Virus Genes Don't Come from Host Genes

There's a school of thought that says that viruses originated as escaped constellations of host genes. Virus genes have to originate from somewhere. One theory is that they started with host genes.

Trouble is, there's precious little evidence that viral genes originated from host genes, and plenty of evidence to the contrary. It may actually be that host genes came from viruses.

To say viral genes derive from host genes is like saying hemorrhoids derive from earlobes. Any resemblance is, at best, superficial.

In a previous post, I showed data for the relatively large phylogenetic distance between thymidine kinase genes in phages (viruses that attack bacteria) and their hosts. In one case, I showed that prophage genes (genes from viruses that have succeeded in integrating into the host DNA) are more similar to host genes than lytic-lifestyle phages, but even in the case of temperate phages, I think we have to be honest and say that a prophage is still an example of foreign DNA integrating into a host. (Prophage genes can usually be easily identified by their base composition, which differs noticeably from the base composition of host genes.) Once a prophage becomes fully integrated into the host, its DNA (under the influence of the host repairosome) will tend to ameliorate, taking on the base composition and other characteristics of the host DNA, making it superficially similar to host DNA.

What "other characteristics" does ameliorated DNA take on? Consider codon usage patterns. Recall that the genetic code is set up in such a way that most amino acids correspond to more than one codon (three-letter pattern) in the DNA. Leucine, for example, can be encoded six different ways (namely by base patterns CTA, CTG, CTT, CTC, TTA, and TTG). Likewise, alanine can be encoded four ways (GCA, GCG, GCT, or GCC). But specific organisms develop specific patterns of codon use, preferring certain synonyms over others. For example, Clostridium botulinum (the food-poisoning bug) overwhelmingly prefers to use TTA for leucine (rarely using the other 5 synonyms), whereas E. coli strongly prefers to use CTG (choosing it four-to-one over the next-most-used leucine codon). These codon preference patterns are highly specific to a given species and are thought to be related to the numbers and types of available transfer RNAs (tRNA) in the cell, although frankly it's still an open question whether codon usage adapted to tRNA availability or the reverse.

The idea that viruses mutate rapidly and evolve in close harmony with the hosts on which their reproduction depends suggests that virus codon preferences should match those of the host. (This would be particularly true if virus genes come from host genes.) Remember that a virus has no ribosomal machinery and must rely on the host's protein-making equipment in order to survive. Therefore it would make sense for a virus to adapt its codon usage patterns to the patterns most favored by the host equipment.

That's not what we find. When we look at the codon usage patterns of phage T4 (a classic enterobacterial phage) versus E. coli's codon usage, we find that they differ substantially:

Codon usage frequencies for T4 phage (left) and E. coli B (right).

In this graphic, host-cell codon usage frequencies are on the right while corresponding T4 virus frequencies are on the left. Note that the T4 chromosome encodes 274 protein genes, encompassing over 50,000 codons, so the graphic is based on fairly solid numbers; variations from E. coli can't be accounted for simply by statistical noise.

T4's codon preferences are so different from the host cell's, the T4 phage brings with it genes for 8 types of tRNA. But the differences in codon usage go well beyond 8 codons, so the presence of tRNA genes in T4 DNA doesn't, by itself, explain the divergence of the data.

But what about temperate phages, like Fels-2 (a prophage in the Salmonella genome)? Since prophage genes are, in effect, a permanent part of the host genome, we would expect to see some amelioration of codon usage. And in fact, that is what we do see:

Codon usage in Fels-2 phage (left) and Salmonella typhimurium LT2 (right).

Here, we see that the codon usage patterns of Fels-2 and its host are quite similar. The differences are easily accounted for by the fact that Fels-2 has only 47 protein-coding genes, and the amino acid composition of those genes is probably different enough from "average" host genes to sway the usage stats to the degree shown here. Nevertheless, codon usage patterns aren't sufficient to tell us where Fels-2 genes came from originally. That's still an open question. Like the Martians in War of the Worlds, Fels-2 genes probably came from "somewhere else."

Robbie: "What, you mean, like Europe?"

Tom Cruise character: "No, Robbie. Not like Europe."

More Science on the Desktop

Not to keep harping on the amazing power of desktop omics tools, but I thought I'd share a tip for those of you into genome-mining. The tip in a nutshell is that if you gang-load a bunch of FASTA sequences (DNA sequence data) into the FeatView form at http://genomevolution.org, then click the rather inconspicuous button labeled "Phylogeny.fr" at the bottom left of the FeatView page, you'll be taken automatically to http://www.phylogeny.fr, where you'll get a realtime-generated phylogenetic tree based on the sequence data you provided in FeatView, with no effort on your part (it's truly a one-click operation). Copy and paste DNA sequences into FeatView, click one button, and 30 seconds later a tree shows up on your screen, looking (perhaps) something like this:

The reason I made this tree is that I wasn't satisfied with my knowledge of the relatedness of certain weird microorganisms I've recently run into. Namely:

• Ralstonia (which I mentioned yesterday), WEIRD BECAUSE: It turns hydrogen gas and CO2 into plastic.
• Bordetella, a bronchial infection agent; WEIRD BECAUSE: It turns out to be very similar, genetically, to Ralstonia
• Burkholderia, a soil organism (and human and animal pathogen), WEIRD BECAUSE: It has an unexpectedly large amount of genetic similarity to Ralstonia and Polynucleobacter
• Polynucleobacter, a ditch-water bacterium, WEIRD BECAUSE: It can live as an intracellular parasite of freshwater ciliates or it can live independently in soil (making it potentially a great study organism for determining the genetic bases of intracellular symbiosis)
• Thiomicrospira, a very tiny CO2- and sulfur-loving organism, WEIRD BECAUSE: It can only be found near deep-sea thermal vents (see my previous writeup)
• Polaromonas, a relatively newly discovered and still poorly understood bacterium, WEIRD BECAUSE: It is abundant in glacier ice on multiple continents. Plus it has an amazing (and totally unexpected) amount of genetic overlap with our good friend Bordetella, the whooping-cough bug.

