Database analysis of patterns of silent mutations at RT codons 64 to 66 in subtype B and C viruses
compares the variation at the silent third nucleotide position at codons 64 to 66 in HIV-1 subtype B and C viruses. Viruses with the wildtype KKK motif were obtained from NRTI-naïve individuals and viruses with the K65R mutation from NRTI-treated individuals. In the untreated wildtype samples, the two most common variants in subtype B viruses differed from the two most common subtype C variants. The two most common subtype B variants accounted for 97% of the eight possible KKK-encoding nucleotide patterns; six other variants accounted for the remaining 3% of patterns. The two most common subtype C variants accounted for 98% of the eight possible KKK-encoding nucleotide patterns; six variants accounted for the remaining 2% of patterns.
Comparison of Nucleotide Patterns in Subtype B and Subtype C HIV-1 Reverse Transcriptase Codons 64, 65 and 66 in the HIV Drug Resistance Database.
The nucleotide patterns encoding KRK in the subtype B and C isolates from NRTI-treated individuals occurred in proportions similar to the KKK-encoding nucleotide patterns but had a G at the second position of codon 65. Forty individuals had a pre-therapy KKK sample and a post-therapy KRK sample. All but two of the post-therapy samples were identical to the pre-therapy sample except for the presence of an A to G substitution at the second position of codon 65.
UDPS of subtype B and C plasma virus samples from ARV-naïve individuals
shows that UDPS detected K65R in a higher proportion of sequence reads of subtype C plasma samples than in subtype B plasma samples (1.04% vs. 0.25%; p<0.001 Wilcoxon Rank Sum Test). K65R was detected in at least 1.0% of reads in 8/18 (44%) subtype C samples and in 1/27 (3.7%) subtype B samples (p
0.01; Fisher's Exact Test).
Comparison of Ultra-Deep Pyrosequencing (UDPS) Results of Subtype B and Subtype C Clinical HIV-1 Samples.
shows the dominant wildtype KKK nucleotide patterns in the UDPS reads of the 18 subtype C and 27 subtype B viruses and the KRK nucleotide patterns for those samples in which K65R was present in at least 1.0% of UDPS reads. The two most common subtype B wildtype KKK nucleotide patterns matched the two most commonly reported subtype B nucleotide patterns. The most common dominant subtype C wildtype KKK nucleotide pattern matched the most commonly reported subtype C nucleotide pattern.
Dominant Intra-Individual Pattern of Nucleotides Coding for K64, K65 and K66 in 27 Subtype B and 18 Subtype C Isolates from Untreated Individuals: Association with presence of K65R as Determined by Ultra-Deep Pyrosequencing (UDPS)*.
Among the subtype B samples, K65R was detected in more than 1.0% of reads in a sample with a complex mixture of KKK nucleotide variants including AAA-AAG-AAA (the second-most common subtype C pattern) in 38% of reads, AAG-AAA-AAA (the most common subtype B pattern) in 7% of reads, and an uncommon pattern AAG-AAG-AAA in 50% of reads. Despite the fact that the K65R reads in this subtype B sample (1.1% of all reads) were encoded by AAA-AGA-AAA, there were no reads with the closest wildtype variant, AAA-AAA-AAA.
Among the subtype C samples, K65R was detected in at least 1.0% of reads in eight samples, including 7 of 11 with the most common subtype C pattern and the one sample with the second-most common subtype C pattern. Of the four subtype C samples with the most commonly reported subtype B KKK nucleotide variant (AAG-AAA-AAA), none had K65R in 1.0% or more of reads.
The coefficient of variation for the duplicate UDPS runs on the subtype C sequences was 0.23. For eight of the 16 repeated subtype C samples, the proportion of reads with K65R was >1.0% in each of the duplicate runs and for seven of the 16 repeated subtype C samples, the proportion of reads with K65R was <1.0% on both runs. The median proportion of reads with K65R was similar in the forward (0.93%) and reverse (1.2%) UDPS reads.
Clonal analysis of two subtype C viruses for which more than 2.0% of UPDS reads contained K65R
To determine whether the high frequency of K65R reads in subtype C viruses reflected a natural occurrence of this mutation at low levels in subtype-C-infected individuals or a technical artifact of either PCR or UDPS, we performed clonal dideoxynucleotide sequencing on the two subtype C viruses with the highest proportions of K65R reads. shows that, although 133 of 5186 UDPS reads of sample SCR268 had K65R, none of 265 limiting dilution clones of the same sample had the mutation (p
0.001; Fisher's Exact Test). Similarly, although 57 of 3613 UDPS reads of sample 9635 had K65R, none of the 254 limiting dilution clones we sequenced of this sample had the mutation (p
0.02, Fisher's Exact Test). In contrast, there was no statistically significant difference in the number of UDPS vs. molecular clones for sample SCR268 (6 of 278 vs. 133 of 5186; p
0.8) or 9635 (3 of 278 vs. 57 of 3613; p
0.8). Although limiting dilution sequencing did not detect K65R, it did detect each of the other 33 nucleotide and three amino acid mutations that were present in more than 1.0% of UDPS reads in these two samples. Taken together, the limiting dilution and molecular cloning sequencing results strongly suggest that PCR error was responsible for the subsequent frequent false positive detection of K65R by UDPS of subtype C samples.
Comparison of Ultra-Deep Pyrosequencing (UDPS) with Limiting Dilution Clonal Sequence on the Two Samples with the Highest Proportion of K65R.
UDPS KKK sequences of site-directed mutants and plasmid clones
shows that a median of 1.2% of 454 UDPS reads (range: 0.7%–1.3%) of the eight clones with the two most common subtype C KKK patterns had K65R. In contrast, the four clones with the two most common subtype B KKK patterns had K65R in a median of 0.15% of reads (range 0.1%–0.3%; P
0.007; Wilcoxan Rank Sum Test). The proportion of UDPS reads containing K65R appeared to be entirely dependant on the KKK pattern in the sample. The subtype from which the surrounding viral sequence was derived did not significantly influence the number of reads with K65R. For plasmid clones with a subtype C nucleotide pattern, UDPS detected K65R in a mean 1.1% of reads (range 0.7–1.3). In contrast, for plasmid clones with a subtype B nucleotide pattern, UDPS detected K65R in a mean of 0.2% reads (range 0.1–0.3).
Ultradeep-Pyrosequencing of PCR-Induced Mutations at Codon 65 in Plasmid Clones with Different KKK Nucleotide Patterns in Subtype B and Subtype C Genetic Contexts.
For clones with three subtype C KKK patterns, cDNA was amplified with PfuUltra II Fusion, as well as Taq/Pwo polymerase (Expand High FidelityPLUS PCR System). The proportion of K65R clones was 16-fold to 80-fold lower in the samples amplified using PfuUltra II Fusion than in the samples amplified with Taq/Pwo ( footnote).