The YMDD and rtA194T Mutations Result in Decreased Replication Capacity in Wild-Type HBV as well as in HBV with Precore and Basal Core Promoter Mutations

Abstract

Background:

A recent study indicated that addition of the hepatitis B e antigen (HBeAg) precore (PC) or basal core promoter (BCP) mutations to wild-type HBV offset the reduced replication of the HBV polymerase rtA194T ±rtL180M+rtM204V mutations. rtA194T was reportedly associated with tenofovir resistance. We investigated these findings in genotype D HBV, where both PC and BCP naturally occur in vivo.

Methods:

A plasmid containing a wild-type 1.3 genome length genotype D HBV laboratory strain was used as a parent for PC, BCP, rtA194T ±rtL180M+rtM204V, rtL180M+rtM204V and rtM204I mutants. Viral replication was evaluated by Southern blot analysis of intracellular HBV core DNA following transient transfection of HepG2 cells. Drug susceptibility was evaluated by quantitative PCR of intracellular HBV DNA.

Results:

PC and BCP mutations each increased HBV DNA replication by approximately 200% over wild-type. rtA194T reduced replication by <40%, whereas rtL180M+rtM204V, rtL180M+rtA194T+rtM204V or rtM204I each reduced by >75% from their respective wild-type, PC or BCP genome backbone (P<0.05). The enhanced replication by PC or BCP offset the reduction by rtA194T; however, the other reverse transcriptase (RT) mutations in PC or BCP backbones remained signifcantly lower than wild-type (P<0.05). Regardless of the backbone, rtA194T ±rtL180M+rtM204V remained susceptible to tenofovir in vitro. rtA194T alone remained susceptible to lamivudine, while rtL180M+rtM204V and rtL180M+rtA194T+rtM204V were resistant.

Conclusions:

PC or BCP mutations increased HBV DNA replication, offset the decreased replication by rtA194T alone, but they did not fully rescue the impaired replication conferred by other RT mutations as compared with wild-type. rtA194T ±rtL180M+rtM204V did not confer tenofovir resistance.

Introduction

Chronic HBV infection exists in two phases: hepatitis B e antigen-positive (HBeAg-positive) and hepatitis B e antigen-negative (HBeAg-negative) [1]. HBeAg-negative virus can emerge in chronic hepatitis B (CHB) patients with HBeAg-positive infection who experienced HBeAg seroconversion accompanied with continued infection or reactivation of hepatitis B [2]. The two major types of HBV mutations that are associated with HBeAg-negative CHB are: the precore (PC) stop codon G1896A mutation [3] and the A1762T/G1764A basal core promoter (BCP) mutations [4]. The PC mutation causes a change in the ε stem-loop structure, resulting in a T and A Watson–Crick base pairing with T1858 at the opposite side of the stem-loop [5,6]. Because genotype A HBV has a C residue at position 1858, the A1896 PC mutation would result in the disruption of the base pairing; therefore, PC mutations very rarely occur in genotype A HBV [7,8]. BCP mutations can occur in all major HBV genotypes (A–D), but are more prevalent in genotypes A and C [7,8].

Mutations in the YMDD motif, associated with lamivudine (LAM) resistance, have been reported to result in lowered virus DNA replication [9 –11]. By contrast, BCP and possibly PC mutations each increased HBV DNA replication levels in vitro [12,13]. However, there are conflicting reports regarding the effect of PC mutations on wild-type (wt) HBV DNA replication in vitro [12 –15]. The mechanism by which PC and BCP could increase HBV DNA replication remains to be understood. Several studies pointed to an inhibitory function of HBeAg on virus DNA replication [15 –17], and point mutations that abolish proper HBeAg expression enhance virus DNA expression [13 ,–17]. The PC mutation also resides within the ε structure [6], causing a T1858:A1896 Watson–Crick base pairing, which may increase packaging efficiency of polymerase– pregenomic RNA complex by stabilizing the stem-loop, leading to increased virus replication [5].

