Uncovering a missing link in viral evolution - how did some get so big?

Vietnamese jungle.

Credit: FLETCHER & BAYLIS/SCIENCE PHOTO LIBRARY

  Out of the dense, tropical rainforest of Northern Vietnam, researchers have discovered a missing link in viral evolution. Through the large-scale screening of trapped mosquitoes, a joint Dutch and Japanese group may have identified the secrets of how one group of viruses - the nidoviruses - got really big. 

  This work potentially answers one of the more prevailing mysteries in viral evolution: how can RNA viruses escape the evolutionary restrictions placed on them by their very high mutation rates and get more complex? If you look closer though, more questions are thrown back than are answered.

  The positive-sense RNA viruses are an extremely large and diverse group of viruses, housing many known - and unknown - human pathogens and indeed many non-pathogenic, environmentally influential microbes. On the whole, these viruses don't get very big (see the graph below); having a genome made of RNA isn't particularly a good thing if you want to have a long genome, encoding lots of complex genes. The enzymes these viruses use to copy their genomes are nowhere near as accurate as their cellular counterparts. And, introducing mutations every few thousands nucleotides is bound to impact on your evolutionary potential and fitness.

 

Distribution of +ve sense RNA virus genome sizes, notice the large gap between the nidoviruses and others. See the newly discovered virus, NDiV lying close to the lower limit.

  But certain members of one group, the nidoviruses (containing the now infamous SARS-coronavirus) are somehow able to greatly expand their genomes and complexity taking the range of positive-sense RNA genome sizes  from 3 kilobases (kb) to 31. The longer the genome you can have, the more genes and proteins you can encode. This thus increases the potential function and complexity of the virus which obviously plays a major role in host adaptation, evasion of immunity and pathogenesis. 

  The reasons just how the nidoviruses achieve comparatively massive size are subject to intense research and interest. Yet to make it all the more complex, the nidoviruses themselves bridge this genome size gap: some of them are 'small', with genomes around 13kb while some are 'large', with genomes around 30kb. Clearly, something is going on with these larger viruses that allows them to get around this apparent mutational block on genome size.

  Using bioinformatic techniques, this capacity to achieve genome sizes of 30kb has been potentially mapped to one particular region of the 'larger' nidovirus genomes: a 3'-5' exoribonuclease found within the polymerase enzyme that facilitates the editing out of incorrect components of RNA and may allow for a decreased mutation rate. If you mess with this enzyme, you also screw up their replication accuracy, as documented here using deep-sequencing. And, as the theory goes, the lower this rate, the bigger the genomes can get. It is believed that this enzyme is a distant relative of cellular proteins, but how it arrived in the nidoviruses is anyones guess.

SARS-coronavirus genome structure and virus particle. This virus is a good model for the larger nidoviruses. The uncovered exoribonuclease is found within ORF1b.

  This is where the mosquitoes come in, while searching for unknown human pathogens in Vietnamese jungles, the researchers found something they altogether didn't expect (grab the paper here). Although they found a new virus (named Nam Dinh virus after the province it was found in - NDiV), when sequenced, it was found to be a novel member of the nidoviruses and probably didn't cause any disease in humans. But strangely - when examined more closely - it didn't fit into the pre-defined 'small' and 'large' groups: it's genome lay between the small and large groups of viruses at around 20kb. .

  The team also determined that this virus encoded those enzymes previously thought to be specific to the large viruses, although it had a genome size much closer to the smaller ones. NDiV was found to be an ancestral lineage of current invertebrate nidoviruses, which themselves are on the small end of the scale. So what does this say about how these positive-sense RNA viruses got so big? And why is this study so important? Especially as just a few months ago, another insect nidovirus was discovered in Côte d'Ivoire, although its genome did not encode an exoribonuclease.

  The discovery of the exonuclease enzyme  - combined with it's genome size - in this newly discovered virus leads to its classification as a viral 'missing link' between the large and small nidoviruses. Yet, while it is closer in size to the smaller viruses, it does encode those enzymes that control mutation rate in the larger viruses, thus, if anything, this discovery expands the lower limit of 'large' nidoviruses. On the other hand, this may be taken as evidence that the exoribonuclease isn't ALWAYS responsible for large genome sizes. But the previously carried out biochemical work clearly shows that the enzyme is responsible for nidovirus replication fidelity. 

  These results are only the first, near-preliminary analysis of this virus and I suspect that a more detailed molecular study may be able to expand on the activity of the 'large' versus 'small' enzymes in nidovirus evolution.The other proteins from this virus will continue to interest researchers studying how these viruses got so big.

The 'Eigen Trap' hypothesis of factors influencing genome size, compexity and mutation rate. RNA viruses may escape all restrictions through the acquisition of the exoribonuclease enzyme. 

Finally, the group lay down their hypothesis that may explain how small RNA viruses can get bigger and more complex - modelled on the scenario of the nidoviruses and shown below as the ' Eigen Trap':

In RNA viruses, the low fidelity of replication severely restricts the size of their genomes, which can encode only relatively simple replication complexes that, hence, suffice to support low-fidelity replication [21], [69]. This low-state trap is known as the “Eigen paradox”. Accordingly, a transition from the “low” to the “high” state may not be accomplished by changing only one element of the triangle, e.g. improving replication fidelity, since such a change would not be compatible with the “low” state of the other two elements [68] [70]. The exclusive presence of ExoN in ssRNA+ viruses above 20 kb supports the logic of the Eigen paradox [68]. It also shows how the paradox could be solved with a single evolutionary advancement, the acquisition of ExoN, which may have relieved the constraints on all three elements of the triangular relationship (Fig. 7), providing a lasting benefit to the virus lineage that acquired ExoN.

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Nga, P., Parquet, M., Lauber, C., Parida, M., Nabeshima, T., Yu, F., Thuy, N., Inoue, S., Ito, T., Okamoto, K., Ichinose, A., Snijder, E., Morita, K., & Gorbalenya, A. (2011). Discovery of the First Insect Nidovirus, a Missing Evolutionary Link in the Emergence of the Largest RNA Virus Genomes PLoS Pathogens, 7 (9) DOI: 10.1371/journal.ppat.1002215