HIV exists as multiple genotypes in a single infected individual referred to as a quasispecies. Here we reproduced a quasispecies by moderate selective pressure using an HIV reverse transcriptase inhibitor. The drug resistant genotype never completely supplanted the drug sensitive genotype, which stabilized at about 20 percent of viral sequences. Single-cell sequencing showed that resistant genotype frequency plateaued when cells were co-infected with sensitive and resistant genotypes, suggesting a sharing of viral proteins in co-infected cells (complementation) which masks genotypic differences. To test if complementation can confer phenotypic drug resistance, we co-transfected fluorescently labelled molecular clones of sensitive and resistant HIV and observed that genotypically sensitive virus from co-transfected cells was drug resistant. Resistant virus preferentially infected cells in tandem with drug sensitive HIV, explaining how co-infections of sensitive and resistant genotypes were initiated. Modelling this effect, we observed that a stable quasispecies could form at the experimental multiplicities of infection for the drug resistant and drug sensitive virus, showing that complementation can lead to a quasispecies in an HIV evolution experiment. ### Competing Interest Statement The authors have declared no competing interest.
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