The rtA194T in combination with the rtL180M+ rtM204V LAM resistance mutation was identified in two HIV/HBV-coinfected patients treated with a combination of drugs including LAM and tenofovir disoproxil fumarate [18], and rtA194T alone or in combination with rtL180M+rtM204V was shown to cause reduced susceptibility to tenofovir (TFV) in vitro [18]. However, the in vitro resistance to TFV reported in these studies could not be reproduced [19]. More recently, it was shown that the rtA194T mutation alone or in combination with LAM resistance rtL180M+rtM204V mutations had reduced virus DNA replication and that addition of PC and BCP mutations to virus containing these mutations could restore replication to levels similar to that of the wt [20]. Based on the in vitro results, the authors speculated that patients with HBeAg-negative chronic HBV infection may be at particular risk for developing drug resistance to TFV [20]. However, one important fact is that PC mutation rarely exist in HBV genotype A [7,8], the genotype that the previous study was conducted on [20]. Because of the contradicting data surrounding rtA194T results, and the inconsistencies of the results regarding the effect of PC and BCP on wt as well as the LAM resistance mutations, we undertook the current investigation on an HBV genotype D HBV strain for which both PC and BCP mutations exist naturally in vivo to analyse the effects of these mutations on HBV DNA replication as well as resistance to TFV and LAM. Attempts were also made toward understanding the mechanism by which the PC mutation may increase HBV DNA replication.

Methods

Cells and compounds

The HepG2 cell line (American Type Culture Collection [ATCC] number HB-8065) used for anti-HBV activity assays was purchased from ATCC (Manassas, VA, USA) and was maintained in DMEM-F12 media supplemented with 10% fetal bovine serum and penicillin/streptomycin solution (Invitrogen Corporation, Carlsbad, CA, USA). TFV and LAM were synthesized by Gilead Sciences, Inc. (Foster City, CA, USA).

Construction of plasmids expressing 1.3-unit length HBV genome

The plasmid pCMVHBV containing an HBV genotype D (subtype ayw) genome was used to generate all mutant viruses [21]. A plasmid vector containing 1.3-unit length HBV genome was first created by partial digestion of pCMVHBV with PsiI to obtain the linearized plasmid. The linearized pCMVHBV was then digested with SfiI to obtain the ∼6 kbp fragment containing the majority of pCMVHBV less the CMV promoter. In parallel, a ∼1 kbp fragment containing ∼0.3-unit length HBV genome from pCMVHBV was obtained by digestion with PsiI and BstZ17I. This fragment was then ligated with the PsiI to SfiI fragment after the SfiI site was blunt-ended with T4 polymerase treatment. The resulting plasmid containing 1.3-unit length HBV genome (p1.3HBV) was verified by sequencing. The 1.3-unit length HBV fragment was obtained by digestion of p1.3HBV with BsrFI (blunt-ended afterwards) and NotI. The 1.3-unit length HBV fragment was then ligated into pFastBac vector (Invitrogen) linearized with SnaBI and NotI to obtain pBac-1.3HBV (p1.3Wt). The plasmid p1.3Wt was subsequently used as a parent to generate the mutant viruses described in this study.

