Impact of immune escape mutations on HIV-1 fitness in the context of the cognate transmitted/founder genome
- Hongshuo Song1,
- Jeffrey W Pavlicek1,
- Fangping Cai1,
- Tanmoy Bhattacharya3, 11,
- Hui Li4,
- Shilpa S Iyer4,
- Katharine J Bar4,
- Julie M Decker6,
- Nilu Goonetilleke7,
- Michael KP Liu7,
- Anna Berg1,
- Bhavna Hora1,
- Mark S Drinker1,
- Josh Eudailey1,
- Joy Pickeral9,
- M Anthony Moody1, 8,
- Guido Ferrari9,
- Andrew McMichael7,
- Alan S Perelson3,
- George M Shaw4, 5,
- Beatrice H Hahn4, 5,
- Barton F Haynes1, 2, 10 and
- Feng Gao1, 2Email author
© Song et al.; licensee BioMed Central Ltd. 2012
Received: 3 June 2012
Accepted: 7 October 2012
Published: 30 October 2012
A modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method.
The T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region.
These findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.
KeywordsHuman immunodeficiency virus type I Viral fitness Cytotoxic T lymphocytes Immune escape mutation Transmitted/founder virus Mathematical model
HIV-1 fitness plays a critical role in virus persistence, transmission, pathogenesis, and disease progression [1–9]. Because of HIV-1 error prone reverse transcriptase and rapid virus turnover and immune selection pressure, a small viral fitness change may have a significant impact on HIV-1 evolution . Strong pressure from cytotoxic T lymphocyte (CTL) responses selects virus mutants, with complete replacement of CTL sensitive viruses within weeks of HIV-1 infection [11, 12]. These CTL escape mutations have been widely studied for their ability to impair viral fitness [13–17]. If fitness is reduced there may be a decrease in viral load, leading to long-term HIV-1 control and decreased probability of transmission to new hosts [7, 9]. Moreover, if less fit viruses are transmitted into new hosts, viral loads may be lower and a better clinical outcome expected , although the effect may not be sustained into chronic infection .
The majority of fitness studies have compared viruses in vitro using either parallel or competition assays [13–19]. In the latter, competition between two viruses is performed in the same culture, and the relative fitness is determined by the dynamic changes of the ratio of viruses over time [17, 20, 21]. The proportion of each virus in the culture is determined by detecting unique artificial markers introduced into the viral genomes or mutations by population sequencing, clone sequencing or real-time PCR [13, 14, 16, 18, 19, 22, 23]. However, there are several factors that may compromise evaluation of viral fitness. A lab-adapted viral backbone isolated from chronically infected individuals may not represent viruses that exist as a quasispecies viral population in vivo. When mutations of interest or gene fragments are introduced into unrelated viral genomes they may have confounding effects on fitness. This may be particularly important since the mutations can have disparate effects on viral fitness in different virus backbones [14, 18]. Recombination between viruses can affect the interpretation of the fitness results [24, 25]. The sensitivity to detect minority variants is low for sequencing based methods [16, 18, 19, 26, 27]. Finally, only two viruses are usually compared in each assay, whereas many variants compete against each other in the HIV-1-infected individuals [13, 14, 16–19]. Therefore, a more reliable, sensitive and reproducible assay is needed.
Combining the unique advantages of parallel allele-specific sequencing (PASS) technology , unmodified HIV-1 T/F genomes  and new mathematical modeling, we have established a new viral fitness assay to determine the impact of CTL escape mutations emerging in early HIV-1 infection on the fitness of the transmitted/founder (T/F) virus. In the current study, we determined the impact of CTL escape mutations on viral fitness in their cognate T/F viral genome. We found that although CTL escape mutants can be associated with significant fitness costs, this can be negated by emergence of compensatory mutations. In addition, the new PASS fitness assay can be used to simultaneously compare viral fitness among multiple viruses in a single assay and determine the influence of recombination on fitness comparisons.
