Although important advances have been achieved in the treatment of individuals infected with the human immunodeficiency virus type I (HIV-1), the development of resistance to current antiretroviral drugs is a topic of major concern . Drug resistance is due to both the error-prone nature of HIV-1 reverse transcriptase (RT) as well as the high replication rate of the virus [2,3]. The persistence of cellular reservoirs of virus and anatomic sanctuary sites ensures the continuing replication of viruses, even in patients with suppressed plasma viremia. Consequently, the eradication of HIV-1 is not considered to be achievable with currently available treatment regimens [4,5].
Nucleoside analog reverse transcriptase inhibitors (NRTIs) of HIV-1 RT function as chain terminators of viral DNA synthesis by virtue of the fact that they lack a 3′-OH group. Numerous combinations of NRTIs have been effectively used in the treatment of HIV-1 infected patients [6,7]. However, resistance has been reported against all currently approved members of this class .
Two distinct mechanisms explain HIV-1 drug resistance to NRTIs. The first involves the diminished recognition of NRTIs by mutated, resistant RT enzymes, so that these drugs are no longer incorporated into the nascent viral DNA chain . The other mechanism involves increased phosphorolytic excision of the incorporated drug [10,11].
The K65R mutation is associated with a low–intermediate level of resistance to tenofovir (TDF) both in vitro and in vivo [12,13]. In addition, K65R is found in fewer than 2% of clinical isolates from dideoxycytidine (ddC) and dideoxyinosine (ddI)-treated patients and shows a two-fold to 10-fold decrease in susceptibility to these antiretroviral drugs [14,15]. The M184V mutation is clinically associated with high-level resistance to lamivudine (3TC) and low–intermediate level resistance to ddI [16–18].
Both mutations share numerous characteristics: (1) they involve similar discriminatory mechanisms with regard to incorporation of relevant NRTIs that would ordinarily inhibit reverse transcription ; (2) both result in decreased viral replication capacity [20,21]; (3) both K65R and M184V-containing mutant enzymes have been associated with zidovudine (ZDV) hypersusceptibility, suppression of resistance to ZDV, and impaired rescue of chain-terminated DNA synthesis [12,22,23].
However, the occurrence of viruses containing both K65R and M184V is infrequent [16,24]. Recently, it has been demonstrated that HIV-1 viruses containing the doubly mutated K65R/M184V RT show severely impaired viral replicative capacity compared to wild-type virus, and this loss of fitness can be correlated with a poor ability of K65R/M184V RT to use natural dNTPs relative to wild-type RT .
In spite of our increased understanding of NRTI resistance, the biochemical basis for diminished replicative capacity associated with some resistance-conferring mutations is not well understood. The present study evaluated molecular mechanisms associated with efficiency of (-)ssDNA synthesis in the presence of either the cognate tRNA3Lys primer or the DNA dPR primer, resistance to relevant NRTIs and ZDV hypersusceptibility and viral replicative capacity in the context of each of the K65R and M184V mutations and the combination of both.
Materials and methods
Reverse transcriptase enzymes and other HIV-1 proteins
Recombinant wild-type and mutated HIV-1 RTs (K65R, M184V and K65R/M184V) were prepared as previously described . HIV-1 nucleocapsid (NC) protein was purified as described .
Tenofovir disoproxil fumarate (TDF) and tenofovir diphosphate (TFV-DP) were kindly provided by Gilead Pharmaceuticals (Foster City, California, USA). ZDV and ddI were purchased from Sigma-Aldrich Inc. (Oakville, Ontario, Canada). ZDV triphosphate (ZDV-TP) and ddATP were purchased from Trilink Biotechnologies (San Diego, California, USA) and Invitrogen (Burlington, Ontario, Canada), respectively. The 3TC and 3TC triphosphate (3TC-TP) were generously donated by GlaxoSmithKline (Research Triangle Park, North Carolina, USA).
To study the rescue of chain-terminated DNA synthesis, both a DNA primer (PPT-18) and DNA template (PPT-57) were derived from the polypurine tract (PPT) of the HIV-1 genome . To study the efficiency of initiation of (-)ssDNA synthesis, an HIV-1 RNA (pHIV-PBS) containing the region spanning the 5′-untranslated region to the primer binding site (PBS) was in-vitro transcribed and purified as previously described . Human placenta tRNA3Lys was purchased from Bio S&T (Montreal, Quebec, Canada). Alternatively, an 18-nucleotide DNA (dPR), complementary to the PBS, was utilized to prime reverse transcription. Oligonucleotides were purchased from Invitrogen (Burlington, Ontario, Canada).
