Do giant viruses have a CRISPR-like immune system or a protein restriction factor?

Zamilon virophageA battle is brewing between two research groups in Marseille, France that are involved in the discovery and study of giant viruses. Didier Raoul and colleagues believe that they have discovered a CRISPR-like, DNA based defense system in mimivirus that confers resistance to virophage (paper link). Claverie and Abergel disagree: they think that the defense system involves proteins, not nucleic acids (paper link).

Virophages are DNA viruses that can only replicate in cells infected by giant viruses like mimivirus. Their name, which means ‘virus eater’, comes from the observation that they inhibit mimivirus replication. A specific virophage called Zamilon was discovered that can inhibit the replication of lineage B and C mimivirus but not lineage A.

Examination of the DNA sequences of 60 different mimivirus strains revealed that the genomes of lineage A contained a 28 nucleotide sequence identical to Zamilon virophage. This sequence was not found in any lineage B mimivirus and in only one out of 19 lineage C mimiviruses. In addition, a 15 nucleotide subset of this sequence is repeated four times in the lineage B and C mimivirus genomes.

Near the 15 nucleotide Zamilon-derived repeated sequences in lineage B and C mimivirus genomes are genes encoding several proteins related to components of the bacterial CRISPR-Cas system. These include a nuclease, an RNAse, and an ATP-dependent DNA helicase.

The CRISPR system provides defense against invading DNA. When a foreign DNA, such as a bacteriophage genome, enters a bacterial cell, some is fragmented and integrated into the CRISPR locus as a ’spacer’ (sequences in the foreign DNA are called ‘protospacers’). Following transcription, CRISPR RNAs (crRNA) are processed by a multiprotein complex to produce ~60 nucleotide RNAs. When the spacer of a crRNA base pairs with a complementary sequence in an invading DNA molecule, CRISPR-associated endonucleases cleave the DNA. The integration of the sequences of the invading DNA into the host cell genome, from which they can be mobilized in the form of crRNAs, provides a form of “memory” and acquired immunity. It should be noted that there are six known types of CRISPR systems that differ in their components and mechanisms.

Because the CRISPR-Cas system is an adaptive immune system that protects bacteria and Archaea from virus infections and invasion of foreign DNA, the authors propose that they have discovered a new adaptive immune system that protects mimiviruses from virophage infection. They call this system mimivirus virophage resistance element, or MIMIVIRE.

The authors provide experimental support for their hypothesis by showing that silencing the genes encoding the endonuclease, the helicase, and the repeated insert using siRNA allows Zamilon replication in mimivirus-infected cells.

Claverie and Abergel think that Raoult and colleagues are wrong (paper link). They provide three reasons to dispute their findings, and ‘propose a simpler protein-based interaction model that explains the observed phenomena without having to extend the realm of adaptive immunity to the world of eukaryotic viruses, a revolutionary step that would require stronger experimental evidences.’

The first problem is that mimivirus and Zamilon virophage replicate in the same location in the infected cell, making a CRISPR-like defense system difficult to conceptualize. In contrast, CRISPR sequences reside in the bacterial genome, from which RNAs are produced that target the destruction of invading DNAs elsewhere in the cell.

The second problem is that the Zamilon sequences in the mimivirus genome are not regularly spaced or flanked by recognizable repeats, a hallmark of the CRISPR system (the name stands for ‘clustered regularly interspersed short palindromic repeats). However it should be noted that type VI CRISPR systems have no CRISPR locus and likely function via mechanisms that are different from other CRISPR systems.

Finally, Claverie and Abergel argue that there is no way for the proposed nucleic acid defense system to distinguish between the virophage and the virophage sequences in the mimivirus genome. In some CRISPR systems this discrimination is achieved by protospacer adjacent motifs (PAMs), short (2-5 nt) sequences next to the invader protospacer sequences that are recognized by the endonuclease complex guided by the crRNA. PAMs are not present in the bacterial genome, sparing it from endonucleolytic cleavage. Nevertheless, non PAM-based mechanisms of discriminating invader from host are known, for example, in the type III CRISPR system.

If Raoult and colleagues have not discovered a CRISPR-like mimivirus defense system, then why would silencing the genes encoding CRISPR-like proteins allow Zamilon replication? Claverie and Abergel think that it is not the 15 nucleotide Zamilon repeats that are important to mimivirus, but the encoded amino acids: Asp-Asn-Glu-Ser (DNES in one letter code). They believe that DNES is a motif present in proteins that block Zamilon replication by as yet unidentified mechanisms.

Many cellular proteins have been identified that interfere with virus replication, such as those encoded by interferon induced genes (ISGs). DNES containing proteins that inhibit Zamilon replication would be conceptually analogous, except that they are encoded by a virus, not the host.

Claverie and Abergele appear to have a strong case that mimivirus defense against Zamilon virophage is mediated by protein, not nucleic acid, but further experimentation is certainly needed to support their position. Nevertheless they recognize that the discovery by Raoult and colleagues “remains fascinating even if it falls short of demonstrating the existence of a CRISPR-Cas-like adaptive immune system”.

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

Brent Johnson on virophage

Virophage is the name coined for viruses such as Sputnik and Mavirus that can only replicate in cells infected with a helper virus, whose replication they inhibit. I’ve never liked the name – it means virus eater – and neither does Brent Johnson, a virologist at Brigham Young University:

“I believe the term ‘virophage’ is unfortunate because it implies one virus is infecting another virus and eating it. The small virus isn’t infecting another virus, it’s just using it to assist in replication, which is consistent with the needs of a defective virus.” he explains. He prefers calling the giant viruses “megaviruses,” and considers the name “Megavirus-Associated Virus (MAV) more consistent with currently accepted virus nomenclature.”