The p1.3PC containing the stop codon HBeAg mutation was created by making a G to A mutation at genome position 1896 at the 1-unit length as well as the 0.3-unit length locations using primer overlapping extension mutagenesis [22] onto p1.3Wt. Using the same approach, the LAM resistance mutations rtL180M+rtM204V were created on p1.3Wt by C to A and A to G mutations at genome positions 667 and 739, respectively. The rtM204I mutation was generated with a G to T mutation at genome position 741. The rtL180M+rtM204V and rtM204I mutations were subsequently transferred to p1.3PC genome background by cloning the SanDI to BstZ17I fragment (containing rtL180M+rtM204V or rtM204I) into p1.3PC. The BCP mutations were created by making an A to T mutation at genome position 1762 and G to A at 1764 of the 1-unit length as well as the 0.3-unit length locations using Quikchange kit (Agilent, Santa Clara, CA, USA) onto p1.3Wt as well as p1.3Wt-rtL180M+rtM204V and p1.3Wt-rtM204I, respectively. The rtA194T as well as rtL180M+rtA194T+rtM204V mutations were also created using the Quikchange kit to introduce these mutations into p1.3Wt, p1.3PC and p1.3BCP, respectively. Finally, p1.3Wt-T1858C mutation was generated using the contract service by Genscript (Piscataway, NJ, USA). All constructs were verified by sequencing the entire HBV sequences in each plasmid to confirm the intended mutation and that no other mutations existed. Constructs are shown in Figure 1.

Figure 1.

Schematic illustration of the 1.3-genome length HBV constructs

Southern blot analysis to evaluate intracellular replicated HBV core DNA

HepG2 cells were seeded into six-well culture plates at 7.5×10⁵ cells/well. At 16 h post-seeding, cells were transfected with 1 μg of plasmid DNA/well using Fugene 6 transfection reagent (Roche, Indianapolis, IN, USA). Transfection efficiency was assessed by cotransfection with pRLuc (Promega, Fitchburg, WI, USA), and the luciferase activity evaluated using a luciferase kit (Promega). The transfected cells were incubated at 5% CO₂ for 7 days with media change every 2–3 days. At day 7, supernatants were harvested and plates were stored at −20°C. Intracellular HBV core DNA extraction was performed using the method of Yamamoto, et al. [23], scaled down to 0.3 ml lysis buffer per well. The revised protocol also utilized 1.25 mg/ml of proteinase K instead of pronase as previously described [23]. Viral DNAs representing one-half of the cell culture well were separated on 1.5% agarose gels and transferred to nitrocellulose membranes using standard Southern blotting procedures. Membranes were hybridized with a ³²P-labeled HBV probe, and viral DNA was quantified using a BioRad Phosphorimager (Hercules, CA, USA). A linearized HBV genome standard at an amount of 25 pg/well was included in each gel, and the relative amount of intracellular HBV replicative intermediates was calculated for each sample based on the standard HBV DNA, and normalized to transfection efficiencies. A plasmid, RTAN, containing the entire HBV genome except reverse transcriptase (RT) [24], was included in each experiment as a negative control. Assays were performed a minimum of 3× for each of the constructs.

HBeAg assay

The DiaSorin ETI-EBK HBeAg plus kit (DiaSorin, Saluggia, Italy) was used to quantify HBeAg levels in cell culture supernatants. A serial dilution of the HBeAg-positive control was used to generate a standard curve. The manufacturer's experimental protocol was followed. The absorbance was read at 450 nm with 630 nm as reference wavelength. The relative HBeAg level was evaluated by normalizing the absorbance readings of each sample to that of supernatant of p1.3Wt in each experiment.

In vitro drug susceptibility

Freshly confluent HepG2 cells were trypsinized and resuspended in culture media. Plasmid DNA and FuGENE®6 reagent (Roche) were mixed at 100 ng DNA to 0.3 μl Fugene6/well and diluted into a total volume of 10 μl in serum-free media. The DNA and Fugene6 mix was then mixed wit resuspended HepG2 cells at 5×10⁴ cells/well ratio and aliquoted into a 96-well culture plate. The cells were then fed fresh medium containing serial dilutions of TFV at 200, 100, 50, 25, 12.5, 6.25, 3.1, 1.6, 0.8, 0.4 and 0 μM, or LAM at 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 and 0 μM. There were four replicate wells at each drug concentration.

