ResearchBlogging.orgTHE MICROVIRUSES are positive-strand DNA viruses with very small genomes, typified by ΦX174 with 5,400 base pairs and nine genes. Cramming this many genes into that short a sequence requires overlapping reading frames, with gene B contained inside gene A, and gene E contained inside gene D.1 These nested genes are frame-shifted compared to the gene that contains them.

Procapsid assembly occurs by a shell of F protein pentamers interspersed with G spike proteins assembling with the aid of external scaffolding protein D tetramer and internal scaffolding protein B. Scaffolding protein B is absolutely required for procapsid assembly. Chen and coworkers have removed this requirement by selecting for sequential mutations that reduce and finally eliminate viral dependence upon protein B.2

Since B is contained within protein A, the researchers could not proceed as usual by completely deleting the B gene. Instead, it was silenced with the introduction of a stop codon causing early termination of gene transcription. This NullB mutant was then grown in the presence of progressively shorter exogenous B protein. The first mutant used could be truncated significantly since the first 40% of the B protein is unordered, and the first 53 amino acids can be eliminated with cold-sensitivity as the only detrimental result. Culture in the presence of this truncated protein resulted in selection in generation 1 for a mutation in the external scaffolding protein allowing growth at 28°C. The B gene was then further truncated, by a total of 58 amino acids. Another round of selection favored two mutations in generation 2, one in the packaging protein A and the other in H protein, enabling easier protein incorporation. In the next round of selection the B protein was truncated by 67 amino acids. This favored selection for up-regulation of the external scaffolding protein. The result was a decrease in lag phase before virion formation in generation 3.

The final three rounds of selection went more rapidly, with large deletions in the B gene. The B protein was truncated by 97 amino acids, resulting in cold sensitivity and a very long lag phase. Selection under these conditions favored a mutation causing increased external scaffolding protein affinity for coat protein at low temperatures, increasing virion reproduction in generation 4. A final truncation by 100 amino acids resulted again in cold sensitivity, which was abolished during culture by further up-regulation of external scaffolding protein expression in generation 5.

Further attempts at selection discovered the fact that this strain was now completely independent of protein B. A mere six mutations removed the requirement for protein B.

In a related type of microvirus, the Gokushoviruses, there is no internal scaffolding protein. However, this species is limited to bacteria that live inside eukaryotic cells. These bacteria are not victim to many types of viruses, so the Gokushoviruses do not have to compete much with other viruses. Other microviruses infect free-living bacteria. These bacteria are hosts to many much larger, more complicated viruses. Many of these have evolved to take steps that prevent superinfection by other viruses once they enter a bacterial cell. Microviruses, with their limited genomes with only a handful of genes, are not able to prevent superinfection or overcome other viruses’ defenses. Instead, they cope by have a very rapid lifecycle, infecting a bacterial cell and producing virions within 5 minutes of infection. Scaffolding protein B helps to achieve this goal by enabling rapid virion assembly.

While this reversal of requirement for scaffolding protein B does not follow the route of B gene evolution, it shows that apparent “irreducible complexity” is not impossible to overcome evolutionarily. A series of six mutations was required to eliminate the need for protein B, and a similar set of gradual modifications could return this requirement, producing an apparently irreducibly complex system.

SANGER, F., AIR, G. M., BARRELL, B. G., BROWN, N. L., COULSON, A. R., FIDDES, J. C., HUTCHINSON, C. A., SLOCOMB, P. M., SMITH, M. (1977). Nucleotide sequence of bacteriophage ΦX174 DNA. Nature, 265, 687-695. DOI: 10.1038/265687a0

CHEN, M., UCHIYAMA, A., FANE, B. (2007). Eliminating the Requirement of an Essential Gene Product in an Already Very Small Virus: Scaffolding Protein B-free ΦX174, B-free. Journal of Molecular Biology, 373(2), 308-314. DOI: 10.1016/j.jmb.2007.07.064