1 Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, NN10, 9500 Euclid Avenue, Cleveland, OH 44195, USA
2 Laboratori de Retrovirologia, Fundacio irsiCaixa, Hospital Universitari Germans Trias I Pujol, Badalona, Spain
3 ViroLogic Inc., South San Francisco, CA, USA
Correspondence
Miguel Quiñones-Mateu
quinonm{at}ccf.org
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ABSTRACT |
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INTRODUCTION |
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Multiple methods have been used to measure HIV-1 fitness in vitro (Clavel et al., 2000; Nijhuis et al., 2001
; Quiñones-Mateu & Arts, 2001
), yet few studies have directly compared different methods to measure HIV-1 fitness (Grant et al., 2001
; Prado et al., 2002
). These studies have suggested that the highly impaired replication capacity of drug-resistant HIV-1 pol mutants may allow the use of recombinant viruses to estimate overall HIV-1 fitness and its relationship to drug resistance. However, small differences in virus fitness have been more apparent using growth competition experiments than single-cycle assays (Bleiber et al., 2001
; Prado et al., 2002
). A recent study comparing the individual contribution of PR- and RT-encoding regions to fitness in drug-resistant HIV-1 primary isolates suggested that other genomic regions outside of drug-targeted genes may compensate for the drug-resistant substitutions in pol (Bleiber et al., 2001
). In addition, new antiretroviral drugs are being developed that target other regions of the viral genome, which may not be included in these recombinant pol constructions. For example, HIV entry inhibitors that involve viral envelope glycoproteins and their cellular receptors are now under clinical evaluation (Moore & Stevenson, 2000
). Therefore, the use of HIV-1 clinical isolates instead of recombinant viruses may help elucidate the interdependence between different viral genes, their effect on virus fitness (i.e. virulence, transmission and pathogenicity) (Quiñones-Mateu et al., 2000
), multidrug resistance to PI and RTI (Brenner et al., 2002
; Quiñones-Mateu et al., 2002
) and potential resistance to multi-target antiretroviral therapy (e.g. directed against pol and env).
In the absence of a consensus method for quantifying virus replication capacity, many studies have employed different techniques to assess HIV-1 fitness (Quiñones-Mateu & Arts, 2001). New technologies such as real-time PCR have also been applied to dual virus detection. de Ronde et al. (2001)
used real-time nucleic acid sequence-based amplification PCR and molecular beacons to quantify individual mutant viruses in a mixture. Lu & Kuritzkes (2001)
developed a recombinant marker virus assay to perform growth competition experiments between two RT recombinant viruses which were quantified using real-time PCR for the corresponding marker. Several studies have described the use of TaqMan real-time PCR as a sensitive technique to measure and quantify plasma HIV-1 RNA or proviral HIV-1 DNA (Lewin et al., 1999
; de Baar et al., 2001
; Desire et al., 2001
; Hance et al., 2001
; Zhao et al., 2002
). However, TaqMan real-time PCR has not been used to detect two different HIV-1 primary isolates in growth competition experiments in order to estimate virus fitness.
It is important to note that the term virus fitness' is commonly employed in both in vitro studies and the clinical setting (Nijhuis et al., 2001; Quiñones-Mateu & Arts, 2001
). For example, most studies describing the diminished replicative capacity of HIV-1 drug-resistant strains have referred to this phenotype as virus fitness. However, in vivo virus fitness depends on multiple virus and host factors (Quiñones-Mateu & Arts, 2001
), while replication capacity is the intrinsic capacity of the virus to replicate efficiently in an ideal environment (i.e. cell culture) (Nijhuis et al., 2001
). Therefore, and in order to distinguish between both approaches, we have denominated ex vivo virus fitness' to those values estimated using HIV-1 isolates in growth competition experiments to differentiate them from replication capacity (in vitro virus fitness) values obtained using pol recombinant viruses. In the present study, we have developed a rapid method for quantifying two different proviral HIV-1 genomes in a mixture using TaqMan real-time PCR, which specifically differentiates subtype B HIV-1 isolates from subtype A or CRF01_AE HIV-1 strains. This method, comprising independent growth competition experiments between a subtype B HIV-1 isolate and two non-subtype B viruses followed by the TaqMan real-time PCR assay, was sensitive, reproducible and allowed a wide dynamic range of detection to measure HIV-1 fitness. We compared our new assay with a single-cycle replication capacity assay to quantify the reduction in fitness of drug-resistant recombinant viruses, demonstrating consistent results between both methods.
