Student Theses and Dissertations

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Bieniasz Laboratory

Abstract

Genetic robustness describes a capacity to maintain function in the face of mutation. RNA viruses, like human immunodeficiency virus-1 (HIV-1), experience extremely high mutations rates in vivo, which contribute to the evolvability that permits HIV-1 to evade immune defenses. However, the compact nature of the HIV-1 viral genome, in which small or overlapping proteins are often multifunctional and essential, often necessitates a strong pressure to conserve functional roles, which is at opposition with immunological pressures to diversify. Under such conditions, and in consideration of the relatively rapid replication cycle of HIV-1, natural selection for robustness would be predicted to be beneficial. At the same time, robustness can come at the cost of fitness, and it is unclear whether robustness or fitness would constitute the dominant selective force in the natural setting. Within a given virus, genetic robustness is expected to vary amongst proteins, in accordance with specific function. Therefore, to more accurately examine genetic robustness within HIV-1, we selected one protein in which the competing needs to both conserve function yet diversify sequence is particularly acute. HIV-1 capsid (CA) is under strong pressure to preserve roles in viral assembly, maturation, uncoating and nuclear import, but is also under intense immunological pressure to diversify, particularly from adaptive immune responses. To evaluate the genetic robustness of HIV-1 CA, we generated a large library of random single amino acid substitution CA mutants. Surprisingly, after measuring the replicative fitness of the CA library mutants, we observed HIV-1 CA to be the most genetically fragile protein analyzed with such an approach, with 70% of random CA mutations resulting in nonviable, replicationdefective viruses (<2% of WT fitness). While CA is involved in several steps of HIV-1 replication, analyses of conditionally (temperature-sensitive) and constitutively nonviable mutants indicated that the biological basis for the genetic fragility, or lack of robustness, was principally the need to organize accurate and efficient assembly of mature viral particles. Examination of the CA library mutations in naturally occurring HIV-1 subtype B populations indicated that all mutations occurring at a frequency >3% in vivo had fitness levels that were >40% of WT in vitro. Moreover, protective CTL epitopes were observed to preferentially occur in regions of HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. Overall, these results suggest the extreme genetic fragility of CA may explain why immune responses to Gag, as opposed to other viral proteins, correlate with better prognosis in HIV-1 infection. To evaluate whether the fragility we observed in CA was unique to CA or a general property of HIV-1 proteins, and to more firmly establish causes and consequences of genetic robustness or fragility, we evaluated the robustness of another HIV-1 protein, integrase (IN). Again, a large library of unbiased single amino acid mutants was created. In contrast to CA, IN appeared comparatively robust, with only 35% of 156 single amino acid mutations resulting in nonviable viruses. However, when these nonviable mutants were mapped onto a model of the HIV-1 intasome, we noted a striking localization of nonviable mutants to certain IN subunit interfaces. To this end, single mutants affecting both particle production and IN expression in virions could be mapped to even more specific regions within the intasome model. Furthermore, after examining the prevalence of the IN library mutations in an in vivo cohort of subtype B IN sequences, mapping to the intasome helped explain why some in vitro library mutations with high fitness (>40% of WT) never occur in vivo, and also revealed that residues that are highly variable in vivo are more likely to occur in regions in the intasome with more exposed surface area. Despite the relative robustness of HIV-IN, our analyses with an intasome model demonstrate that localized regions of fragility, namely certain subunit interfaces, exist. Understanding the genetic robustness of HIV-1 is particularly important given the continued need for novel therapeutic interventions. Not only has the evaluation of HIV-1 CA and IN robustness revealed particularly fragile regions in both proteins, which may prove ideal targets, but comparisons of their genetic robustness is suggestive of the direction future therapeutic research should take.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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