Article Figures & Data
Figures
- FIG. 1.
nef synonymous-sequence tag ASPCR. (A) Tagged recombinant HIV-1C backbones were constructed by introduction of four synonymous nucleotide substitutions into the nef gene of the MJ4 infectious clone. (B to D) Allele-specific quantitative PCR using a common reverse primer paired with a forward primer specific for the nefWT (B) or nefSYN (C) tag allowed efficient and sensitive quantification over a wide range of nefWT (D)- and nefSYN (E)-tagged template concentrations.
- FIG. 2.
The nefSYN sequence tag does not affect RRC. MJ4-nefWT and MJ4-nefSYN viruses exhibited similar growth kinetics over a 7-day dual infection (A) with the log-linear growth phase occurring from day 2 to day 6 postinfection (B). The relative frequency of the two viruses varied within a narrow range over the log-linear phase (C). The RRC was quantified by determining the ratio of mutant to wild-type viruses, in this case nefSYN to nefWT (log transformed), calculating the change in the ratio over each time point, and taking the mean of the values.
- FIG. 3.
The RT M184V mutation causes a reduced RRC. The MJ4-RT V184-nefSYN virus exhibited reduced growth kinetics relative to the MJ4-RT M184-nefWT virus (A), which affected the relative frequencies of the two viruses over time (B).
- FIG. 4.
Quantification of RRC. The mean RRC (dots) with 95% confidence interval (CI) (whiskers) is shown for the benchmark RT M184V mutation (A), the HLA-B57-associated capsid escape mutations expressed individually (B) and in multiples (C), and the putative compensatory mutation CA H219Q individually and with the associated CA T242 mutations (D). The horizontal line denotes the “wild-type” RRC, which is equal to 1.00 by definition.
Tables
- TABLE 1.
Differential quantification of mixed plasmid template samples by nef synonymous ASPCR
Sample composition (%)b nef synonymous-tag ASPCRa Copies/μl % of totalc pMJ4-nefWT pMJ4-nefSYN pMJ4-nefWT pMJ4-nefSYN pMJ4-nefWT pMJ4-nefSYN 99 1 449,719 4,781 99 1 95 5 446,684 24,475 95 5 80 20 367,034 89,644 80 20 70 30 318,377 139,766 69 31 60 40 307,776 198,032 61 39 50 50 239,559 233,320 51 49 40 60 177,828 258,501 41 59 30 70 137,479 304,565 31 69 20 80 92,195 344,423 21 79 5 95 25,119 440,471 5 95 1 99 4,063 425,677 1 99 ↵ a nef synonymous tagged templates were quantified in parallel nefWT- and nefSYN-specific quantitative PCRs.
↵ b Mixed samples consisted of known quantities of linearized pMJ4-nefWT and pMJ4-nefSYN plasmid standards.
↵ c ASPCR-derived template frequencies in the sample calculated from copy number values.