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METHODS |
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Growth competition experiments to estimate HIV-1 fitness.
All dual infection/competition experiments were carried out as described previously (Quiñones-Mateu et al., 2000, 2002
) with minor modifications. Briefly, each subtype B HIV-1 primary isolate or recombinant virus was competed against two different non-subtype B HIV-1 control strains (HIV-1A-92UG029 and HIV-1AE-CMU06) in a 1 : 1 initial proportion using an m.o.i. of 0·01 IU per cell (Fig. 1
A). Of these virus mixtures, 1 ml was incubated with 1x106 PBMCs for 2 h at 37 °C and 5 % CO2. Subsequently, the cells were washed three times with 1x PBS and then resuspended in culture medium (1x106 ml-1). Cells were washed and fed with medium after 4 days. Supernatants and cells were harvested at day 8 and stored at -80 °C for subsequent analysis.
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To quantify the proportion of both HIV-1 variants in the mixture, three sets of subtype-specific primers and probes were designed (Fig. 1B). Subtype B-specific primers, Bgag-S (5'-GGAGCTAGAACGATTCGCAG-3', nt position 906), Bgag-AS3 (5'-TCTTACTTTTGTTTTGCTCTTCC-3', nt position 1104) and probe pBgag-ROX (5'-ROX-TACTGGGACAGCTACAACCATCCCTT-BHQ-3', nt position 968), where 6-carboxy-X-rhodamine (ROX) is the reporter fluorochrome and black hole quencher (BHQ) corresponds to the quencher. This primer/probe combination was designed to recognize a conserved region within the subtype B HIV-1 gag gene. In addition, two sets of primers and probes were designed to recognize conserved regions in the HIV-1 env genes of clade A and the circulating recombinant form CRF01_AE. Subtype A-specific primers were A2env-S (5'-CATAATAGTACAACTTACCAAGC-3', nt position 7073), A2env-AS2 (5'-ATAGTACAACTTACCAAGCCTG-3', nt position 7338) and probe pA2envFAM (5'-FAM-CAGAAGAAGGATACATATAGGAAGAGGACA-TAMRA-3', nt position 7133). CRF01_AE-specific primers were E1env-S (5'-TTACTATGGGACCAGGACAC-3' nt position 7144), E1env-AS (5'-GCCAAGTACCATTAACCAAGTT-3', nt position 7403) and probe pE1envFAM (5'-FAM-CGGGAGGGGATCTAGAAATTACAATGCATCA-TAMRA-3', nt position 7318). FAM and TAMRA correspond to the 6-carboxyfluorescein report fluorochrome and the 6-carboxytetramethylrhodamine quencher, respectively.
These three sets of primer/probe combination allowed subtype-specific PCR amplification and hybridization so that cross-hybridization between subtype B probes and subtypes A or AE did not occur. PCR products corresponding to three primary isolates (i.e. HIV-1B-92US076, HIV-1A-92UG029 and HIV-1AE-CMU06) were used to verify the sensitivity and specificity of these oligonucleotides. Each 25 µl TaqMan real-time PCR mixture contained 10 µl DNA (usually 1/20 of the DNA extract), 1x PCR buffer (Invitrogen), 6 mM MgCl2, 0·2 mM dNTPs, 0·2 µM of each primer, 0·4 µM of probe and 1·25 U Platinum Taq DNA polymerase (Invitrogen). PCR conditions consisted in one cycle of denaturation (95 °C for 10 min), followed by 40 cycles of amplification (95 °C for 15 s, 63 °C for 1 min and 56 °C for 1 min). All real-time PCR amplifications, data acquisition and analysis were performed using the Smart Cycler System, software version 1.2d (Cepheid).