If you're not familiar with how bacterial classification works, let's just say it's a mess. There's a long historical tradition of classifying microorganisms based on a hodgepodge of ad hoc methods involving everything from physical appearance under the microscope (especially after staining with crystal violet), to the habitat of the organism, to its ability to metabolize various substances, its ability to make spores, adaptation to oxygen or lack of oxygen, serological characteristics, etc. It's always been an error-prone system, resulting in many misclassifications and later corrections, owing to its inconsistency and basic irrationality, to put it bluntly. With the advent of molecular genetic techniques, it's now possible to create accurate phylogenies based on little more than DNA sequence differences, usually involving the 16S ribosomal RNA (more here).

Freshwater ciliates (like this Euplotes) are
home for Polynucleobacter endosymbionts.

As big an advance as ribosome-based phylogeny is, it's pretty far from ideal (IMHO), mainly because it ignores phenotypes. In fact it's pretty far removed from anything at all having to do with an organism's ecology, metabolism, mode of living, etc. What are we really measuring when we measure relatedness according to a 16S ribosomal yardstick? Just the rate of random mutation accumulation in a pretty uninteresting cell artifact. I'd rather have a yardstick that's tied to phenotypic reality than to a slow-to-change, "highly conserved" piece of cold dead scaffolding.

So to create my own "family tree" of two dozen or so microbes, I said to hell with 16S ribosomes and decided to use, as my yardstick, genetic variation in the GroEL gene, which codes for the 60-kiloDalton heat-shock protein. I chose this protein (or rather, the gene for it) as my phylo-yardstick for a number of reasons. First, the DNA sequence is sizable, at about 1643 nucleotides (making it somewhat bigger than the 16S rDNA). It's important to have a large yardstick gene when looking for faint genetic signals. Secondly, this protein is essentially universal in prokaryotes. It's ubiquitous but not necessarily highly conserved, in the same sense that rRNA is highly conserved. ("Highly conserved" is not what you want. Think about it. Taken to the extreme, a "highly conserved" sequence is invariant. It never changes. And is therefore useless for phylogenetics.) Thirdly, the GroEL heat-shock protein has multiple intracellular touchpoints: It's known to interact with GroES, ALDH2, and dihydrofolate reductase, and it's involved in signal tranduction (it's induced not just by heat but by hydrogen peroxide). Not to overlook the obvious, but it is also a touchpoint protein for any enzyme that can be repaired by the 60kDa heat shock protein. That's probably dozens if not hundreds of enzymes. Why is that important? Think about it: A protein that is sensitive to the 3D conformational requirements of other proteins has to evolve in response to the needs of all the proteins it services. A thermophile (Thermomicrospira)  is going to need a different heat-shock repair system than a psychrophile (Polaromonas). A salt-lover needs a different one than a freshwater-lover. GroEL has to reflect, in its own structure, the many shifting requirements of the host proteome. These considerations make GroEL a highly appropriate basis gene for phylogenetic analysis.

And frankly, I think the GroEL-based phylo-tree phylogeny.fr spit out for me (see illustration further above) speaks for itself. It's a remarkably informative (and accurate) tree. GroEL evolutionary differences not only accurately grouped endosymbionts together, soil organisms together, aquatic organisms, etc., it also correctly grouped the "enteric-alike" Erwinia with E. coli and Shigella, and it cannily put Polaromonas with soil organisms (rather than aquatics), which I think is correct, based on recent Polaromonas isolates being found in soil rather than snow. Likewise, it's good to see Bdellovibrio (a freshwater bug) clustered with Polynucleobacter (which is symbiotic with a ciliate protozoan), with Thiomicrospira (the saltwater hydro-vent organism) a very nearby out-node.

If you get an infection while in a hospital, pray
it's not Clostridium difficile, which is often deadly.

A harder call to make is Clostridium difficile, which is present in 1% to 5% of non-ill people's intestines. Is it an enteric (a la E. coli)? Definitely not. The Clostridia (botulism, tetanus, etc.) are spore-forming soil bacteria. Their placement in the tree not far from the soil-dwelling spore-former, Bacillus thuringensis, is thus eminently correct. Bacillus is a proximal out-node relative to Clostridium, which is understandable in that Bacillus is aerobic whereas Clostridia are strict anaerobes.

Buchnera (an aphid symbiont) comes at an odd location, much further away from the insect-dwelling Wolbachia than I would have predicted, but then again Buchnera's host feeds on cold sap where Wolbachia's hosts typically feed on warm blood. All the organisms around Wolbachia in the tree are hemophiles.

Our good friend Bordetella (of pertussis fame) is placed firmly in the soil group. I think that's real and significant. When you start to look at Bordetella's high DNA sequence similarity with Ralstonia and Burkholderia, it would be surprising, actually, if it fell anywhere else in the tree.

Honestly, when I took Bacterial Ecology 201 in college, many years ago, it was under duress and I hated the experience. But now, decades later, I'm starting to like it. With tools like those available for free at http://genomevolution.org and http://www.phylogeny.fr, what's not to like?