Good viruses visiting bad neighborhoods

Marco VignuzziWhat would happen to an RNA virus if its genome were placed in a bad neighborhood? The answer is that fitness plummets.

RNA virus populations are not composed of a single defined nucleic acid sequence, but are dynamic distributions of many nonidentical but related members. In the past I have referred to these populations as quasispecies but that is no longer the preferred term: mutant swarms or heterogeneous virus populations should be used instead.

The term for all possible combinations of a viral genome sequence is sequence space; for a 10,000 nucleotide genome this would be theoretically 410,000 different genomes – a huge number, more than the atoms in the universe. Any RNA virus population occupies only a fraction of this sequence space, in part because many mutations are deleterious. Studies have shown that viral genomes occupy specific parts of sequence space, called neighborhoods, and movement to different neighborhoods is important for viability. If the viral genome is placed in a bad neighborhood – one that is detrimental for virus fitness – the ability to explore sequence space is restricted.

An example of the effect of changing viral sequence space is shown by a study in which hundreds of synonymous mutations (they did not change the amino acid sequence) were introduced in the capsid region of poliovirus (link to paper). Such rewiring, which placed the virus in a different sequence space, reduced viral fitness and attenuated pathogenicity in a mouse model. In other words, the viral genome was placed in a bad neighborhood, from where it could not move to other neighborhoods needed for optimal replication and pathogenesis. While the genome rewiring did not affect the protein sequence, it might have had deleterious effects on RNA structures or codon or dinucleotide frequency. For example, introduction of codon pairs that are under-represented in the human genome can produce less fit viruses.

A recent study avoids these potential issues by introducing changes in the viral genome that do not affect protein coding, RNA structures or codon or dinucleotide frequency, yet place the viral genome in a different sequence space (link to paper). All 117 serine/leucine codons in the capsid region of Coxsackievirus B3 were changed so that a single nucleotide mutation would lead to a stop codon, terminating protein synthesis and virus replication (this virus is called 1-to-Stop). The serine codons were changed to UUA or UUG; one mutation changes these to the terminators UAA, UGA, or UAG. Another virus was made in which two mutations were needed to produce a stop codon (NoStop virus).

1-to-Stop viruses replicated normally, but when mutagenized, they had significantly lower fitness than wild type or NoStop viruses. Extensive passage of the virus in cells, which would be expected to cause accumulation of mutations, had the same effect on fitness. When a high fidelity RNA polymerase was introduced into 1-to-Stop virus, it replicated like wild type virus. Similar results were obtained with an influenza virus when one of its 8 genome segments was rewired to produce 1-to-Stop and NoStop counterparts.

The 1-to-Stop Coxsackieviruses were attenuated in a mouse model of infection. Furthermore, mice infected with 1-to-Stop virus were protected against replication and disease after challenge with wild type virus. These observations suggest that rewiring viral RNA genomes could be used to design vaccines.

These findings show that recoding a viral genome places it an different sequence space than wild type virus, in which single mutations can lead to inactivation of viral replication. This new neighborhood is unfavorable (‘bad’) because the virus cannot readily move to other neighborhoods to accommodate the effects of mutation.

For more discussion of viral sequence space and rewiring viral genomes, listen to the podcast This Week in Evolution #24: our guest is Marco Vignuzzi (pictured), senior author on the second paper discussed here.

From cell proteins to viral capsids

Origin of virusesWe have previously discussed the idea that viruses originated from selfish genetic elements such as plasmids and transposons when these nucleic acids acquired structural proteins (see A plasmid on the road to becoming a virus). I want to explore in more detail the idea that the structural proteins of  viruses likely originated from cell proteins (link to paper).

Three ideas have emerged to explain the origin of viruses: 1. viruses evolved first on Earth, before cells, and when cells evolved, the viruses became their genetic parasites; 2. viruses are cells that lost many genes and became intracellular parasites; 3. viruses are collections of genes that escaped from cells. Missing from these hypothesis is how nucleic acids became virus particles – that is, how they acquired structural proteins. It seems likely that viral structural proteins originated from cellular genes.