HIV-1 isolates used to test the TaqMan real-time PCR assay.
To test our method to detect two different HIV-1 strains in a mixture, HIV-1 isolates were obtained from two different sources. First, four HIV-1 strains with different levels of susceptibility to the PI amprenavir (APV) (i.e. collected during serial passages of HIV-1IIIB in the presence of increasing concentrations of the drug) were obtained from a previous study (Table 1) (Prado et al., 2002
). Second, four HIV-1 primary isolates with distinct patterns of drug-resistance mutations in pol were obtained from three different HIV-1-infected individuals treated at the Hospital Universitari Germans Trias I Pujol in Badalona, Spain (Table 2
) (Cabana et al., 1999
).
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HTA for detection of two HIV-1 env fragments in a mixture.
To evaluate the new TaqMan real-time PCR assay, nested PCR products (C2-C4 of env) from selected growth competition experiments were analysed using HTA, as described previously (Quiñones-Mateu et al., 2000, 2002
; Ball et al., 2003
).
Construction of PR and RT HIV-1 recombinant viruses.
To analyse the contribution of different HIV-1 genomic regions to virus fitness, different HIV-1 recombinant viruses were constructed (Fig. 1C). PR and RT recombinant viruses were prepared as described previously (Gutierrez-Rivas et al., 1999
; Mas et al., 2000
). Briefly, proviral DNA was extracted from lysed PBMCs from three different patients with different susceptibility to PI and RTI (i.e. longitudinal samples F96 and F98 and two cross-sectional samples, J94 and A94) (Table 2
) (Cabana et al., 1999
). Full-length PR- or RT-encoding sequences were amplified by nested PCR. PCR products were then co-transfected with a PR-deleted (pHIVProBstEII) (Maschera et al., 1995
) or an RT-deleted (pHIVRTBstEII) (Kellam & Larder, 1994
) HIV-1HXB2-based clone into SupT1 cells, as described previously (Gutierrez-Rivas et al., 1999
). Culture supernatants were harvested when the HIV-1 p24 antigen concentration surpassed 20 ng ml-1. The complete PR-encoding sequence and the first 750 nt of the RT-encoding sequence of the progeny viruses were determined and compared with the genotype of the initial PCR products.
Estimation of drug susceptibility and virus replication capacity.
A rapid recombinant assay was used to measure the drug susceptibility and replication capacity of recombinant viruses (PhenoSense HIV) (Petropoulos et al., 2000; Prado et al., 2002
). Briefly, this assay involved the use of a replication-incompetent HIV-1 vector in which a luciferase gene has been inserted into a deleted portion of env (Petropoulos et al., 2000
). A PCR fragment derived from patient plasma samples or virus stocks (containing the 3' end of gag and both PR- and RT-encoding regions) (Fig. 1C
) was introduced into this vector and co-transfected with a murine leukaemia virus env gene construction. HIV-1 3'Gag/PR/RT vectors were prepared as libraries to capture most of the viral sequence pool heterogeneity contained in the patient. The susceptibility (IC50 values) of these recombinant vectors to a panel of PI and RTI was compared to a reference vector containing the PR and RT sequences derived from HIV-1NL4-3. In addition, the relative replication capacity of these viruses was determined by normalizing the luciferase activity produced at 72 h post-infection in the absence of drug, with the luciferase activity expressed by cells infected with the parental HIV-1NL4-3 strain to give relative light units. Replication capacity measurements were expressed as a percent of the mean of a large wild-type HIV population.
Estimation of ex vivo HIV-1 fitness from growth competition experiments.
To determine the fitness of HIV-1 primary isolates and recombinant viruses, the final ratio of the two viruses produced from each dual infection/growth competition experiment was determined by HTA and/or TaqMan real-time PCR assay and compared to production in monoinfections, as described previously (Quiñones-Mateu et al., 2000, 2002
). Briefly, a relative fitness (w) value for each virus in the competition was estimated by the production of each individual HIV-1 strain in the dual infection. Total relative fitness was calculated as the average of the two relative fitness values, corresponding to the competition between each subtype B HIV-1 isolate or recombinant virus and each one of the non-subtype B HIV-1 control strains. The total relative fitness was then compared and expressed as a percentage of a wild-type subtype B HIV-1 primary isolate (HIV-1B-92US076, taken as 100 %) (Quiñones-Mateu et al., 2002
).
