A bag of genes and surprises

Abstract

Giant viruses continue to yield fascinating discoveries from ancient eukaryotic immune defenses to viruses’ role in the global carbon cycle.

The identification of the first giant virus shook up the field of virology in 2003 and challenged common ideas about the early evolution of viruses and eukaryotes (La Scola et al, ²⁰⁰³). Since, more giant viruses from different host species have been discovered, along with virophages that are viral parasites of giant viruses. It has also become increasingly clear that giant viruses and their parasites are not just another curiosity from an ecological niche but do play an important role in eukaryotic evolution and also perhaps in global marine carbon cycles. Notwithstanding, the evolution and ecology of giant viruses has become a fascinating field of study in itself.

A bumpy start

In fact, it started with an error: the first known virus, Mimivirus, had been observed in its amoebae host in 1992, but had been mistaken for a bacterium on account of its huge size. It was discovered in a Gram stain during research into legionnaires disease and so thought to be a Gram‐positive bacterium that was incorrectly named Bradfordcoccus, after Bradford, UK, where the amoeba was isolated. Mimivirus (short for “mimicking microbe”) is roughly 400 nm in capsid diameter with protein filaments extending a further 100 nm or more from the surface (Fig 1). Its genome comprises 1.2 million base pairs and 1,018 coding genes, compared with an absolute maximum of 375,000 base pairs and a few hundred genes for the more closely related DNA viruses. In fact, it is more comparable to small bacteria such as Mycoplasma genitalium, which is 450 nanometres in diameter and has a genome just half the size, coding for 482 proteins.

Figure 1.

Electron micrograph of a mimivirus particle. From Ghigo et al (2008).

… it started with an error: the first known virus, Mimivirus, had been observed in its amoebae host in 1992, but had been mistaken for a bacterium on account of its huge size.

In terms of hosts, giant viruses have been found in various protists beyond amoeba, including green algae where various viral genes are closely related to host genes that perform vital eukaryotic functions (Schvarcz & Steward, 2018). “The giant viruses themselves mostly occur in algae and heterotrophic protists, but we expect more cases in lower animals such as sponges and worms”, said Matthias Fischer at the Max Planck Institute for Medical Research in Heidelberg, Germany. Even if so, however, there may always be some doubt over the evolutionary origin, given that, as Fischer pointed out, there is no fossil record to provide clues. Yet, the weight of consensus is falling on just one of the two competing hypotheses of how giant virus evolved.

The evolution of giant viruses

The first was the reductive model, according to which the genome of an ancestral parasitic cell shrunk in size until it became dependent on the host’s metabolism. The presence of genes carrying vital cellular functions, such as components of DNA translation, in virtually all known giant viruses, is at least consistent with this model. The problem with this hypothesis though is that it assumes the existence of large ancestral genomes with all the genes from which the current reduced sets were derived. This is known as the “Garden of Eden” assumption and has fallen out of favour as an explanation of how viruses could have evolved from cellular ancestors.

The alternative explanation is the expansion model, by which giant viruses expanded and diversified their genome and accordingly size through gene duplications and horizontal gene transfer. This model is in line with metagenomic studies of the giant virus discoveries in recent years, which indicate that indeed massive gene exchange occurred with a variety of organisms.

Whichever model is correct, it is clear that giant viruses are confined to eukaryotes and as such are distinct from bacteria‐infecting phages. Given this distinction, one surprising discovery was that some of the genes found in giant viruses are highly likely to have come from bacteria. “My guess is that the same amoebae “like” to swallow bacteria and such transfer then occurs via co‐infection”, commented Michal Linial from the Hebrew University of Jerusalem in Israel.

… one surprising discovery was that some of the genes found in giant viruses are highly likely to have come from bacteria.

Shaping eukaryote evolution

A far greater number of genes though have been transferred in either direction with eukaryotic hosts thinks Mohammad Moniruzzaman at Virginia Polytechnic Institute in Blacksburg, USA. “Effectively, thousands of genes can be transferred through endogenization by giant viruses”, he explained. “Some of these genes can be ‘co‐opted’ by the host eukaryote so that they become beneficial for the host. Many giant viruses also encode mobile elements, and after endogenization, these mobile elements can influence the genome structure of the host eukaryote”.