Fitness comparison in the single passage assay
The CTL escape mutation T242N has been reported to cause a significant fitness loss in vitro using the laboratory adapted NL4-3 virus backbone in several studies [14–16]. We first sought to investigate if the T242N mutation rendered the virus less fit than T/F in a single passage assay. The equal amounts of each virus were mixed to infect primary CD4+ T cells, and culture supernatants were harvested daily to determine the relative fitness of the compared viruses. After cDNA was made using viral RNA extracted from the cell culture supernatants, the proportion of each virus in the culture was determined by PASS. The numbers of detected viral genomes increased exponentially from day 2 to day 4, similar to the replication dynamics determined by measuring p24 concentrations (Figure 2B). We then analyzed an average of 600 (200 to 1400) viral genomes in each sample to determine the proportion of each virus in the viral population.
TK was the predominant virus (53%) as early as at day 14 and was fixed at day 592 in the virus population (Figure 1A), suggesting that the virus with both mutations was strongly selected in vivo. To determine if the CTL escape mutation in TK caused fitness loss, we compared the TK and T/F viruses. The percentages of the T/F and TK viruses (60% and 40%, respectively) did not change over time (Figure 3D). Importantly, the proportion of each virus during the culture was similar to that in the inoculum stock (57% and 43% for T/F and TK, respectively). These results showed that TK was approximately as fit as T/F (s ij = 0.01 ± 0.01).
Taken together, in the single passage assay, no significant differences in relative fitness between any pair of viruses were observed, although the viruses exponentially increased in the supernatant. This is at odds with the previous observation that a virus with the CTL escape mutation T242N was less fit than the WT virus [14–16]. However, when T242N was compared to T/F or NIA, the proportion of T242N in the culture decreased by two fold compared to that in the inoculum stock (Figure 3A and 3C), suggesting that T242N was less fit than both T/F and NIA. These results indicated that the fitness differences among these viruses were not accurately measured in the single passage assay.
Fitness comparison through multiple passages of compared viruses
Since the fitness loss caused by the T242N mutation was better revealed after multiple rounds of passages , and discordant fitness results have been observed between single-passage and multiple-passage fitness assays , we sought to investigate whether the fitness cost caused by the T242N mutation in the T/F virus can be more accurately determined by repeatedly passaging the cell-free viruses to fresh CD4+ T cells. Cell-free viruses harvested 3 or 4 days after infection were sequentially passaged four times to fresh CD4+ T cells. The proportion of each virus in the culture was determined by PASS as in the single passage assay. We analyzed an average of 636 (135 to 2197) viral genomes in each sample to determine the proportion of each virus in the samples.
When T242N and NIA, which was naturally selected in vivo, were compared, NIA was also more fit than T242N. During four passages, NIA continuously increased in the viral population (from 74% to 91%) while T242N was gradually outcompeted (from 26% to 8%), although the proportion of each virus in the inoculum was similar (43% T242N and 57% NIA) (Figure 5B). As a result, T242N was 37% less fit than NIA (s ij = -0.37 ± 0.14). We then compared T/F and NIA and found that the proportion of each virus did not change throughout all passages (Figure 5C), indicating that the fitness of both viruses was similar (s ij = -0.03 ± 0.03). Taken together, our results confirmed that the T242N mutation alone in the TW10 CTL epitope caused significant fitness loss in the multiple passage assay. However, two additional mutations in the same epitope restored the viral fitness to the wild type virus level.
We also compared the TK and T/F viruses to determine if the CTL escape mutation in TK caused fitness loss after multiple passages. The proportion of each virus was similar to that in the inoculum stock (57% and 43% for T/F and TK, respectively) throughout passages, suggesting both viruses were similarly fit (Figure 5D).