Statistical methods.
Pearson product moment correlation coefficient was used to determine the strength of association or correlation between different methods or techniques to measure virus fitness. All statistical tests were performed using SIGMASTAT, version 2.03 (SPSS).
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RESULTS |
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HIV-1 competitions and estimation of virus fitness: comparison between HTA and TaqMan real-time PCR assays
We have described previously the use of HTA to identify two HIV-1 isolates in a mixture and estimate their virus fitness (Quiñones-Mateu et al., 2000, 2002
; Ball et al., 2003
). In this study, we have refined our dual competition assay by using TaqMan real-time PCR assay to accurately detect and measure two HIV-1 isolates in a growth competition experiment. However, several controls were needed to validate this new assay as an accurate method to quantify virus production and fitness. Four different competitions were established between two subtype B HIV-1 strains (i.e. HIV-1HXB2 and HIV-192US076) and the two non-subtype B HIV-1 primary isolates used as controls in our assay (i.e. HIV-1A-92UG029 and HIV-1AE-CMU06, see Methods). Detection and quantification of virus production was performed by HTA and TaqMan real-time PCR assay (Fig. 3
). In this case, both subtype B viruses outcompeted the non-B control isolates. Both techniques, HTA and TaqMan, were able to distinguish clearly between subtype B and non-clade B virus production. However, in all cases, the TaqMan assay was more sensitive than HTA and detected smaller amounts of HIV-1 control isolates (Fig. 3
). This increase in the sensitivity to detect low levels of virus production in a mixture using TaqMan real-time PCR allowed a better quantification and estimation of HIV-1 fitness. Therefore, all subsequent competition experiments were analysed using the TaqMan method of detection.
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DISCUSSION |
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Fitness of a virus is best defined in growth competition experiments (Holland et al., 1991). Our new assay involves dual infections with two different HIV-1 strains in a single culture, followed by quantification of both viruses using TaqMan real-time PCR. This technique combines the sensitivity of PCR amplification and the quantification of hybridization tests, eliminating laborious post-PCR handling of samples for quantification. Similar to the initial development of many other HIV-1-related techniques (e.g. diagnostic tests, plasma HIV-1 RNA quantification, antiretroviral drugs or vaccines), we have designed a method to specifically measure virus fitness of subtype B HIV-1 strains. Subtype B HIV-1 isolates were competed against two different non-clade B HIV-1 controls (i.e. HIV-1A-92UG029 and HIV-1AE-CMU06) in order to obtain a relative fitness value. Initial attempts using universal PCR primers and clade-specific probes in a single genomic region for all three subtypes showed a low detection level of the virus falling behind in the competition (data not shown). A preferential PCR amplification of the most fit (most abundant) virus was observed, limiting the sensitivity of the assay. To circumvent this problem, detection and quantification of both viruses in the mixture was carried out using three different subtype-specific HIV-1 primer/probe sets labelled with two different report fluorochromes (i.e. gag oligonucleotides for the subtype B query sequences and env oligonucleotides for the subtype A or CRF01_AE HIV-1 controls). All three subtype-specific TaqMan probes were able to distinguish and quantify the corresponding HIV-1 strain from a mixture with two other HIV-1 subtypes. A lower detection level (102103 proviral DNA copies per reaction) was observed with the TaqMan detection method than with the heteroduplex tracking assay (Fig. 3
). With a broad linear dynamic range of detection (103107 HIV-1 copies) and a strong linear correlation between the threshold cycles and the number of virus copies, this TaqMan real-time PCR assay should be useful to detect low levels of virus production in a competition, which may improve measurement of ex vivo HIV-1 fitness.