Moniruzzaman added that an important step now is to understand how mobile elements associated with giant viruses shaped the genome structure of eukaryotes. It is well established that retrotransposons and other viral genetic elements have played a big part in the structural genome evolution in higher eukaryotes, including plants and mammals, but it has yet to be determined how important the contribution of giant viruses was lower down the evolutionary chain. A recent study of green algae has made some progress in this direction (Moniruzzaman et al, ^2020a,b). “That 2020 Nature paper helped reveal the role giant viruses may have played in structuring the genomes of green algae that could identify previously unseen forces in the evolution of these important plant species”, said Karen Weynberg at the University of Queensland in Brisbane, Australia.

It is certainly clear that giant viruses have been significant in the functional evolution of eukaryotes through gene exchange, as Frank Aylward, Moniruzzaman’s supervisor at the Virginia Polytechnic Institute, pointed out. “Given the size and complexity of giant virus genomes, their ability to endogenize creates many opportunities for the evolution of genomic novelty in their eukaryotic hosts”, he commented. “Smaller viruses have a very restricted set of genes that their genomes encode, but giant viruses encode genes involved in metabolism, nutrient transport, cytoskeletal dynamics, and many other important processes (Moniruzzaman et al, ^2020a,b)”. These findings challenge the long‐standing assumption that eukaryotes do not experience gene transfer events on the same scale as bacteria. “The endogenization of giant viruses shows that this is not necessarily true”, Aylward argued. Among the genes that were swapped back and forth are genes for glycolysis and the TCA cycle, as well as nutrient transport. Moniruzzaman also cited a recent study which argues that various functions in algal genomes—sugar and amino acid metabolism, and nutrient transport—had been contributed by giant viruses (Nelson, 2021).

It is certainly clear that giant viruses have been significant in the functional evolution of eukaryotes through gene exchange…

Taxonomy and parasites

There is still some confusion and disagreement over how giant viruses should be defined, as the term has been applied rather loosely and colloquially, according to Moniruzzaman. “Some consider a virus ‘giant’ if it can be viewed with a light microscope only, which is unusual for viruses since traditionally they were thought to be too small for this, that is visible only with an electron microscope like Tobacco Mosaic Virus”, Aylward said. “Others have used genome size as a cut‐off, with ‘giant’ viruses having genomes greater than 300 or 500 kilobase pairs, depending on the study”. Nonetheless, there has been convergence towards a taxonomic framework for viruses in general, with giant viruses assigned to the phylum Nucleocytoviricota. “Many recent studies refer to all viruses within this phylum as ‘giant viruses’ based on their common ancestry, even if some are smaller than the traditional thresholds described above”, Aylward added. “I have recently been using this definition, since Nucleocytoviricota do have quite large genomes and virions, even if some are smaller than others”.

Another intriguing aspect of giant viruses is the presence of smaller, co‐infecting viruses that can protect the host against normal destruction by lysis. These viruses were discovered by Curtis Suttle at the University of British Columbia, Canada, and Matthias Fischer in 2008 and are called virophages because they rely on the replication machinery of a giant virus for their own replication (Fischer & Suttle, 2011). They were studying the Cafeteria roenbergensis virus (CroV; Fig 2), which infects the marine bicosoecid flagellate Cafeteria roenbergensis, and found surprisingly that the virus seemed to infect its host better when it aged, contrary to what normally happens. The explanation only emerged when they detected the second much smaller virus in the infecting system. “There was so much virophage present that it inhibited the giant virus’s replication”, Fischer recalled. “But if you kept the sample in the fridge a couple of years the virophage decays much faster than the more stable giant virus. That’s why the older virus worked better”.

Figure 2.

Cryo‐electron micrograph of four CroV particles. Scale bars represent 2,000 Å. From Xiao et al (2017).

