We describe the natural appearance of low-level K65R variants in South African NRTI-naive adults and infants with subtype C infections at frequencies higher than those detected for subtype B and AE quasispecies. The absence of evidence for mother-infant K65R transmission supports a spontaneous emergence of K65R that was nearly 4-fold more prevalent in acutely infected infants than in chronically infected women. However, incomplete infant viral load data prevented us from ascertaining whether greater viremia, typically associated with rampant, acute infection, could have contributed to increased K65R expression.
The most striking finding with subtype C viruses was the frequent appearance of frameshift mutations at codon 65, which suggested that the region is a mutagenesis “hot spot.” This transcription error has recently been demonstrated in vitro for subtype C and was suggested to be the possible result of sequence-induced RT slippage and pausing, leading to nucleotide dislocation [8
]. An earlier evaluation of the same mothers using an M184V-specific assay found that the M184V quasispecies frequency was very similar to that of subtype B (data not shown), further indicating that the extent of mutations observed around codon 65 was not a result of indiscriminate mutagenesis in subtype C. The only K65R variant identified in the subtype AE population could not be explained by the same dislocation mechanism reported for subtype C [8
], because there was no adjacent guanosine in codon 65, and, furthermore, the absence of codon 65 frameshifts also implied a less-volatile genomic region.
Although additional screening did not identify intact K65R clones in mothers of infants with K65R, we did not screen the number of clones that could have compensated for random selection error. Therefore, it is possible that intact variants were present above the target frequency in those mothers but were missed in the random clone analyses. Furthermore, the years sampled for each subtype varied, and unrecognized thymidine analog resistance may have been transmitted in the contemporary subtype C and AE populations. However, the absence of other mutations provided no indication that our results may have been biased by cases of transmitted drug resistance. Although recent reports have indicated that PCR generates spurious low-level K65R with subtype C [10
], we found intact variants in infants at frequencies above the reported artifact frequencies. Moreover, if the observed codon 65 errors are indeed template driven, they would be expected to occur with lower-fidelity HIV RT in vivo.
The data suggest that subtype C generates a higher quasispecies frequency of codon 65 mutations. This finding may help explain what has been reported as greater selection of K65R in subtype C–infected subjects receiving ART, mostly from the use of stavudine [2
]. However, longitudinal studies of K65R at baseline and during therapy are needed to ascribe clinical significance to low-level quasispecies variants. The detection of subtype-specific spontaneous frameshift variants may confound resistance screening; thus, data from point mutation testing must be analyzed carefully.