Giant viruses have been unearthed in several of the world’s most mysterious locations

In recent years, giant viruses have been unearthed in several of the world’s most mysterious locations, from the thawing permafrost of Siberia to locations unknown beneath the Antarctic ice.

But don’t worry, “The Thing” is still a work of science fiction. For now.

In a new study, a team of Michigan State University scientists shed light on these enigmatic, yet captivating giant microbes and key aspects of the process by which they infect cells.

With the help of cutting-edge imaging technologies, this study developed a reliable model for studying giant viruses and is the first to identify and characterize several key proteins responsible for orchestrating infection.

Giant viruses are bigger than 300 nanometers in size and can survive for many millennia.

For comparison, the rhinovirus – responsible for the common cold – is roughly 30 nanometers.

“Giant viruses are gargantuan in size and complexity,” said principal investigator Kristin Parent, associate professor of Biochemistry and Molecular Biology at MSU.

“The giant viruses recently discovered in Siberia retained the ability to infect after 30,000 years in permafrost.”

The outer shells – or capsids – are rugged and able to withstand harsh environments, protecting the viral genome inside.

The capsids of the species analyzed in this study – mimivirus, Antarctica virus, Samba virus and the newly discovered Tupanviruses – are icosahedral, or shaped like a twenty-sided die.

These species have a unique mechanism for releasing their viral genome. A starfish-shaped seal sits atop one of the outer shell vertices. This unique vertex is known as the ‘stargate.’ During infection, the ‘starfish’ and ‘stargate’ open to release the viral genome.

During the study, several roadblocks needed to be addressed. “Giant viruses are difficult to image due to their size and previous studies relied on finding the ‘one-in-a-million’ virus in the correct state of infection,” Parent said.

To solve this issue, Parent’s graduate student Jason Schrad developed a novel method for mimicking infection stages. Using the university’s new Cryo-Electron Microscopy microscope and the university’s Scanning Electron Microscope, Parent’s group subjected various species to an array of harsh chemical and environmental treatments designed to simulate conditions a virus might experience during the infection process.

“Cryo-EM allows us to study viruses and protein structures at the atomic level and to capture them in action,” Parent said. “Access to this technology is very important and the new microscope at MSU is opening new doors for research on campus.”

The results revealed three environmental conditions that successfully induced stargate opening: low pH, high temperature and high salt. Even more, each condition induced a different stage of infection.

With this new data, Parent’s group designed a model to effectively and reliably mimic stages of infection for study. “This new model now allows scientists to mimic the stages reliably and with high frequency, opening the door for future study and dramatically simplifying any studies aimed at the virus,” Parent said.

The results yielded several novel findings. “We discovered that the starfish seal above the stargate portal slowly unzips while remaining attached to the capsid rather than simply releasing all at once,” Parent said. “Our description of a new giant virus genome release strategy signifies another paradigm shift in our understanding of virology.”

With the ability to consistently recreate various stages of infection, the researchers studied the proteins released by the virus during the first stage. Proteins act as workers, orchestrating the many biological processes required for a virus to infect and hijack a cell’s reproductive capabilities to make copies of itself.

“The results of this study help to assign putative – or assumed – roles to many proteins with previously unknown functions, highlighting the power of this new model,” Parent said. “We identified key proteins released during the initial stages of infection responsible for helping mediate the process and complete the viral takeover.”

As for future study? “The exact functions of many of these proteins and how they orchestrate giant virus infection are prime candidates for future study,” Parent said.

“Many of the proteins we identified matched proteins that one would expect to be released during the initial stages of viral infections. This greatly supports our hypothesis that the in vitro stages generated in this study are reflective of those that occur in vivo.”

That many of the different giant virus types studied responded similarly in vitro leads the researchers to believe they all share common characteristics and likely similar proteins.

Whether giant viruses are capable of infecting humans ­- unlike the coronavirus ­- is an evolving topic of discussion amongst virologists.

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he discovery of giant viruses infecting protists, sometimes called giruses, pioneered by the isolation of Acanthamoeba polyphaga mimivirus (APMV), is one of the most unexpected and spectacular breakthroughs in virology in decades (Claverie, 2006; Claverie and Abergel, 2010, Claverie et al., 2009, Claverie et al., 2006, Koonin, 2005, La Scola et al., 2003, Raoult et al., 2004, Van Etten et al., 2010, Van Etten, 2011).

The giant viruses shatter the textbook definition of viruses as “filterable” infectious agents because their virions do not pass bacterial filters and obliterate all boundaries between viruses and cellular life forms in terms of size.

Indeed, not only are the particles of giant viruses larger than the cells of numerous bacteria and archaea but also the genomes of Pandoraviruses, the current record holders at approximately 2.5 Mb (Philippe et al., 2013), are larger and more diverse in gene content than many bacterial and archaeal genomes, from both parasites and free-living microbes (Koonin and Wolf, 2008).

The recent identification of Pandoraviruses and Pithoviruses (Legendre et al., 2014) that are not only huge, by the standards of the virology, but also possess a previously unseen, asymmetrical virion structure, shows that the true diversity of giant viruses has been barely tapped into.

The unexpected, “cell-like” features of giant viruses led several researchers to propose fundamental concepts that go far beyond the study of these particular viruses and beyond virology in general.

The foremost of these conceptual developments is the proposition that giant viruses represent a “fourth domain of life” that is distinct from but comparable to the three cellular domains, bacteria, archaea and eukaryotes (Claverie et al., 2006, Colson et al., 2012, Colson et al., 2011, Desnues et al., 2012, Legendre et al., 2012, Raoult et al., 2004).