Determination of 50% inhibitory concentrations of reverse transcriptase activity
The 50% inhibitory concentration (IC50) values for TFV-DP, ddATP and 3TC-TP were determined as previously described .
Rescue of chain-terminated DNA synthesis with ATP
The ZDV-terminated primer strands were detected as previously described . The excision of the ZDV-terminated primer was initiated by adding a mix containing 100 μmol/l dTTP, 10 μmol/l dGTP, 100 μmol/l ddATP and 3.5 μmol/l ATP (pretreated with inorganic pyrophosphatase). DNA synthesis was monitored in time-course experiments. Samples were resolved in an 8% polyacrylamide–7M urea gel followed by exposure on X-ray films. Band intensities were analyzed by molecular imaging.
Synthesis of (-)ssDNA
The efficiency of (-)ssDNA synthesis was determined as described . Briefly, 20 nmol/l tRNA3Lys were heat-annealed to 40 nmol/l pHIV-PBS. Alternatively, 9 μmol/l NC was added to place the tRNA3Lys onto the HIV-1 RNA template. Then, 150 nmol/l wild-type or mutated RT and 6 mmol/l MgCl2 were added. Reactions were initiated with 10 μmol/l dNTPs and monitored by incorporation of [α-32P]-dCTP. Aliquots were removed at various times and quenched with 95% formamide–40 mmol/l ethylenediamine tetraacetic acid. Samples were resolved in a 5% polyacrylamide–7M urea gel and analyzed as described above.
Cells and viruses
The MT-4 cell line was grown in RPMI 1640 medium/2 mmol/l L-glutamic acid/10% fetal calf serum and penicillin/streptomycin as described . Cord blood mononuclear cells (CBMCs) were isolated by Ficoll-Hypaque centrifugation, stimulated with phytohemagglutinin (PHA) and interleukin (IL)-2 and depleted of CD8+ suppressor cells as previously described . pNL4-3 derived wild-type viruses and viruses containing the K65R, M184V and K65R/M184V mutations were amplified in MT-4 cells as described .
Multiple round infection assays
We incubated 106 MT-4 cells with aliquots of virus equivalent to 10 ng of p24 at 37°C for 2 h. The cells were washed and maintained in 10 ml of medium. CBMC infections were performed as described . Culture fluids were collected at various times to determine levels of RT activity as described .
Single round infection assays
Either 106 MT-4 cells or 2 × 106 CBMCs were infected with DNase I-treated wild-type or mutated HIV-1 at a multiplicity of infection of 0.0001 as described . At 12, 24 and 48 h after infection, cells were washed with phosphate-buffered saline and re-suspended in 200 μl. DNA was extracted using a QIAamp DNA mini kit (Qiagen Inc., Mississauga, Ontario, Canada) according to the manufacturer's instructions and quantified by optical density at 260 nm. Reverse transcribed DNAs were quantified by real-time polymerase chain reaction (PCR) as previously described .
Phenotypic resistance assays
To determine the sensitivity of wild-type, K65R, M184V and K65R/M184V-containing viruses to relevant RT inhibitors, 2 × 106 MT-2 cells were infected with 1 × 105 tissue culture infective dose (TCID50) virus stock as previously described . The cells were then washed and plated in increasing concentrations of NRTI. The IC50 values were determined based on RT activity in culture supernatants as described above.
Drug susceptibility in cell culture and cell-free assays
Cell culture and clinical data have shown that viruses containing the K65R and M184V mutations display moderate to high-level resistance to 3TC and ddI [9,38,39]. We infected MT-2 cells with wild-type or mutated viruses and observed hypersusceptibility to ZDV (range 0.5 to 0.7-fold resistance), low-to-moderate-level resistance to TDF (4.3-fold, 1.1-fold and 2.2-fold for K65R, M184V and K65R/M184V-containing viruses, respectively), moderate-level resistance to ddI (11-fold, 2.7-fold and 17-fold for K65R, M184V and K65R/M184V-containing viruses, respectively) and finally, high-level resistance to 3TC (six-fold, > 500-fold and > 500-fold for K65R, M184V and K65R/M184V-containing viruses, respectively) (Table 1).
