Abstract
Human immunodeficiency virus type-1 (HIV-1) fitness has been associated with virus entry, a process mediated by the envelope glycoprotein (Env). We previously described Env genetic diversification in a Zambian, subtype C infected, slow-progressor child (1157i) in parallel with an evolving neutralizing antibody response. Because of the role the Variable-3 loop (V3) plays in transmission, cell tropism, neutralization sensitivity, and fitness, longitudinally isolated 1157i C2-V4 alleles were cloned into HIV-1NL4-3-eGFP and -DsRed2 infectious molecular clones. The fluorescent reporters allowed for dual-infection competitions between all patient-derived C2-V4 chimeras to quantify the effect of V3 diversification and selection on fitness. ‘Winners’ and ‘losers’ were readily discriminated among the C2-V4 alleles. Exceptional sensitivity for detection of subtle fitness differences was revealed through analysis of two alleles differing in a single synonymous amino acid. However, when the outcomes of N = 33 competitions were averaged for each chimera, the aggregate analysis showed that despite increasing diversification and divergence with time, natural selection of C2-V4 sequences in this individual did not appear to be producing a ‘survival of the fittest’ evolutionary pattern. Rather, we detected a relatively flat fitness landscape consistent with mutational robustness. Fitness outcomes were then correlated with individual components of the entry process. Env incorporation into particles correlated best with fitness, suggesting a role for Env avidity, as opposed to receptor/coreceptor affinity, in defining fitness. Nevertheless, biochemical analyses did not identify any step in HIV-1 entry as a dominant determinant of fitness. Our results lead us to conclude that multiple aspects of entry contribute to maintaining adequate HIV-1 fitness, and there is no surrogate analysis for determining fitness. The capacity for subtle polymorphisms in Env to nevertheless significantly impact viral fitness suggests fitness is best defined by head-to-head competition.
Highlights
Human immunodeficiency virus type I (HIV-1) is a positivesense RNA virus that replicates via error-prone reverse transcription and undergoes inter-strand recombination, introducing approximately 3.461025 mutations/base pair per replication cycle [1,2]
In experiments using subtype C envelope glycoprotein (Env) C2-V4 sequences from an slowprogressor child, we show the capacity to detect ‘winners’, ‘losers’, and ‘ties’ in individual competitions, including detection of fitness differences between extremely subtle polymorphisms
These approaches are often incapable of elucidating subtle fitness differences between Human immunodeficiency virus type-1 (HIV-1) variants as might be anticipated to exist in genetically related viruses derived from a patient over the course of infection, or in samples from a quasispecies at a single time-point [15,45,46,48]
Summary
Human immunodeficiency virus type I (HIV-1) is a positivesense RNA virus that replicates via error-prone reverse transcription and undergoes inter-strand recombination, introducing approximately 3.461025 mutations/base pair per replication cycle [1,2]. These diversification mechanisms result in HIV-1 populations behaving as large, dynamic groups of related, yet genetically distinct, organisms whose evolutionary characteristics can be modeled as a quasispecies [3]. Large dynamic populations of subtle sequence variants allow HIV-1 continue to replicate despite potent environmental selection imposed by treatment and the immune system
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