Most drug resistance mutations impair virus replication.6,7
Moreover, some mutations that confer resistance to 1 drug also hypersensitize the virus to 1 or more other drugs.8–11
Therefore, it might be expected that the accumulation of multiple drug resistance mutations in the genome of a single virus would occur uncommonly and that multidrug resistance might instead result from the emergence of multiple virus lineages within a patient, each with resistance to different combinations of antiretroviral drugs. Indeed, several studies have shown that in less heavily treated persons or in persons undergoing treatment interruptions, viruses with “incomplete” or intermediate resistance patterns are more commonly detected than those with more complete resistance patterns.12,13
This study, however, shows that most individual clones from heavily treated patients contain either all or most of the mutations detected by population-based sequencing. Whether this finding is restricted to viruses from persons as heavily treated as those described in this study is not yet known. Although viruses from persons who have not been subjected to prolonged selection pressure may harbor virus clones lacking the large numbers of mutations described here, we postulate that viruses exposed to prolonged therapy may undergo extensive purifying selection. The genomic stability of such isolates may result from an interlocking of primary and compensatory drug resistance mutations that limits reversion through unfit intermediate amino acid variants.14
It is essential that our study not be misinterpreted to suggest that population-based sequencing provides a complete picture of the quasispecies within an individual. Several studies have shown that sequencing multiple clones often detects mutations that are not detected by population-based sequencing.15–19
Indeed, we found 17 examples of mutations that were detected by clonal but not population-based sequencing. The main clinical implication of the insensitivity of population-based sequencing is that genotypes must be interpreted within the context of the past treatments received by the person whose virus is sequenced. Because of the increased cost associated with sequencing multiple clones, however, it is unlikely that population-based sequencing will be replaced by the sequencing of large numbers of clones. Moreover, the 2 currently US Food and Drug Administration (FDA)—approved tests for HIV genotypic resistance testing—TruGene (Bayer Diagnostics, Emeryville, CA) and ViroSeq (Celera Diagnostics, Alameda, CA)—use population-based sequencing.
Phenotypic drug susceptibility testing is usually also performed using the population of viruses within a plasma HIV-1 sample. Our drug susceptibility results obtained testing individual virus clones complement our sequence data by showing that multidrug resistance is often a property of individual clones as well as of the population of viruses present within an isolate.
The mutations that were not detected by clonal sequencing were predominantly at positions at which the population-based sequence contained electrophoretic evidence of a mixture of wild-type and mutant residues. Therefore the presence of multiple “pure” mutations in a population-based sequence indicates mutations that are likely to be colinear, whereas the presence of mixtures indicates mutations that may or may not be colinear.
It has been proposed that most HIV-1 genomes and, possibly, most HIV-1 genes are defective because of high-level viral mutagenesis.20,21
Nevertheless, 45 of the 51 transfected clones in this study were replication competent, showing that most RT genes, even those with multiple drug resistance mutations, are replication competent.
In conclusion, this study shows that individual clones in plasma virus samples from persons treated with multiple RT inhibitors contain most of the mutations present within the population-based sequence and may be resistant to most available RT inhibitors. The potential benefit of using a large number of NRTIs (eg, as part of a mega—highly active antiretroviral therapy [HAART] regimen) in this population is therefore likely to result from the impaired replication of viruses containing multiple RT inhibitor-resistant mutations rather than from the effects of different drugs acting on different virus sub-populations.