Extreme Genetic Fragility of the HIV-1 Capsid

Suzannah J Rihn,Paul D Bieniasz,Saskia E Bakker,Frazer J Rixon,Nick J Loman,Robert J Gifford,Mudathir Alim,Sam J Wilson,David Bhella,Jeremy Luban

doi:10.1371/journal.ppat.1003461

Abstract

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.

Highlights

IntroductionGenetic robustness is defined as the ability of a biological entity (e.g. a protein or organism) to maintain function in the face of mutations [1,2]
Genetic robustness is defined as the ability of a biological entity to maintain function in the face of mutations [1,2]
capsid protein (CA) encoding sequences were amplified by error-prone PCR and cloned using a TOPO TA cloning Kit to generate a library with an estimated complexity of 16104 clones

Summary

Introduction

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 [1,2]. Viruses that replicate via RNA intermediates using non-proofreading polymerases exhibit high mutation rates, suggesting that robustness should be advantageous to them [3]. While the robustness or fragility of RNA viruses has been investigated in several studies [4,5,6,7,8,9], most reports characterize robustness by treating an entire viral genome as a single biological entity. Viruses encode a variety of proteins that execute a range of functions to enable replication, and the genetic robustness of individual proteins is expected to vary within a given virus. Proteins that perform complex or multiple functions that are highly dependent on accurate structure (e.g. enzymes), should tend to be more genetically fragile, i.e less tolerant of mutation, and exhibit greater sequence conservation than those that do not (e.g. proteins that provide peptide binding sites to recruit other proteins)

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