An analysis of the sequence an structure of major virion proteins has identified likely ancestors in cellular proteins. Following are some examples to illustrate this conclusion.

A very common motif among viral capsid proteins is called the single jelly roll, made up of eight beta strands in two four-stranded sheets. Many cell proteins have jelly role motifs, and some form 60-subunit virus-like particles in cells. The extra sequences at the N-termini of viral jelly roll capsid proteins, involved in recognizing the viral genome, likely evolved after the capture of these proteins from cells.

The core proteins of alphaviruses (think Semliki Forest virus) has structural similarity with chymotrypsin-like serine proteases. The viral core protein retains protease activity, needed for cleavage from a protein precursor.

Retroviral structural proteins also appear to have originated from cell proteins, with clear homologies with matrix, capsid, and nucleocapsid proteins. The matrix Z proteins of arenaviruses are related to cellular RING domain proteins, and the matrix proteins of some negative strand RNA viruses are related to cellular cyclophilin. There are many more examples, providing support for the hypothesis that viruses evolved on multiple instances by recruiting different cell proteins.

Given this information on the origin of viral capsid proteins, we can modify the three hypotheses for the origin of viruses into one. Self-replicating, virus like nucleic acids emerged in the pre-cellular world and from the emerged the first cells. The replicating nucleic acids entered the cells, where they replicated and became genetic parasites. At some point these genetic elements acquired structural proteins from the cells and became bona fide virus particles. As cells evolved, new viruses emerged from them.

It is important to point out that the genes do not always flow from cells to viruses. We know that viral proteins can be returned to cells, where they serve useful functions. One example is syncytin, a retroviral protein used for the construction of the mammalian placenta.

A plasmid on the road to becoming a virus

Origin of virusesPlasmids have been discovered that can move from cell to cell within membrane vesicles in a species of Archaea (link to paper). They provide clues about the origin of virus particles.

Electron microscope analysis of the culture medium from Halobrum lacusprofundi R1S1, an Archaeal strain from Antarctica, revealed spherical particles which were subsequently shown to contain a 50,000 base pair circular double-stranded DNA molecule. When added to H. lacusprofundi, the purified membrane vesicles entered the cells and the DNA replicated.

Nucleotide sequence analysis of the plasmid within the membrane vesicles revealed 48 potential protein coding regions and an origin of DNA replication. None of these proteins showed any similarity to viral stuctural proteins, leading the authors to conclude that these particles are not viruses.

Many of the proteins encoded in the plasmid DNA were found in the membrane vesicles. Some of these are similar to cell proteins known to be involved in the generation of membrane vesicles. However no DNA polymerase-like proteins are encoded in the plasmid. These data suggest that the plasmid encodes proteins that generate, from the membranes of the cell, the vesicles needed for their transport to other cells. However, replication of the plasmid is carried out by cellular DNA polymerases.

It is likely that the plasmid-containing membrane vesicles are precursors of what we know today as virus particles. It is thought that viruses originated from selfish genetic elements such as plasmids and transposons when these nucleic acids acquired structural proteins (pictured; image credit). Phylogenetic analyses of the structural proteins of many enveloped and naked viruses reveal that they likely originated from cell proteins on multiple occasions (link to paper).

The membrane-encased Archaeal plasmid seems well on its way to becoming a virus, pending acquisition of viral structural proteins. Such an early precursor of virus particles has never been seen before, emphasizing that science should not be conducted only under the streetlight.

TWiV 454: FGCU, Zika

Sharon Isern and Scott Michael return to TWiV for a Zika virus update, including their work on viral evolution and spread, and whether pre-existing immunity to dengue virus enhances pathogenesis.


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Ancient proteins block modern viruses

T7-like virusCould ancient host proteins contribute to the replication of a modern virus? The answer is, not very well (link to paper).

Viruses are obligate intracellular parasites, which means that they have to get inside of a host cell to produce more viruses. The genomes of all viruses, even the biggest ones, do not encode anywhere near the number of proteins that are needed to replicate. The cell provides thousands of proteins that are involved in energy production, membrane synthesis, protein synthesis, transport, and so much more.