To test our assay further, we compared the virus fitness of drug-resistant HIV-1 isolates and autologous pol recombinant viruses using three different assays: (i) virus growth kinetics; (ii) growth competition cultures followed by TaqMan detection; and (iii) single-cycle replication capacity. An equal resolution of the differences in virus fitness between APV-resistant viruses was observed when growth competition experiments or single-cycle replication capacity assays were performed (Fig. 4). A similar correlation was obtained when virus fitness of HIV-1 primary isolates with different patterns of drug resistance in pol was analysed using both methods (Fig. 5
). Thus, it seems possible that a considerable decrease in HIV-1 fitness due to selection and accumulation of drug-resistant mutations in pol may overcome the effect of other viral genomic regions allowing the use of 3'Gag/PR/RT recombinant viruses to estimate replicative capacity in viruses from patients treated with PI and/or RTI. Similar results have been published recently describing consistent outcomes between growth competition experiments and single-cycle replication assays (Resch et al., 2002
; Prado et al., 2002
). However, differences in the magnitude of the fitness loss of some PR-resistant mutants were attributed to intrinsic variations of the assay used (Resch et al., 2002
).
In this study, we observed a discrepancy between growth competition/TaqMan and single-cycle replication assays when the virus fitness of an HIV-1 isolate harbouring the multi-NRTI-resistance 151 mutation complex (virus A94) was analysed (Fig. 5A). Interestingly, contradictory results have been obtained when the fitness of viruses carrying these RT mutations (i.e. 62V, 75I, 77L, 116Y and 151M) was studied using different methods. For example: (i) no effect on virus fitness, based on virus growth kinetics (Maeda et al., 1998
); (ii) fitness impairment, using single infections and competition experiments (Garcia-Lerma et al., 2000
); or (iii) an increase in virus fitness, comparable to the wild-type control when growth competition experiments were used (Kosalaraksa et al., 1999
). All these studies analysed the virus fitness of this mutation complex in a HIV-1HXB2 backbone. Here we identified a reduction in virus fitness of the A94 virus using growth competition/TaqMan assay (HIV-1 isolate), while no effect was observed with the single-cycle assay (recombinant virus). Based on these discrepancies, it is evident that amino acid changes outside the 3'Gag/PR/RT region used in this and other studies to generate recombinant viruses may have a significant impact on the overall fitness of viruses containing the 151 mutation complex.
Finally, a previous study described the individual contribution of different HIV-1 genes to virus fitness (i.e. mutant PR and RT) (Bleiber et al., 2001). Singular recombinant clones, carrying PR, RT and PR/RT from drug-resistant virus isolates, showed a marked impairment on virus fitness using virus growth kinetic assays. However, the fitness of the parental drug-resistant HIV-1 isolates was similar to the wild-type control (Bleiber et al., 2001
). In our study, we extended this analysis by comparing the virus fitness of four HIV-1 primary isolates and the contribution of PR, RT and 3'Gag/PR/RT fragments to the overall fitness in the absence of drug. Both assays, growth competition/TaqMan and single-cycle replication, generated similar results with a strong significant correlation (r=0·72, P=0·01; Pearson product moment). Even more interesting, a difference in virus fitness relative to the HIV-1 isolate was observed when recombinant viruses carrying only the PR or RT gene were analysed, including the wild-type F96 strain, stressing the importance of using HIV-1 isolates or at least the whole genomic region implicated (e.g. 3'Gag/PR/RT) to measure fitness of drug-resistant variants.
In conclusion, we have developed an ex vivo system to measure HIV-1 fitness based on growth competition experiments followed by TaqMan real-time PCR. Our results showed that both methods, growth competition/TaqMan and single-cycle replication assay, were able to detect differences between viruses with highly impaired replication capacities due to mutations in pol and gag. However, consequent with the actual trend of multi-target antiretroviral therapies (e.g. PR, RT, integrase and entry inhibitors), this new assay may be useful to measure virus fitness of subtype B HIV-1 primary isolates or recombinant viruses carrying pol and env.
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ACKNOWLEDGEMENTS |
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Received 28 January 2003;
accepted 22 April 2003.