Virophages and Giant Viruses: Redefining Life on Earth

By Wang Jia Hua

Viruses are classically defined as obligate intracellular parasites which require a host cell for replication, as they lack their own metabolic machinery for energy generation and protein synthesis. They possess an astounding genomic and morphological diversity and are the most abundant biological entities within the biosphere (Mougari et al., 2019). Currently, viruses are known to infect virtually all cellular life forms, including bacteria, archaea, and eukaryotes. Accordingly, viruses also differ in their strategies for genome expression and replication within their specific host cells (Mougari et al., 2019). Over the past two decades, however, the discoveries of giant DNA viruses and the virophages that parasitize on them challenge the conventional notions of a virus while posing vast ecological and evolutionary implications.

In 2003, the first giant virus, Acanthamoeba polyphaga mimivirus (APMV), was successfully isolated from an amoebal coculture in a water-cooling tower of a hospital in England (La Scola et al., 2003). It was aptly named mimivirus (from mimicking microbe virus) as it resembled a bacterium with its large virion and genome size. Remarkably, APMV has a capsid size of 500 nm with protein fibrils reaching 140 nm in length, and contains a dsDNA genome of 1.2 megabase pairs (Mb) encoding 979 putative proteins (La Scola et al., 2003; Raoult et al., 2004). This greatly exceeds the minimum of 4 genes required by a virus as exemplified by the Escherichia viruses MS2 and Qβ (Prescott, Harley & Klein, 1993). Consequently, a new taxonomic family Mimiviridae was established with APMV as the founding member in view of its exceptional features. According to the International Committee of Taxonomy of Viruses (ICTV), there are presently two genera within this family, namely Mimivirus and Cafeteriavirus (Lefkowitz et al., 2018). Cafeteria roenbergensis virus (CRoV) is the only member in the latter genus and infects the marine biflagellate C. roenbergensis (Mougari et al., 2019). As more mimivirus strains were discovered in the following years, they were classified by phylogenomics into three lineages – A, B, and C. APMV, Acanthamoeba polyphaga moumouvirus, and Megavirus chiliensis were the prototypes (i.e. ancestral forms) of the respective lineages (Mougari et al., 2019). 

Interestingly, while sampling for additional amoeba-infecting mimiviruses using the same coculture protocol, two other families of giant viruses (Pandoraviridae and Pithoviridae) were unexpectedly discovered (Abergel, Legendre & Claverie, 2015). Both families exceed Mimiviruses in size: Pandoraviruses have a virion size of around 1 by 0.5 µm and a genome size of over 2.5 Mb (Philippe et al., 2013), while Pithoviruses are currently the largest known viruses with virion sizes of 1.5 by 0.5 µm, albeit with smaller genomes (Legendre et al., 2014). They are, however, not yet formally classified by the ICTV owing to their recent discoveries. The astonishing sizes and complexity of these giant viruses blur the line between what is considered a virus or a cellular life form. While giant viruses display many phenotypic and genotypic features of a typical virus, they also share major differences from other viruses. 

Apart from some bacteriophages which encode tRNAs, viruses do not contain genes for translation system components. This results in an unequivocal reliance on host translation machinery for propagation which forms the hallmark of what separates viruses from cellular life forms (Koonin & Yutin, 2019). In contrast, APMV encodes key components such as translation factors, six tRNAs and, in particular, four aminoacyl-tRNA synthetases (aaRS) – unprecedented in a virus (Raoult et al., 2004). Amazingly, other related giant viruses (e.g. Klosneuviruses) encode nearly all translation system components except for the ribosome itself (Schulz et al., 2017). Major differences between giant viruses and other viruses also include the presence of a complex mobilome (i.e. entire set of all mobile genetic elements, MGE) and a broad host spectrum (Sharma et al., 2016). Notably, giant viruses are visible under a light microscope, have a larger physical and genome size than some microbes, and contain genes that even some small obligate intracellular bacteria lack. In fact, both DNA (genome) and RNA (mRNA, tRNA) could be detected in their purified virions (Colson et al., 2017). As a result, they can be seen to exist at the boundary between living and non-living, sparking debates as to whether (giant and, by extension, all) viruses could, in principle, belong in the tree of life.