For more discussion, see the article by Marsha Stone in the July 2011 Microbe.

TWiV 128: Virologists in the mist

gorillaHosts: Vincent Racaniello, Alan Dove, Dickson Despommier, and Welkin Johnson

Vincent, Alan, Dickson and Welkin review how a virus regulates the severity of mucocutaneous leishmaniasis, virophage control of antarctic algal host-virus dynamics, and human metapneumovirus infection in gorillas.

Click the arrow above to play, or right-click to download TWiV #128 (67 MB .mp3, 92 minutes).

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Weekly Science Picks

Welkin – Walter Reed Collection, University of Virginia
Dickson – Bacteria-phage antagonistic coevolution in soil (Science)
Alan –
The Artful Amoeba
Vincent – Potential bacteriophage applications (Microbe)

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

Virophages engineer the ecosystem

organic lake virophageLast week we discussed the second known virophage, but we didn’t have any explanation of why such viruses might evolve. This week we have the discovery of a third virophage, hints of many more, and a hypothesis for what they might be doing in the global ecosystem.

The newest virus eater is called Organic Lake virophage (OLV), for the body of water in Antarctica where it was identified. Antarctic Lakes are well suited for metagenomic analyses (nucleotide sequences produced from environmental samples) because they are dominated by microbes and typically sustain few multicellular eukaryotes. For example, a metagenomic study of Lake Limnopolar, another Antarctic lake, revealed many novel eukaryotic and ssDNA viruses.Nucleotide sequence analysis of water samples taken from Organic Lake in 2006 and 2008 revealed the presence of three abundant phycodnaviruses. These are large, dsDNA-containing viruses that infect algae. Among the sequences was a novel virophage, called Organic Lake virophage (OLV), related to Sputnik virophage, but with a larger circular dsDNA genome (26,421 bp, pictured). OLV was found in water samples taken two years apart, indicating that it is a stable part of the Organic Lake ecosystem.

Because phycodnaviruses are related to mimivirus, it is assumed that OLV replicates in phycodnavirus-infected cells and inhibits the viral production. However, an experiment to test this relationship was not done. Six of the OLV genes are related to genes found in the lake phycodnaviruses, suggesting that gene exchange between virus and virophage has occurred, likely during co-infection of the same host.

When the Sputnik virophage infects mimivirus-infected amoebae, the yield of the larger virus is decreased by 70%, and there is also a threefold inhibition of virus-induced cell lysis. The authors speculated that the Organic Lake virophage could therefore influence the composition of the microbial community. To test this idea, they conducted mathematical modeling of the effect of virophage on a population of microbes that is infected with a lytic virus. Without the virophage, there are cycles of virus reproduction, cell death, and cell growth (blooms). The effect of adding OLV to the equation is that the frequency of blooms increases. The authors suggest that the virophage has this effect by reducing mortality of the host algal cell caused by phycodnaviruses. Antarctic lakes have long cycles of daylight and darkness, and a decrease in phycodnavirus cell killing caused by virophages may be essential for maintaining stability of the microbial food web.

This hypothesis makes perfect sense, and would explain why virophages evolved, but it is based in part on data on how Sputnik virophage interferes with mimivirus. It will be necessary to confirm that OLV indeed inhibits the replication of phycodnaviruses. Furthermore, proving that OLV affects microbial communities in lakes will require extensive field studies.

Perhaps even more interesting than the notion that virophages may engineer ecosystems is finding them in a broader range of environments. Virophage sequences were found in water samples from nearby Ace Lake, and a search of the Global Ocean Survey database revealed virophage sequences in a hypersaline lagoon and an ocean upwelling in the Galapagos islands, a Delaware Bay estuary in New Jersey, and a freshwater lake in Panama.

It is well known that cell killing by viruses has a major impact on ocean ecology. By regulating virus-induced cell lysis, virophages might also have a major effect on aquatic ecology. This possibility makes me wonder if there are virophages of animal viruses that might regulate viral pathogenesis.

Yau S, Lauro FM, Demaere MZ, Brown MV, Thomas T, Raftery MJ, Andrews-Pfannkoch C, Lewis M, Hoffman JM, Gibson JA, & Cavicchioli R (2011). Virophage control of antarctic algal host-virus dynamics. Proceedings of the National Academy of Sciences of the United States of America PMID: 21444812

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 125 – TWiV infects FiB

icosahedron light

Hosts: Vincent Racaniello, Dickson DespommierAlan DoveRich Condit, and Marc Pelletier

This Week in Virology and Futures in Biotech join together in a science mashup to talk about a virophage at the origin of DNA transposons, and unintended spread of a recombinant retrovirus.

Click the arrow above to play, or right-click to download TWiV #125 (59 MB .mp3, 81 minutes).

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Links for this episode:

Weekly Science Picks

Marc – JotNot
Dickson – New bunyavirus in China (NEJM)
Rich – Listening to the Deep Ocean Environment (LIDO) recording of the Hatsushima earthquake (ScienceDaily article) – thanks Bridget!
Alan –Walter and Ina: A Story of Love, War, and Science
Vincent – Icosahedral light fixture (thanks, Eric!)

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