Mavirus particles (white arrows).

There are other examples of viruses that depend on a second, different virus for replication. For example, satellites are small, single-stranded RNA molecules 500-2000 nucleotides in length that replicate only in the presence of a helper virus. The satellite genome typically encodes structural proteins that encapsidate the genome; replication functions are provided by the helper. Most satellites are associated with plant viruses, and cause distinct disease symptoms compared with those caused by helper virus alone. Some bacteriophages and animal viruses have satellites. E. coli bacteriophage T4 is a satellite that requires bacteriophage T2 as a helper, while the adeno-associated viruses within the Parvoviridae are satellites requiring adenovirus or herpesvirus helpers. The hepatitis delta virus genome is a 1.7 kb RNA molecule that requires co-infection with hepatitis B virus to provide capsid proteins.

The difference between Sputnik, Mavirus, and satellites is that the latter do not interfere with the replication of helper viruses. Indeed, Sputnik was termed a virophage by its discoverers because its presence impairs the reproduction of another virus. The name is derived from bacteriophage – the name means ‘bacteria eater’ (from the Greek phagein, to eat). The idea is that one virus impairs the replication of the other – ‘eating’ the other virus.

In addition to their unique effect on their helper viruses, it appears that virophages have other stories to tell. The Sputnik DNA genome contains genes related to those in viruses that infect eukaryotes, prokaryotes, and archaea. Virophages may therefore function to transfer genes among viruses. The relationship of Mavirus to DNA transposons is also intriguing. DNA transposons are a kind of ‘jumping gene’, a piece of DNA that can move within and between organisms. They are important because they can change the genetic makeup of living entities, thereby influencing evolution. It is possible that DNA transposons evolved from ancient relatives of Mavirus, which would give virophages a particularly important role in the evolution of eukaryotes.

Fischer MG, & Suttle CA (2011). A Virophage at the Origin of Large DNA Transposons. Science (New York, N.Y.) PMID: 21385722

TWiV 93: Our infectious inbox

Hosts: Vincent Racaniello, Alan Dove, and Rich Condit

On episode #93 of the podcast This Week in Virology, Vincent, Alan, and Rich answer listener questions about lab procedures, prokaryotes, endogenous retroviruses, the iPad and teaching, prions, mimivirus, splitting water with viruses, and the polio outbreak in Tajikistan.

Click the arrow above to play, or right-click to download TWiV #93 (76 MB .mp3, 105 minutes)

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Weekly Science Picks

Alan – Southern Fried Science
Rich –
Tree of Life web project
Vincent – Dickson Despommier at Big Think

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.

The virus and the virion

The illustration at left depicts a virion – the infectious particle that is designed for transmission of the nucleic acid genome among hosts or host cells. A virion is not the same as a virus. I define virus as a distinct biological entity with five different characteristics. Others believe that the virus is actually the infected host cell.

The idea that virus and virion are distinct was first proposed by Bandea in 1983. He suggested that a virus is an organism without a cohesive morphological structure, with subsystems that are not in structural continuity:

Viruses are presented as organisms which pass in their ontogenetic cycle through two distinctive phenotypic phases: (1) the vegetative phase and (2) the phase of viral particle or nucleic acid. In the vegetative phase, considered herein to be the ontogenetically mature phase of viruses, their component molecules are dispersed within the host cell. In this phase the virus shows the major physiological properties of other organisms: metabolism, growth, and reproduction.

According to Bandea’s hypothesis, the infected cell is the virus, while the virus particles are ‘spores’ or reproductive forms. His theory was largely ignored until the discovery of the giant mimivirus, which replicates its DNA genome and produces new virions in the cytoplasm within complex viral ‘factories’. Claverie suggested that the viral factory corresponds to the organism, whereas the virion is used to spread from cell to cell. He wrote that “to confuse the virion with the virus would be the same as to confuse a sperm cell with a human being”.

If we accept that the virus is the infected cell, then it becomes clear that most virologists have confused the virion and the virus. This is probably a consequence of the fact that modern virology is rooted in the study of bacteriophages that began in the 1940s. These viruses do not induce cellular factories, and disappear (the eclipse phase) early after cell entry. Contemporary examples of such confusion include the production by structural virologists of virus crystals, and the observation that viruses are the most abundant entities in the seas. In both cases it is the virion that is being studied. But virologists are not the only ones at fault – the media writes about the AIDS virus while showing an illustration of the virion.

Those who consider the virus to be the infected cell also believe that viruses are alive.

…one can conclude that infected eukaryotic cells in which viral factories have taken control of the cellular machinery became viruses themselves, the viral factory being in that case the equivalent of the nucleus. By adopting this viewpoint, one should finally consider viruses as cellular organisms. They are of course a particular form of cellular organism, since they do not encode their own ribosomes and cell membranes, but borrow those from the cells in which they live.

This argument leads to the assumption that viruses are living, according to the classical definition of living organisms as cellular organisms. Raoult and Forterre have therefore proposed that the living world should be divided into two major groups of organisms, those that encode ribosomes (archaea, bacteria and eukarya), and capsid-encoding organisms (the viruses).

BANDEA, C. (1983). A new theory on the origin and the nature of viruses Journal of Theoretical Biology, 105 (4), 591-602 DOI: 10.1016/0022-5193(83)90221-7

Forterre, P. (2010). Defining Life: The Virus Viewpoint Origins of Life and Evolution of Biospheres, 40 (2), 151-160 DOI: 10.1007/s11084-010-9194-1

TWiV 77: Non-nuclear proliferation

Hosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich revisit circovirus contamination of Rotarix, then discuss poxvirus-like replication of mimivirus in the cell cytoplasm, and whether seasonal influenza immunization increases the risk of infection with the 2009 H1N1 pandemic virus.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $50 off a Drobo or $100 off a Drobo S.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #77 (60 MB .mp3, 83 minutes)

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Rich The Way We Work by David Macaulay
Alan DimDim
Vincent Polio: An American Story by David Oshinsky

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.