Frequent recombination in fitness assay can be assessed by PASS
A better understanding of the fitness cost of mutations associated with immune escape can have important implications for understanding viral pathogenesis, transmission and vaccine development [1–9]. To accurately determine fitness of viruses present in vivo, we developed a new PASS fitness assay with unmodified HIV-1 genomes that were identified in vivo. Using this assay, we have precisely determined the impact of mutations in the cognate T/F virus genome. Although much more effort is required to generate T/F IMCs to study viral fitness than to use existing IMCs from unrelated viruses, we have generated over 20 IMCs and identified a number of CTL cell escape mutations through analysis of longitudinal follow-up sequences . They can serve as ideal candidates for further study of the impact of immune escape mutations on the fitness of cognate viral genomes. We determined the fitness cost of two CTL escape mutations (R355K in Env and T242N in Gag) in the context of other mutations in the cognate viral genomes (TK and NIA, respectively). Both CTL escape mutations were present together with other mutations that were within or outside the CTL epitopes. Interestingly, both mutant viruses were as fit as the T/F virus. The CTL escape mutation T242N alone had a significant fitness cost as previously shown by others [14–16]. It has been shown that compensatory mutations, within or outside the T cell epitopes, can partially restore the fitness loss caused by T cell escape mutations [7, 14, 15, 32, 33]. Here, we demonstrate that fitness loss by CTL escape mutations can be completely restored by the mutations in the same epitope in their cognate virus genome. The TK virus with both the I64T mutation in Tat/Rev and the R355K CTL escape mutations in Env represent a naturally selected virus in vivo. Although the TK virus had two mutations, it was as fit as the T/F virus. No fitness loss of the TK virus also explained why it outcompeted the T/F in vivo and dominated the viral population under the CTL selection pressure only 14 days after the date when the T/F virus was inferred. More importantly, this represents another case, like the NIA virus, demonstrating that the overall viral fitness is not impaired by the presence of both a CTL escape mutation and an additional mutation in the T/F viral genome.
These observations may have important implications for understanding pathogenesis and vaccine development. Many mutations that were not associated with immune responses are often identified in the viral genome and their biological functions have not been well elucidated . Results from other studies and ours indicate that such mutations, especially those reversion mutations, may play an important role in compensating fitness loss caused by the CTL escape mutations, so the virus can survive the unfavorable environment in the presence of immune selection pressures. Alternatively, they may represent adaptations that favor virus replication in the host target cell environment.
In two comparison pairs (T/F verses T242N and NIA verses T242T), no difference in replication rates were observed for the compared viruses in the single passage assay even though the viruses increased exponentially in the cell culture medium. However, the fitness loss by the T242N mutation was clearly demonstrated when the cell free viruses were passaged multiple times as shown in this study and a previous report . This may be explained by the following reasons. First, the T242N mutation affects the viral replication through interacting with cyclophilin A . Since the viral entry was not impaired by the T242N mutation, the T242N was marginally less fit than T/F in the single passage assay. The p24 concentration of T242N was only less than two fold lower than that of the wt or T/F virus at the end of the culture when virus replication plateaued as shown in a previous study  and by our result (Figure 2B). Second, the infectious viruses generated during the first 2 days of culture were far fewer than those in the inoculum (90,000 infectious units (IU)/ml versus 200 IU/ml). Thus, it is unlikely that the majority of the new infections during the four-day culture were initiated by the cell-free viruses newly generated in the single passage culture. Third, cell-to-cell infection was about 100-1000 time more efficient than the cell-free virus in culture . Thus, the subsequent infection is most likely established through the cell-to-cell infection in the single passage culture. If the efficiency of the cell-to-cell infection was equal for the compared viruses with the identical env gene, the proportion of each virus might not change over time in the culture. As a result, the relative fitness determined in the single passage culture would be similar between the compared viruses. However, by passaging the cell-free viruses to the fresh CD4+ T cells, the cell-free viruses would compete with each other multiple times. Since the more fit virus accounted for a larger portion of the viral population at each passage, they would infect more cells in each subsequent passage as shown in this study and a previous report . Importantly, the dynamics of proportion changes for each compared virus between the inoculum and the first passage was consistent with the viral replication slope during the subsequent passage(s), suggesting that the initial changes in viral proportion from the inoculum to the first passage represented intrinsic viral fitness differences and could be further confirmed by subsequence passages (Figure 5). Since virus growth was not in exponential stage throughout passaging, previous models that assume constant exponential growth could not be used to determine relative fitness between compared viruses. Thus, we developed a new mathematical model that does not assume constant exponential growth to measure relative fitness of HIV-1 strains after multiple rounds of passages. Taken together, results from this and other studies indicate that multiple passages are needed to better determine the fitness difference between compared viruses if the proportions of viruses change obviously between the inoculum and the first passage, but no difference in replicative slope is observed during the first passage (Figure 3A and 3C). However, if the proportions of viruses do not change from the inoculum to the culture throughout the first passage, this indicates that the compared viruses are similarly fit and additional passages may not be necessary (Figure 3B and 3D). When the difference in replication slope is clearly demonstrated over time during the first passage as shown in our previous study , the relative fitness can be determined without further passaging.