The discovery of these giant viruses has contributed to the emergence and revival of ground-breaking yet provocative notions that giant viruses are possible descendants (though reductive evolution) of a putative cellular ancestor that belonged to a fourth domain of life, apart from bacteria, archaea and eukaryotes. This was suggested following phylogenetic and phyletic studies of universal informational genes (e.g. RNAP, DNAP) common to all three domains of life that are also present in the large genomes of giant viruses, in place of the usual ribosomal genes used in phylogenetic reconstruction (Boyer et al., 2010). However, this has been highly contested on both technical and biological grounds. Some argue that the fourth branch of life hypothesis is artefactual owing to lateral gene transfers or convergent evolution (Sharma et al., 2016), and others claim that the universal genes were captured primarily from eukaryotic hosts on multiple occasions and not inherited from a fourth domain (Koonin & Yutin, 2019). Currently, this remains a major issue of controversy between virologists and evolutionists alike.  

Curiously, giant viruses were discovered to be themselves parasitized by another group of viruses named virophages. Virophages are classified under the family Lavidaviridae (or large-virus dependent or associated viruses) which consists of two genera: Sputnikvirus (containing two mimivirus-dependent viruses, Sputnik and Zamilon) and Mavirus (containing only Cafeteriavirus-dependent mavirus). All isolated virophages are non-enveloped with small icosahedral capsids 35-74 nm in diameter, and have genomes ranging from 17-19 kb in length (Paez-Espino et al., 2019). Unlike other host-parasite systems, virophages require two distinct hosts for successful replication – namely a susceptible host cell and a permissive host virus. Specifically, virophages rely on the host cell for energy, metabolites, and other essential systems while hijacking the cytoplasmic virion factory produced by a coinfecting giant virus (Duponchel & Fischer, 2019). Moreover, some virophages (e.g. Sputnik) cause partial or complete inhibition of host virus growth and may protect the host cell from infection. In particular, the presence of Sputnik results in abortive forms, characterized by thickening of the capsid layer, and irregular capsid assembly of the host virus which reduce the yield of the giant virus progeny by around 70% (La Scola et al., 2008). Zamilon, however, does not appear to affect host virus replication or infectivity which raises questions regarding its parasitic status as a virophage (Mougari et al., 2019). Arguably, virophage discovery can be perceived as additional evidence of a unique, cell-like character of giant viruses, and even of their status as life forms.

Additionally, there seems to be some form of coevolution observed as a result of the tripartite host cell, giant virus, and virophage interactions. Notably, Sputnik is capable of infecting all three lineages of mimiviruses while Zamilon has a narrower host range of only lineages B and C. Recent studies revealed that the resistance of lineage A mimiviruses to Zamilon is linked to the presence of a multigene-containing operon named mimivirus virophage resistance element (MIMIVIRE), which contains endogenized virophage sequences (Levasseur et al., 2016). MIMIVIRE-associated genes encode a helicase and nuclease which aid in degrading foreign genetic materials and is likened to the to the CRISPR-Cas system, although this nucleic-acid-based immunity remains controversial (Levasseur et al., 2016). The intimate interaction between virophages and giant viruses is further supported by the discovery of integrated virophages (or provirophages) and a previously unknown class of MGE known as transpovirons in the genome of the Lentille virus from the Mimivirus genus. Transpovirons are linear DNA elements (~7 kb) consisting of six to eight protein-coding genes, of which two are homologous to virophage genes (Desnues et al., 2012). These MGEs form part of the complex mobilome of giant viruses and contribute significantly to interviral gene transfer.

While the extent of the ecological impact possessed by virophages is currently unknown, modelling approaches suggest that through influencing the population dynamics of giant viruses and protists alike, virophages exert ecosystem effects on nutrient cycling and primary production. Additionally, they may play a critical role in shaping the genomic landscape of their hosts through gene exchange and recombination. Finally, the study of virophages and giant viruses, and their respective genomes and life cycles, is still in its infancy, and further metagenomic and phylogenetic studies must be conducted to fully understand their evolutionary origins.

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