Previous cell-free resistance data have shown that RTs harboring the K65R mutation display 10-fold resistance against ddC and about 12-fold decreased susceptibility to TFV-DP as compared to wild-type RT [14,30,40]. In addition, M184V-containing RTs have been shown to possess an approximately 136-fold reduction in susceptibility to 3TC-TP, and a two-fold reduction in susceptibility to ddATP [30,35]. In addition, pre-steady state kinetics with single nucleotide incorporation assays have demonstrated that K65R, M184V and K65R/M184V-containing RTs display 4.4, 0.4 and 1.7-fold decreased susceptibilities to TFV-DP, respectively . Our cell-free RT susceptibility assays showed that K65R and M184V-containing RT displayed 9.3-fold and 1.0-fold decreased susceptibility to TFV-DP, respectively, in comparison with wild-type RT. The simultaneous presence of K65R and M184V dramatically resensitized these enzymes to TFV-DP by 5.1-fold in comparison with wild-type RT. Thus, the order of susceptibility to TFV-DP was wild-type = M184V > K65R/M184V > K65R (Table 1).
We also studied the resistance profile of wild-type and mutated RTs to ddATP. Our data show that K65R RT and M184V RT exhibited 6.2-fold and 1.5-fold decreased susceptibility to ddATP, respectively, in comparison with wild-type RT. Addition of the M184V mutation in the context of K65R further decreased susceptibilities to ddATP by 9.8-fold in comparison with wild-type RT. As a result, the order of susceptibility to ddATP was wild type > M184V > K65R > K65R/M184V (Table 1). Similarly, with regard to resistance to 3TC-TP; our data show that K65R RT, M184V RT and K65R/M184V RT displayed 9-fold, 130-fold and 128-fold resistance in comparison with RT, respectively (Table 1).
The K65R and M184V mutations in reverse transcriptase cause a major reduction in the efficiency of excision of zidovudine-monophosphate from newly synthesized viral DNA
The K65R, L74V and M184V mutations have been shown to decrease the rates of excision of ZDV-monophosphate (MP) from ZDV-terminated primers [10,11,41–43]. We wished to determine the effects of the combination of K65R and M184V on the efficiency of excision of ZDV-MP from viral DNA in gel-based assays (Fig. 1a). We found that the level of ZDV-terminated primer at 45 min was 60% for wild-type RT and 75% for M184V RT, respectively (Fig. 1b). In addition, K65R RT showed similar levels of impaired rescue of ZDV-MP from chain-terminated primers in comparison with M184V RT in the presence of ATP as a pyrophosphate donor. In contrast, K65R/M184V RT displayed 92% of ZDV-terminated primer at 45 min (Fig. 1c). Thus, the efficiency of excision of ZDV-MP was wild-type > K65R ≅ M184V > K65R/M184V (Fig. 1d).
Efficiency of (-)ssDNA synthesis
To study the underlying mechanisms potentially associated with the low replicative capacity of viruses harboring the K65R or M184V mutations, we evaluated the efficiency of initiation of (-)ssDNA synthesis using a gel-based system as described  (Fig. 2a). Earlier findings by our group had pointed to a decrease in the total amount of tRNA3Lys primer extension as well as decreased release of pausing at position +3, catalyzed by the L74V- and M184V-containing RTs, compared with wild-type RT . Notably, the K65R RT showed a slight reduction in the total amount of tRNA3Lys extended primer. However, K65R/M184V-containing RTs displayed the maximum decrease in product formation and accumulation at the +3 pausing site (Fig. 2b). Therefore, the order of efficiency of (-)ssDNA synthesis was wt > K65R > M184V >> K65R/M184V. We also restricted the synthesis of (-)ssDNA to the initiation stage by adding ddATP to terminate the reaction at position +6 (Fig. 2c). The results described in Fig. 2d and e show that release from the pausing site at position +3 was somewhat compromised when the singly mutated M184V and K65R RTs were studied. Strikingly, K65R/M184V RT not only displayed the highest accumulation of products at the +3 pausing site, but also a shift at higher time points, pointing to a dramatic impairment of this kinetic mechanism. We also studied the efficiency of (-)ssDNA synthesis primed with the DNA primer dPR (Fig. 3a). We found that the level of full-length (-)ssDNA at 24 min was 24% for wild-type RT, 15% for M184V RT, 20% for K65R RT and 9% for the doubly-mutated K65R/M184V RT (Fig. 3b and c).