Viral fitness is generally determined by either directly sequencing the bulk PCR products or by detecting markers that are incorporated at different locations of the viral genome [16, 18, 19, 26, 27]. Thus, the impact of recombination on viral fitness analysis during culture has not been fully elucidated. Since the high frequency recombination has been observed in culture [24, 25], it is likely that recombination has a significant impact on viral fitness assays, although recombination was considered low in a previous study . The PASS assay can detect multiple sites in the same viral genomes, and the linkage analysis of these sites allows detection of recombination among a limited number of mutations within a small region (≤ 2 kb) in the viral genome. The emergence and gradual increase of the recombinants to as high as 35.4% of the viral population in vitro suggest recombinant viruses are more fit than both parental viruses. Interestingly, all three recombinants (V247I, I64T and R355K) detected in vitro in this study were also identified in vivo (Figure 1A and 1C), suggesting that those recombinant viruses are naturally present in HIV-1-infected individuals. The V247I virus was detected as the predominant virus before the CTL escape mutation T242N was detected and then fixed together with the T242N mutation in the viral population. Since V247I was a reversion mutation, the emergence and domination of this virus suggest that it is also more fit than the T/F virus in vivo although this still needs to be experimentally confirmed. The accumulation of more fit viruses with V247I mutation can also readily compensate the fitness cost of the T242N mutation when it is selected later. Two other recombinants (viruses with either I64T or R355K mutation) were only detected in vivo at low frequencies shortly after infection and then quickly replaced by the TK virus (Figure 1C), suggesting they are less fit in vivo under selection pressure. This suggests that although both recombinants are more fit than the T/F and TK viruses in vitro, the TK virus, under the immune selection pressure, has a higher replication advantage over both of the recombinants in vivo.
Although it was possible that the one base difference could be caused by mutation, the odds for one mutation to occur at a particular site in different experiments at various time points was much smaller than recombination between two existing parental viruses. The point mutation rate for HIV has been estimated as ~ 2.2-5.4 × 10-5 per base per replication in vitro[36, 37]. Because we were interested in mutation to a specific nucleotide, we divided the point mutation rate by 3 (0.73 -1.8 × 10-5), and because either of the two possible bases could mutate, the probability of the observed sequence occurring by mutation was 1.4 - 3.6 × 10-5. In contrast, recent estimates of the recombination rate were 1-1.4 × 10-5 per site per generation [38, 39]. Thus if the recombination target is 13 nucleotides between T/F and NIA, the expected recombination rate is 1.8 × 10-4 per generation. Thus, it is about 5-12 times more likely than a point mutation to a specific nucleotide at one or the other site. In the case of the comparison between T/F and TK, the recombination target is 1258 base long and thus the recombination probability is about 1.8 × 10-2 per generation. This is 500-1200 times more likely to be due to the recombination than the point mutation.
The mutations detected in the recombinant genomes were the same as those selected by CTL or reversion mutations in vivo. There is a possibility that they represented reversion mutations during the in vitro culture. However, analysis of 51 SGA sequences did not show the reversion mutation (I64T) after the T/F virus was passaged six times. In addition, analysis of over 2000 viral genomes from the co-culture of the NIA and T242N viruses (both with the T242N mutation) at passage 4 did not show the wild type base at position 242. This is in good agreement with the in vivo observations, in which the T242N mutation reverted back to the wild type base after months of infection [8, 32, 40–42]. These results demonstrate that mutations detected in the recombinant genomes were the results of the recombination between parental viruses, not the results of reversion or random mutations during the time period of the assay. HIV-1 is known to be highly recombinogenic [24, 25, 38, 39, 43]. Results from this study indicate that the recombination frequently occurs between compared viruses in competition fitness assays and should be considered for accurate estimation of viral fitness. If the different nucleotides are few and within 1.5 kb in the compared viral genomes, the impact of recombination on the fitness analysis can be determined by the PASS fitness assay.
