Date Published: June 20, 2013
Publisher: Public Library of Science
Author(s): Suzannah J. Rihn, Sam J. Wilson, Nick J. Loman, Mudathir Alim, Saskia E. Bakker, David Bhella, Robert J. Gifford, Frazer J. Rixon, Paul D. Bieniasz, Jeremy Luban.
Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies.
Genetic robustness is defined as the ability of a biological entity (e.g. a protein or organism) to maintain function in the face of mutations , . More robust proteins or organisms tolerate higher mutation rates while less robust (more ‘brittle’ or ‘fragile’) proteins or organisms are intolerant of mutation and are more likely to lose function or be driven to extinction by high mutation rates. Viruses that replicate via RNA intermediates using non-proofreading polymerases exhibit high mutation rates, suggesting that robustness should be particularly advantageous to them . Indeed, under some conditions, viral populations that exhibit high robustness at the expense of high fitness might be favored over those that have high fitness but low robustness –. In other words, robustness/fragility might be a more potent selective force than fitness under some circumstances.
The goal of this study was to generate a reasonably sized sample of random mutations in the CA protein that might arise naturally during HIV-1 replication, and examine their biological effects. In so doing, we could determine the genetic robustness of HIV-1 CA and correlate the effect of amino acid substitutions in vitro with their occurrence in natural viral populations. Moreover, such a large library constitutes a resource for investigating various functions and properties of the HIV-1 capsid. Our results uncover a rather extreme genetic fragility in the HIV-1 CA protein, with a large fraction (∼70%) of individual, random amino acid substitutions resulting in non-viable viruses (<2% of WT fitness). Tables 4 and 5 place these findings in context. Specifically, Table 4 summarizes all previous studies of randomly mutagenized HIV-1 proteins (and domains thereof), as well as all other randomly mutagenized viral proteins, and full viral genomes. Table 5 lists the genetic robustness of selected non-viral proteins (it is not inclusive of all random mutagenesis studies but we were unable to find any more fragile than those listed). Viruses (particularly ss(+) RNA viruses) not only have much higher mutation rates than cellular organisms, but also have comparatively low tolerance to mutation (Table 4,5) , . Even when compared to viral genomes or proteins (e.g. enzymes, Table 5) that exhibit low robustness, HIV-1 CA is more genetically fragile than any other protein or virus for which this property has been measured. Source: http://doi.org/10.1371/journal.ppat.1003461