Among the viral factors that regulate the extent of HIV-1 reverse transcription, the NC protein has been described to play a major role by virtue of its chaperoning activity [45–49]. To further understand the observed reduction in the total amount of tRNA3Lys extended primer as well as the decrease release from +3 pausing site by the K65R/M184V RT compared to wild-type RT, we determined the efficiency of the initiation of the (-)ssDNA synthesis in the presence of NC, thus mimicking a more physiological scenario. Our results indicate that K65R/M184V RT pausing products distribution at position +3 and +5 was notably different from that of wild-type RT at early time points (Fig. 3d). At 5 min in this reaction, wild-type RT displayed 13 and 15% of newly synthesized (-)ssDNA at pausing positions +3 and +5, respectively (Fig. 3e). In contrast, K65R/M184V RT showed 43 and 40% of accumulation at the aforementioned pausing sites (Fig. 3f).
Viruses harboring the K65R/M184V mutations exhibit severely compromised replication compared to wild-type viruses in a single round of infection
Recently, L74V-, M184V- and L74V/M184V-containing enzymes have been shown to display diminished efficiency of initiation of minus and plus-strand DNA synthesis . The latter finding is consistent with the observations in this paper on the reduced efficiency of initiation of (-)ssDNA synthesis of RTs containing K65R/M184V. Moreover, both K65R/L74V- and K65R/M184V-containing RTs have been associated with diminished ability to use natural dNTPs relative to wild-type RT [25,50]. To study possible correlations between the decreased levels of (-)ssDNA synthesis by the doubly mutated K65R/M184V RT and the described diminished replicative capacity associated with K65R/M184V-containing viruses, we evaluated the efficiency of the reverse transcription reaction using real-time PCR in a single round of infection in MT-4 cells. Quantification of the data showed that wild-type viruses and M184V and K65R-containing viruses produced peak levels of (-)ssDNA at 12 h after infection as previously described , although K65R/M184V-containing viruses were severely impaired in this regard (Fig. 4a). Additionally, the level of (-)ssDNA synthesis at 24 h after infection for mutated viruses compared to wild-type virus was 89% for K65R, 88% for M184V and 15% for K65R/M184V (Fig. 4c).
An even more dramatic effect was found in regard to reverse transcribed DNAs synthesized after the first strand transfer, which were measured at 24 h after infection. Levels of synthesis for mutated viruses compared to wild-type virus were 56% for K65R, 35% for M184V and 14% for K65R/M184V (Fig. 4b and c).
We also evaluated viral replication of wild-type and mutated viruses in a single round of infection in CBMCs, which are known to exhibit reduced intracellular dNTPs pools . Maximal differences were found at 48 h after infection, at which time wild-type viruses produced at least two-fold more (-)ssDNA than any of the mutated viruses (Fig. 4d). The level of (-)ssDNA synthesis for mutated viruses in comparison with wild-type viruses was 60% for K65R, 32% for M184V and 17% for K65R/M184V (Fig. 4f). Moreover, these differences were further augmented in comparison with wild-type for reverse transcribed DNA detected after the first strand transfer, namely 50% for K65R, 22% for M184V, and 5.5% for K65R/M184V (Fig. 4e and f).
Viral growth kinetics in multiple rounds of infections
We were also interested in studying viral growth kinetics of wild-type and mutated viruses to assess possible correlations with results obtained in our single-round infection assays. Thus, we infected the MT-4 cell line as well as CBMCs. Viral growth kinetics were performed in MT-4 cells to assess the contribution of increased dNTP pools. Unsurprisingly, we found faster viral growth kinetics for both wild-type and mutated viruses compared to those seen in infected CBMCs. Furthermore, wild-type viruses and M184V-containing viruses reached maximum RT activity levels at 4 days after infection; in contrast, K65R-containing viruses showed a delay of 6 days after infection but the RT levels were comparable with those of wild-type viruses. As expected, K65R/M184V-containing viruses displayed the highest decrease in RT activity at that time point. (Fig. 4g).
On the other hand, infections performed in CBMCs show that peak levels of RT activity in culture supernatant were attained with wild-type viruses at 7 days after infection (Fig. 4h). In addition, M184V and K65R-containing viruses replicated very similarly to each other but to a lesser extent than wild-type viruses. Interestingly, only after the addition of freshly obtained CBMCs did K65R/M184V-containing viruses display detectable levels of RT activity in the culture supernatant at 12 days after infection.