Cells were treated with fresh media containing drug every 2-3 days for 1 week, after which the drug media was discarded and the cell monolayer was immediately used for extraction of intracellular HBV DNA replicative intermediates, or stored at −20°C for later use. Extraction of HBV replicative intermediate was performed by lysing cell monolayers at 4°C for 5 min with 100 μl/well of 0.33% NP-40. Cell lysates were then centrifuged for 5 min at 1,500 rpm. 50 μl/well of each supernatant was transferred to a fresh 96-well plate containing 6.5 μl/well of 10X Turbo DNase buffer (Applied Biosystems). 10 μl DNase mix was then added to each well (1X Turbo DNase buffer and 1U Turbo DNase enzyme) and the 96-well plate was incubated at 37°C for 30 min. After 30 min, 6 μl of DNase inactivation reagent was added to each well and incubated for 2 min. The DNase-treated 96-well plate(s) were centrifuged for 5 min at 3,000 rpm. 35 μl/well of DNase-treated supernatant was added to a new 96-well plate containing 65 μl of QuickExtract Solution (Epicentre Biotechnologies, Madison, WI, USA) and then incubated at 65°C for 6 min followed by 98°C for 2 min. Following extraction, the intracellular HBV DNA was quantified by real-time PCR. Each PCR well contained the following: 10 μl of the final cell lysate, 0.9 μM primer HBVF (5′-CCG TCT GTG CCT TCT CAT CTG-3′), 0.9 μM primer HBVR (5′-AGT CCA AGA GTY CTC TTA TGY AAG ACC TT-3′), 0.1 μM HBV probe (5′-6FAM CCG TGT GCA CTT CGC TTC ACC TCT GC BHQ1–3′), 0.9 μM primer AMPF (5′-GTA TGC GGC GAC CGA GTT-3′), 0.9 μM primer AMPR (5′-TGC TAT GTG GCG CGG TAT T-3′), 0.1 μM AMP probe (5′-VIC TCT TGC CCG GCG TCA MGBNFQ-3′) and Taqman® Universal PCR Master Mix (Applied Biosystems) in a final volume of 50 μl. The PCR was conducted using the LightCycler 480 (Roche) with the following sequence of conditions: incubation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. HBV DNA copy numbers were calculated based on a serial dilution of standard HBV DNA included in each PCR run, and the average reading from four replicate wells at each drug concentration was used to calculate 50% effective concentration (EC₅₀) values. The assay had a variation of <2-fold. The susceptibility assay for each construct was independently tested at least 3×.

Data analysis

The average HBV DNA level of four replicate wells for each drug concentration was normalized to that of the no drug control, and regression analyses of antiviral data were performed with TableCurve 2D software (Systat Software, Chicago, IL, USA) and best-fit equations were used to calculate EC₅₀ values.

Results

rtA194T and LAM resistance mutations decreased HBV DNA replication in wild-type as well as in the PC or BCP genome backbones

All of the constructs generated by site-directed mutagenesis led to HBV DNA replication (Figure 2A); the average levels of replicated HBV DNA from at least three independent experiments are summarized in Figure 2B. For RT mutations on the wt genome backbone, the rtA194T mutation resulted in an approximately 40% reduction in virus DNA replication, rtL180M+rtM204V and rtM204I mutations each caused >75% reductions, and rtL180M+rtA194T+rtM204V further reduced the replication level. All the differences were statistically significant (P<0.05, Student's t-test; Figure 2B). Addition of the PC or BCP mutations to the wt genome backbone each led to approximately a twofold increase in virus DNA replication (P<0.05, Student's t-test). As a result, all RT mutations created in the PC or BCP genome backbone appeared to have higher replication levels than each of their corresponding mutations on the wt backbone. However, only the increased replication of PC-rtL180M+rtM204V, PC-rtA194T and PC-rtL180M+rtA194T+rtM204V reached statistical significance when compared with the same RT mutations on the wt backbone (P<0.05, Student's t-test). In addition, the rtA194T mutation alone in the PC or BCP backbones had replication levels comparable to that of wt, whereas the DNA replication of rtL180M+rtM204V, rtM204I and rtL180M+rtA194T+rtM204V in the PC or BCP genome backbone remained significantly lower than that of wt (Figure 2B).