There are several advantages of the new PASS fitness assay. First, no modifications in the viral genome are needed. Second, the mutations of interest are directly measured. Third, fitness can be simultaneously compared for multiple viruses. Fourth, recombinant viruses between targeted mutations can be detected and the influence of recombinant viruses on fitness can be accurately evaluated. Fifth, it is highly sensitive for minority variants present at 0.01% - 0.1% in the population . Sixth, the detection efficiency is equally efficient for compared viruses because of the identical match between the primers and all compared viral genomes. Finally, the viruses representing those in vivo are used to study viral fitness, and mutations evolved from the T/F virus and identified through genetic and immunological analysis are introduced back to the cognate T/F viral genome. Using this assay, we have also found that a neutralizing antibody (nAb) escape mutant identified six months after infection was less fit than the virus without the nAb escape mutation . Thus, this method can be a useful tool to precisely measure the impact of the mutations on viral fitness in the cognate viral genome and if viral fitness plays an important role in viral set points, transmission and pathogenesis of HIV-1.
Analyses of the impact of CTL escape mutations on viral fitness in their cognate viral genome reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations , and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. A number of advantages of the PASS fitness assay make it a powerful approach for assessing the impact of composite mutational events on viral fitness by analyzing T/F genomes and their evolved progeny.
Infectious molecular clones and viral stocks
The infectious molecular clone (IMC) for the CH77 T/F virus was chemically synthesized in a previous study . The mutations were introduced into the CH77 IMC using site-directed mutagenesis kits (Stratagene Santa Clara, CA). The virus stocks were generated by transfecting the IMCs into 293T cells as previously described .
Purification of CD4+T cells
Peripheral blood mononuclear cells (PBMC) were obtained through leukophereses from healthy donors under clinical protocols approved by the Duke University Institutional Review Board. PBMCs were isolated using the Ficoll-Hypaque density gradients and lymphocytes were isolated by elutriation using standard techniques. CD4+ T cells were negatively selected from PBMCs or lymphocytes on an autoMACS Pro Separator using the CD4+ T cell Isolation Kit II (Miltenyi Biotec, Auburn, CA). Purity of the CD4+ T cells was verified by staining with: CD16 (FITC), CD14 (PE), CD56 (PE-Cy5), CD4 (PE-Cy7), CD8 (APC), CD3 (AF700), CD19 (APC-Cy7) and CD45 (PacificBlue) and analyzed on an LSR II (BD Bioscience, San Diego, CA). All CD4+ T cell preparations were ≥ 95% positive for both CD3 and CD4. Purified CD4+ T cells were cryopreserved for later use.
Competitive virus culture
Cryopreserved CD4+ T cells were thawed and stimulated for 3 days in RPMI1640 containing 10% fetal bovine serum (FBS), interleukin 2 (IL-2) (32 IU/ml; Advanced Biotechnologies, Columbia, MD), soluble anti-CD3 (0.2 μg/ml; eBioscience, San Diego, CA) and anti-CD28 (0.2 μg/ml; BD Bioscience, San Diego, CA). After stimulation, 50 μl of cell suspension (1×106 cells) was seeded into each well of a 96-well plate, and infected with the virus mixture stock containing two or more viruses (5 ng p24 of each virus). Since the p24 concentrations and the TCID50 titers were similar among all viruses stocks, equal number of m.o.i. (~0.0003) was used for each virus. After absorption at 37°C for 4 hours, the cells were washed 3 times with RPMI 1640. The infected cells were cultured in a 24-well plate with 600 μl of RPMI 1640 containing 10% FBS and IL-2 (32 IU/ml). In the single passage assay, the culture supernatant was harvested daily and replaced with fresh medium. The virus replication kinetics was monitored by determining the p24 concentration in the supernatant using the p24 ELISA kit (PerkinElmer, Waltham, MA). Multiple-passage infection was performed by passaging the viruses repeatedly to fresh CD4+ T cells. The first round of infection was done as in the single-passage infection described above. The supernatant was harvested at day 3 or day 4 at the peak of the p24 production, and 200 μl of the supernatant was used to infect fresh CD4+ T cells (about 10 ng p24 per 106 cells). The viral replication at each passage was monitored by measuring the p24 concentration. All infections were performed in triplicate.