The difficulty in studying ancient proteins is that none of them exist. But we can make good guesses about what very old proteins might look like, by examining modern proteins, seeing how they vary among organisms, and calculating how they might look like billions of years ago. The field of predicting what ancient proteins might look like is quite active.

Investigators have predicted what ancient versions of a cell protein called thioredoxin might have looked like. They have synthesized such ‘ancient’ thioredoxins and shown that they are stable and active. Thioredoxins are found in nearly all organisms, where they act as antioxidants.

Ancient thioredoxins that have been synthesized include those from the last common ancestors of bacteria; of archaea; and of archaea and eukaryotes (all around 4 billion years old); the last common anestor of cyanobacterial, deinococcus, and thermus groups (about 2.5 billion years old);  the last common ancestor of gamma-proteobacteria; of eukaryotes; and of fungi and animals (around 1.5 billion years old).

These ancient thioredoxins work in a modern E. coli. This bacterium has two thioredoxin genes, and if they are both deleted, growth occurs, but very slowly. If genes encoding ancient thioredoxins are introduced into these mutated bacteria, they can compensate for the growth deficiency. The older thioredoxins (4 billion years) compensate less well than ones that are closer in time (1.5 billion years).

It’s amazing that an ancient protein can work in a modern E. coli. But could ancient thioredoxins support viral growth?

Thioredoxin from E. coli is an essential part of the DNA polymerase complex of the bacteriophage T7 (pictured – image credit). This virus does not form plaques on E. coli lacking the two thioredoxin genes. The only ancient thioredoxin gene that allows phage T7 plaque formation is from the last common ancestor of cyanobacterial, deinococcus, and thermus groups, which is about 2.5 billion years old and has 57% amino acid identity with the E. coli enzyme. But the effienciency of plaque formation was very poor – about 100 million times worse than on regular  E. coli. None of the older thioredoxins worked.

Why would an ancient thioredoxin work for E. coli but not for bacteriophage T7? Over billions of years, thioredoxin evolves but it must still be able to carry out its function for E. coli. The viruses that infected bacteria 4 billion years ago were very different from contemporary viruses, and so the ancient thioredoxin does not work for modern viruses. Today’s thioredoxin could change so that it would not support T7 replication – as long as the enzyme still works for E. coli.

The authors of this work view it as a proof of principle: that virus growth is not supported by an ancient version of a modern protein required for virus replication. They would like to apply this approach to produce plants that are resistant to viruses, which have serious effects on global agricultural productivity.

I think the work is amazing not only because an ancient protein can be made, but it supports growth of the host and not that of a virus. It might therefore be possible to reconstruct the host-virus arms race, starting from ancient proteins. In this race, the gene encoding an essential cell protein can evolve so that it no longer supports virus replication. Next, the viral genome changes to adapt to the altered cell protein. And so the game goes back and forth.

The authors have shown that they can select mutant bacteriophage T7 isolates that replicate in the present of an ancient thioredoxin. This result suggests that it might be possible to reconstruct host-virus arms races beginning with an ancestral host protein. If we can make an ancient protein, could we also make an ancient virus? Why not?

TWiV 450: Ben tenOever and RNA out

Ben tenOever joins the TWiVoli to discuss the evolution of RNA interference and his lab’s finding that RNAse III nucleases, needed for the maturation of cellular RNAs, are an ancient antiviral RNA recognition platform in all domains of life.


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TWiV 440: I hardly noumeavirus

No problem not being nice to Dickson in this episode, because he’s absent for a discussion of a new giant virus that replicates in the cytoplasm yet transiently accesses the nucleus to bootstrap infection.

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TWiV 433: Poops viruses and worms

The lovely TWiV team explore evolution of our fecal virome, and the antiviral RNA interference response in the nematode C. elegans.

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TWiV 432: Conjunction junction, what’s your function?

The TWiVites discuss Zika virus seroprevalence in wild monkeys, Zika virus mRNA vaccines, and a gamete fusion protein inherited from viruses.

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TWiV 424: FLERVergnügen

Trudy joins the the TWiVlords to discuss new tests for detecting prions in the blood, and evidence showing that foamy retroviruses originated in the seas with their jawed vertebrate hosts at least 450 million years ago.

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