It took four years of research to make this discovery, but the wait was worthwhile, Fischer said, because it led to the discovery of an ancient system of adaptive immunity. “The host population survives much better with the virophage present”, he explained. “So, the virophage is considered a mutualist of the host cell”. At the same time, it is also a parasite of the giant virus. “These virophages have adopted completely the lifestyle to parasitize these giant viruses”, Fischer continued. “They use the same transcription signals, promoter signals and transcription terminator sequences, and only replicate when the giant virus is present. So, they use the transcription machinery of the giant virus, rather than of the host like adenoviruses and others of similar size”.

This observation led to another puzzle, which was how this process could be stable given the requirement for co‐infection by two viruses that appeared to have to come together almost by chance. “It seems at first glance like an unstable evolutionary direction, especially when you look at these heterotrophic protists, which are all over the ocean but only at very low abundance”, Fischer said. This evolutionary riddle was solved with the discovery that the virophage had gained the ability to integrate into the host genome. This enables it to wait until the host is eventually infected by a matching giant virus, at which point it could then replicate.

Indeed, the virophage is completely silent in the integrated state and is only activated upon subsequent infection by the giant virus. Fischer pointed to a comparable system in bacteria whereby bacteriophages can become integrated into their host genome as prophages. “But the prophage in bacteria needs to be actively repressed and so needs a repressor protein”, Fischer commented. “That is not the case for virophages, which are not repressed but need activating by an incoming giant virus”. As the virophage protects the eukaryote host from lysis in the event of infection by the corresponding giant virus, it was interpreted as an ancient form of adaptive immunity. “We now hypothesize that this acts as an antiviral defence system in these protists, which can acquire virophages of different sorts”, he explained. “Then when infected by a giant virus that is compatible with one of the stored virophages, that elicits a response and produces virophage particles that save the host from complete lysis”.

As the virophage protects the eukaryote host from lysis in the event of infection by the corresponding giant virus, it was interpreted as an ancient form of adaptive immunity.

He does not however see this as a precursor to more advanced adaptive immunity that evolved in vertebrates, but as a different system. “It follows the same theme but we can’t say it’s the same version, any more than the Crisp /CAS system in bacteria”. Fischer is now determined to take the virophage system towards a similar footing. “That is basically the main theme we’re pursuing, trying to test the hypothesis in natural protist populations for evidence of virophages already integrated”.

… it now turns out that marine viruses play a major role regulating populations of unicellular plankton upon which half of the oxygen production and carbon sequestration on Earth depends.

The process of accumulating virophages itself has implications for genome evolution, because the protists need to excise retroviruses that are no longer needed once a particular giant virus has ceased to be a threat. There is already evidence that integrated virophages can be removed from genomes, according to Fischer, in order to prevent host genomes becoming bloated with unwanted DNA.

Many open questions

There are still other questions to be resolved over virophage and giant virus evolution. The discoveries of four separate families of giant viruses within the larger and loosely defined nucleo‐cytoplasmic large DNA viruses (NCLDV) taxonomic group also hold a lesson for virology, according to Chantal Abergel from the Aix‐Marseille University in France. She noted that investment in virology research had been guided by demands to protect humans, animals or crops from infection. Giant viruses, by posing no threat to any of those three groups, appeared of less interest, and yet it now turns out that marine viruses play a major role in regulating populations of unicellular plankton upon which half of the oxygen production and carbon sequestration on Earth depends (Fischer et al, ²⁰¹⁰).

Abergel is also critical of efforts to compartmentalize viral and cellular evolution in general and establish whether one or the other came first. “We humans need to put things in clear boxes and close them”, she said. “But that is not how life functions. You need to have communications between the boxes. These giant viruses have the same building blocks as cells but operate in a different way. At some point, cells made a big evolutionary leap and continued to advance. […] But it’s reciprocal and viruses are evolving because of the cell as well”, said Abergel. If there is one lesson from the discovery of giant viruses, it is that science can still learn a lot from them and their role in evolution, which is not just important for gaining new insights into early evolution but also for addressing very contemporary problems, such as the role of marine plankton in the carbon cycle.

EMBO reports (2021) 22: e53464.