Although viruses containing both the K65R and M184V mutations are observed rarely among clinical isolates [16,24], this combination of mutations in HIV-1 RT is not well understood with regard to resistance to relevant NRTIs and viral replicative capacity. Recently, it was shown that RTs containing the K65R, M184V and K65R/M184V mutations displayed 8.6-fold, 30-fold and 180-fold resistance to 3TC-TP, respectively, in comparison with wild-type RT and also displayed 4.4-fold, 0.4-fold and 1.7-fold resistance to TFV-DP, respectively, in comparison with wild-type RT . Moreover, pre-steady-state kinetics have shown that discrimination by K65R RT may be due to a decreased catalytic rate of incorporation (kpol) of NRTI-TP in comparison with wild-type RT, with modest effect on binding affinity (Kd). Alternatively, discrimination by M184V RT may be the result of decreased binding affinity of NRTI-TP in comparison with wild-type RT, with only a small effect on the catalytic rate of incorporation. When combined, these mutations within RT exhibit both decreased binding affinity and decreased catalytic rate of incorporation of NRTI-TP in comparison with wild-type RT. Our data and the greater degree of resistance to most NRTIs displayed by K65R/M184V RT reinforce this point .
In addition, HIV-1 viruses harboring the K65R, L74V and M184V mutations have been shown to display hypersusceptibility to ZDV due to an impaired rescue of ZDV-MP chain-terminated primers compared to wild-type RT [31,42,53,54]. Moreover, K65R RT has been shown to decrease the binding or incorporation of ZDV-TP as well as the unblocking of ZDV-MP, even at physiological concentrations of the next inhibitory nucleotide in comparison with wild-type RT . Here, we have shown that the K65R/M184V RT displayed a major reduction in ZDV-MP unblocking, suggesting that, when present together, these mutations may have an additive effect in this regard. Interestingly, both K65R and thymidine analogue mutations (TAMs) have been shown to exhibit bidirectional phenotypic and genotypic antagonism, which may explain the negative association of these mutations in genotype databases and the infrequent emergence of K65R in patients receiving ZDV [55–57].
The synthesis of (-)ssDNA has been described as a rate-limiting step in the reverse transcription reaction. Our data show that K65R- and M184V-containing RTs display slightly decreased efficiency of (-)ssDNA synthesis, as described [35,40]. We have also shown that K65R/M184V RT displays the lowest efficiency of initiation of (-)ssDNA synthesis, as reflected by accumulation of products at position +3 and +5 as well as a decrease in the total amount of tRNA3Lys extended primer even in the presence of NC. Recent data have shown that the use of NC to promote complex formation between tRNA3Lys primer and the RNA template led to qualitative differences between types of primer/template complexes formed in the presence or absence of saturating concentrations of NC .
Furthermore, RTs containing both the K65R and M184V mutations produced more (-)ssDNA when primed with the dPR DNA primer, suggesting that these enzymes have a decreased ability to use RNA primers, as has been described for L74V/M184V-containing RTs . We suggest that these mechanisms are responsible for the diminished viral replicative capacity observed in tissue culture when K65R/M184V-containing viruses are studied. However, we cannot exclude the possibility that other mechanisms, including host factors, may also play important roles in determining the decreased replicative capacity associated with these viruses. It has been recently shown that mutations that reside at the HIV-1 RT polymerase active site, such as K65R, L74V and M184V, display a high degree of negative correlation between template switching frequency and viral titers .
Clinical data support our observations. The Tonus trial demonstrated that 14 of 14 treatment-naive patients who received once-daily TDF, abacavir (ABC) and 3TC harboured the K65R/M184V mutations and had relatively low viral loads over 48 weeks of treatment . In Gilead Study 903, treatment-naive patients who developed K65R while on TDF/3TC/efavirenz (EFV) did not fully rebound to baseline HIV-1 RNA levels. Conceivably, the additional presence of the M184V mutation in some patients may have contributed to impaired viral fitness .
We now wish to study whether other drug-resistance related mutations in association with K65R or M184V will also lead to decreased efficiency of initiation of (-)ssDNA synthesis as well as decreased viral replicative capacity.
We thank Claudio Smolarz for assistance with digital artwork.
This work was performed by F.A.F. in partial fulfillment of the requirements for a PhD degree from the Faculty of Graduate Studies and Research, McGill University, Montreal, Quebec, Canada.
Sponsorship: F.A.F. is the recipient of a Canadian Institutes of Health Research (CIHR) doctoral fellowship award. This work was supported by grants from CIHR.
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