It seems useful to distinguish the fourth domain concept as a general idea and as a specific hypothesis. As a general notion, the claim that giant viruses represent a fourth domain of life simply refers to the “cell-like” character of these viruses in terms of size of the virions and genomes and, in addition, to the observation that many genes of these viruses have no detectable homologs and so might come from some unknown source.

With these general statements, the fourth domain concept does not make any falsifiable predictions. In contrast, the specific fourth domain hypothesis is steeped directly in the original definition of the three domains of cellular life.

These three domains, bacteria, archaea and eukaryota, correspond to the three major trunks in the unrooted phylogenetic tree of 16S ribosomal RNA (Pace, 1997, Pace, 2006, Pace et al., 1986, Woese, 1987, Woese and Fox, 1977, Woese et al., 1990, Woese et al., 1978) that is topologically consistent with the phylogenies of most of the other (nearly) universal genes that encode primarily components of the translation and the core transcription machineries (Brown and Doolittle, 1997, Brown et al., 2001, Puigbo et al., 2009, Puigbo et al., 2013).

Strikingly, and unlike other viruses, the giant viruses encode several proteins that are universal among cellular life forms, in particular translation system components, such as aminoacyl-tRNA synthetases and translation factors. The presence of these universal genes provides for the opportunity to formally incorporate the giant viruses into the tree of life (Raoult et al., 2004).

The outcome of the phylogenetic analysis of the universal genes is (at least, in principle) readily interpretable: the placement of the viral genes outside the three traditional domains of cellular life is compatible with the fourth domain hypothesis whereas their placement within any of the three domains is not.

Several studies, starting with the original analysis of the mimivirus genome, have reported phylogenetic trees that appeared compatible with giant viruses comprising a fourth domain (Colson et al., 2012, Colson et al., 2011, Nasir et al., 2012, Raoult et al., 2004).

However, such observations could be inherently problematic. Indeed, accelerated evolution of viral genes that is likely to have occurred, especially immediately following the acquisition of the respective genes from the host, has the potential to obscure their affinity with homologs from cellular organisms within one of the recognized domains (a common problem in the analysis of deep phylogenies; Felsenstein, 2004).

A subsequent re-analysis of the phylogenies of several universal genes has failed to find support for the fourth domain hypothesis (Williams et al., 2011).

Notwithstanding their unusual size, genetic complexity and the presence of some universal cellular genes, all giant viruses contain a set of core genes that define an expansive group of eukaryotic double-stranded (ds) DNA viruses that is referred to as Nucleo-Cytoplasmic Large DNA viruses (NCLDV) (Iyer et al., 2001, Iyer et al., 2006, Koonin and Yutin, 2010) or the proposed order Megavirales (Colson et al., 2012, Colson et al., 2013).

Hereinafter we refer to this major group of viruses as “Megavirales” to signal our support of this amendment to virus taxonomy while indicating that the order so far has not been officially adopted by the International Committee for the Taxonomy of Viruses.

The “Megavirales” unite 7 families of viruses infecting diverse eukaryotes, namely Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Marseilleviridae, Phycodnaviridae, and Mimiviridae, as well as the recently discovered giant Pandoraviruses and Pithoviruses that could found new families.

Evolutionary reconstructions have mapped about 50 genes encoding essential viral functions to the putative common ancestor of the “Megavirales” although some of these putative ancestral genes have been lost in certain groups of viruses (Koonin and Yutin, 2010, Yutin et al., 2013, Yutin et al., 2009).

This ancestral gene set does not include genes for components of the translation system or any other genes that might be considered suggestive of a cellular nature of the common ancestor of the “Megavirales” implied by the fourth domain hypothesis.

Phylogenetic analysis of the universal “Megavirales” genes reveals apparent evolutionary relationships between giant and smaller viruses. Specifically, Mimiviruses cluster with the so-called Organic Lake phycodnaviruses and Phaeocystis globosa viruses (Santini et al., 2013, Yutin et al., 2013), Pandoraviruses with Phycodnaviruses, in particular Coccolithoviruses (Yutin and Koonin, 2013), and Pithoviruses with Marseilleviruses and Iridoviruses (Legendre et al., 2014).

Combined with the results of evolutionary reconstructions based on the phyletic patterns of “Megavirales” genes (i.e. matrices of gene presence and absence), these relationships suggest that different groups of giant viruses could have independently evolved from smaller ancestral viruses (Yutin and Koonin, 2013).

There is an obvious tension between the fourth domain of life hypothesis and the monophyly of the “Megavirales”. The fact that giant viruses encode the large set of ancestral “Megavirales” genes, some of which are “virus hallmark genes” without close homologs encoded in cellular life forms (Koonin et al., 2006), constrains the fourth domain hypothesis to a specific version.

Specifically, one would have to postulate that a viral ancestor of the giant viruses reproduced in a host that belonged to a fourth domain of cellular life and acquired numerous genes including some that are universal in cellular life forms. After the fourth cellular domain went extinct, the resulting giant viruses would remain the only “living fossils” of their original hosts.

We sought to formally test the fourth domain hypothesis as comprehensively as possible and additionally to address the origins of the gene repertoires of giant viruses, and their evolutionary relationships with other “Megavirales”.

The results of this phylogenomic analysis effectively falsify the fourth domain hypothesis, reveal diverse origins of the genes of giant viruses, and reaffirm the origin of giant viruses from simpler ancestors.

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More information: Jason R. Schrad et al, Structural and Proteomic Characterization of the Initiation of Giant Virus Infection, Cell (2020). DOI: 10.1016/j.cell.2020.04.032