Colinearity of reverse transcriptase inhibitor resistance mutations detected by population-based sequencing

Abstract

High-level resistance to multiple drugs is often detected by directly sequencing uncloned polymerase chain reaction products (population-based sequencing). It is not known, however, if this method of identifying mutations gives an accurate picture of individual viral genomes. To determine how often multidrug-resistant isolates consist of clones containing every mutation present in the population-based sequence, a mean of 2.8 molecular clones was sequenced from the plasma of 25 heavily treated persons whose population-based sequence contained multiple reverse transcriptase (RT) inhibitor resistance mutations (71 clones). The 25 population-based sequences contained a mean of 5.7 nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations and 1.2 nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations. The 71 clones contained a mean of 5.3 NRTI resistance mutations and 1.0 NNRTI resistance mutations. Sequences of clones closely resembled the population-based sequence: 36 (51%) clones had each of the RT inhibitor mutations present in the population-based sequence, 25 (35%) had all but 1 RT inhibitor mutation, 4 (6%) had all but 2 RT inhibitor mutations, 3 (4%) had all but 3 RT inhibitor mutations, and 3 (4%) had all but 4 RT inhibitor mutations. Phenotypic testing of 29 clones showed that most clones were resistant to nearly all NRTIs and that those with NNRTI resistance mutations were also resistant to multiple NNRTIs. These data show that in heavily treated persons, most RT inhibitor resistance mutations are present in the same viral genomes (colinear) and that multidrug resistance often occurs within individual clones as well as within virus populations.

FIGURE 1

Comparison of population-based versus clonal sequencing for 15 isolates for which 3 or more clones were sequenced. Columns 2 through 19 contain 18 nucleoside reverse transcriptase inhibitor (NRTI) resistance positions. Columns 20 through 29 contain 10 nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance positions; NNRTI resistance mutations at positions 225, 227, 230, 236, and 238 were not observed in this data set. The second row contains the consensus amino acid at each of the drug resistance positions. The population-based sequences are shown in bold. Clonal sequences are shown beneath each population-based sequence. Clones containing identical drug resistance mutations have been collapsed into a single line as indicated by the entry in the first column. A dash indicates that the sequence contained the consensus amino acid. A letter indicates the presence of a mutation. More than 1 letter indicates the presence of a mixture of wild-type and a mutation or a mixture of mutations.