In the competitive fitness assay, the relative fitness is determined by measuring the replication slope of each virus in the culture over time. One advantage of the competitive fitness assay is that the variation of the input of each virus does not influence the results. For example, the relative fitness determined using a higher input of the less fit virus (90%) and a lower input of more fit virus (10%) is similar to those determined using the equal input of both compared viruses (50% for each). Since the use of equal amount of each input virus could better show the replication slopes of the compared viruses, all fitness assays were carried out by using equal amount of p24 of compared viruses in this study.
Viral RNA extraction and cDNA synthesis
Viral RNA was extracted from 50 to 200 μl of culture supernatant using the PureLink Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA). RNA was eluted into 20 μl of RNase free water. The viral RNA (17 μl) was used for cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) with the primer A4-lower: 5’-GAGTAAATTAGCCCTTCCAGTCC-3’ (nt 9082-9104, HXB2) for the tat/env amplicon and the primer A1-lower: 5'-CACAGGAACAAGCAGCCAGGTC-3 (nt 1152-1173) for the gag amplicon. The cDNA was either immediately used for the PASS assay or stored at -20°C for later use.
Determination of percentages of each virus in the culture by PASS
The PASS assay was performed as described previously [28, 30]. In brief, 20 μl of acrylamide gel mix (4%) containing 1 μM acrydite-modified primer, 0.3% diallyltartramide, 5% Rhinohide polyacrylamide gel strengthener, 0.2% bovine serum albumin (BSA), 0.1% ammonium persulfate (APS), 0.1% TEMED (N N N’,N’-tetramethylethylenediamine) and cDNA template (diluted in H2O to a final volume of 17 μl) was casted onto a glass slide which had been treated with bind-silane (Amersham Biosciences, Piscataway, NJ). The PCR reaction mix containing 1 μM primer, 0.1% Tween-20, 0.2% BSA, 1x PCR buffer, 230 μM dNTP mix, 3.5 units of Jumpstart Taq DNA polymerase (Sigma, St. Louis, MO), and H2O (up to 300 μl) was added onto the gel. After sealing with a SecureSeal chamber (Grace Bio-Labs, Bend, OR), the in-gel PCR reaction was performed in a PTC-200 Thermal Cycler under the following condition: 94°C for 3 min; 65 cycles of 94°C for 30 sec, 60°C for 45 sec, and 72°C for 1 min; 72°C for 3 min. The tat/env fragment was amplified by using the PCR primers R-lower: 5' Acry-GGAAGCACCCAGGAAGTCAGC-3' (nt 5862-5882) and R-upper: 5'-GTATCCTCTGATGGGAGGGGCATA-3' (nt 7527-7550), and the amplicons were annealed with the sequencing primer Rev7: 5'-ATGCTACTTACTGCTTTGGTAGAGGCGCTTGATTA-3' (nt 6022-6056) to detect the I64T mutation or the sequencing primer Rev13: 5'-CCTCCTGAGGAATGGTTAAAGACTAT-3' (nt 7299-7324) to detect the R355K mutation. The gag amplicon was amplified by the primers A1-lower: 5' Acry-AGGGGTCGTTGCCAAAGAGTGA-3' (nt 2260-2281) and A1-upper: 5'-CACAGGAACAAGCAGCCAGGTC-3', and the amplicons were annealed with the sequencing primer C1548A: 5'-AAGGGGAAGTGATATAGCAGGATCTACTAGTA-3' (nt 1482-1513) to detect the T242N mutation or G1562A: 5'-TATAGCAGGATCTACTAGTACCCTTCAGGAACAA-3' (nt 1494-1527) to detect the V247I mutation.
After PCR amplification, single-base extension (SBE) was performed with wild type (WT) and mutant bases distinctively labeled with Cy3 and Cy5, respectively, using the primers that annealed immediately upstream of the mutation position to distinguish two compared viruses. When three viruses were compared in the same culture, the gel was re-probed again using an additional sequencing primer. The gel images were acquired using a GenePix 4000B Microarray Scanner (Molecular Devices, Sunnyvale, CA).
The two channel images (Cy5 for the WT base and Cy3 for the mutant base) were first cropped with Picture Window Pro3.5 (Digital Light & Color, Belmont, MA) to remove the edge area containing no specific signals. The cropped images were then analyzed with the Progenesis PG200 software (Nonlinear Dynamics, Durham, NC). After background subtraction, normalization, and spot filter setting, only unambiguous spots at both channels were included for further analysis. The normalized pixel count data at two mutation sites at each spot were exported into an Excel file with a unique identifier. By comparing each spot’s normalized values at both channels, the different viruses were identified based on the base identity, and the percentage of each compared virus in the viral population was then determined. The linkage pattern of two mutations on each viral genome was determined using the Linksys program developed in-house using macros in Excel as previous described . An average of 600 (200 to 1400) viral genomes were analyzed for each sample.
The PASS error rates with DNA and RNA templates were determined in our previous study . Its error rate with the HIV-1 RNA template is 5.5 × 10-5, which is far below the frequencies of the minority virus variants detected in the fitness assay. All PASS PCR and sequencing primers were tested for their specificity using plasmid DNA and viral RNA. Unexpected bases above the error rate were not observed. Thus, the PASS fitness assay was not likely to be affected by false unexpected mutations. Random mutations were generated during 65 cycles of PCR reaction. However, since all the PCR products amplified from a single viral cDNA molecule were sequenced together as a population, these random mutations, which only accounted for a very small portion of the total population, would not be detected.
The simplest analysis of fitness assumes a competition experiment during which each variant is in a phase of exponential growth. In this situation, the concentration of the various forms, c i, grows with time, t, as c i = c i 0 exp(k i t), where k i is the Malthusian growth parameter of this variant and c i 0 is its concentration at time 0. In population genetics, the relative fitness, r ij, of variant i with respect to variant j is often expressed as the difference k i - k j . In many experimental situations, however, factors extrinsic to the individual genotype influence the overall growth rate strongly. To account for this, some authors have chosen to normalize the relative fitness by the growth rate of one of the variants , so that this normalized relative fitness is given as s ij = (k i – k j )/k j = (k i /k j ) - 1.
To find the growth rate k j needed to normalize this, the measured p24 concentration is apportioned among the various forms in the ratio observed in the PASS assay, and the result fit to an exponential function of time.
where c i and c j are the concentrations of the viral variants i and j at the pth passage, and d is the factor by which the viruses at the end of one passage are diluted with fresh medium to start the next passage.
To see this, consider how the values c i and c j at the end of the pth passage are related to the values c i ’ and c j ’ at the end of the previous passage. Since every passage begins by diluting the culture medium by a factor d, the starting concentrations of the two forms at the pth passage are given by c i ’ /d and c j ’ /d respectively. If the average growth rate of the jth form in this passage is k j , we have log [c j /(c j ’ /d)] = k j t, where t is the duration of the passage. From this, we obtain the relation log (c j d p ) – log (c j ’ d p-1 ) = k j t, and a similar relation holds for the ith virus. Taking the ratio of these two relations, one obtains the finite difference equation Δ log (c i d p ) = (1 + s ij ) Δ log (c j d p ), where Δ represents the increase in a quantity during the pth passage. Equation (2) is the unique solution to this finite difference equation if we assume s ij to be the same at each passage.
To fit to this equation to the data, we need the absolute concentrations of the various genomes, whereas the PASS assay is only standardized to provide the relative counts accurately. We therefore use the p24 assay to obtain the total concentration of virions in the culture medium. We have used an estimate of 40000 genomes/pg of p24  to determine the HIV-1 concentration, but the numerical estimates of the normalized selection coefficients is independent of the value of this constant. We obtain the estimate of s ij by numerically maximizing the Poisson likelihood of the observed PASS counts when the logarithms of the underlying concentrations are restricted to the linear form given by Eq. (2). In those cases where we do observe constant exponential growth, the estimates obtained by this method agree with those from fitting Eq. (1) to the data.
Cytotoxic T lymphocyte
- T/F virus:
Parallel allele-specific sequencing
Single genome amplification
Peripheral blood mononuclear cells
Infectious molecular clone
Fetal bovine serum
Multiplicity of infection
Bovine serum albumin
This work was supported by NIH grants to the Center for HIV/AIDS Vaccine Immunology (AI067854) and the Duke Centers for AIDS Research (AI064518). Portions of this work were done under the auspices of the U. S. Department of Energy under contract DE-AC52-06NA25396 and ASP was also supported by NIH grants AI028433 and the National Center for Research Resources and the Office of Research Infrastructure Programs (ORIP) through grant OD011095.
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