Figure 2.

HBV DNA replication of wt, PC and BCP genome backbone containing wt RT and rtL180M+rtM204V, rtA194T, rtL180M+rtA194T+rtM204V or rtM204I mutations

The DNA replication levels of each RT mutation were compared with that of their respective genome backbone and the results are summarized in Figure 3. As indicated, the pattern of the reduction in replication of each RT mutation was very similar when compared with each of their respective genome backbones. Replication of each RT mutation was significantly lower than that of its respective genome backbone, except for BCP-rtA194T, which could be attributed to larger variations among the experimental data.

Figure 3.

Relative levels of HBV DNA replication compared with each respective genome backbone

PC mutation abrogated HBeAg expression, while BCP mutations decreased HBeAg levels in culture supernatant

PC and BCP mutations are known to be associated with HBeAg negativity [3,4]. As shown in Figure 4, constructs with wt HBeAg genome backbone had similar levels of supernatant HBeAg, while constructs with PC genome backbone did not have detectable levels of HBeAg in the supernatant. This result is expected since the PC mutation causes a stop codon at amino acid 28 of HBeAg, thus truncating HBeAg and blocking its secretion [3]. By contrast, BCP mutations only had about 50% reduction in supernatant HBeAg levels. These results are in agreement with previous in vitro data [20].

Figure 4.

Relative HBeAg levels in supernatants of transfected cells

The T1858C mutation did not lead to increased HBV DNA replication

Towards an understanding of the mechanism by which the PC mutation enhanced virus DNA replication, we created the T1858C mutation (Figure 5A) on the wt genome backbone. This mutation would result in a C:G base pairing with G1896, thus stabilizing the stem-loop structure as would the G1896A PC mutation. T1858C will not cause an amino acid change, since CCT and CCC both code for proline at position 15 of the HBeAg. As indicated in Figure 5B, the level of virus DNA replication of T1858C was comparable to that of wt, while PC replicated about twofold greater than wt. Therefore, merely stabilizing the T1858:G1896 base pairing in the ε stem-loop structure did not lead to the increased HBV DNA replication as seen with the G1896A PC mutation.

Figure 5.

The T1858C mutation and its effect on virus DNA replication

rtA194T alone or in combination with rtL180M+rtM204V on wild-type as well as PC and BCP genome background remained susceptible to TFV Constructs containing the rtA194T, rtL180M+ rtM204V and rtL180M+rtA194T+rtM204V in the wt, PC and BCP genome backbone were tested for their susceptibilities to TFV. In addition, pHY92, which expresses a wt laboratory HBV strain [25], and pHY-rtA181V+rtN236T, which expresses the adefovir-associated mutations engineered into pHY92 and which would cause partial cross resistance to TFV [26], were tested in parallel. The EC₅₀ values are summarized in Table 1. None of the tested constructs had EC₅₀ value fold changes >2-fold, which is the upper limit of our assay variation for detection of resistance. In comparison, rtA181V+rtN236T had an average fold increase in TFV susceptibility of 4.2-fold over that of the pHY92 wt parent. Therefore, rtA194T, rtL180M+rtM204V and rtL180M+rtA194T+rtM204V in wt, PC and BCP genome backbones were fully susceptible to TFV.

Table 1.

Susceptibility to tenofovir and lamivudine

Sample	Tenofovir EC₅₀ ±SD, μM	Fold over backbone	Lamivudine EC₅₀ ±SD, μM	Fold over backbone
pHY92	11.6 ±3.5	–	ND	–
rtA181V-rtN236T	48.8 ±13.8	4.2	ND	–
wt	12.5 ±3.8	1.0	3.3 ±1.6	1.0
wt-rtL180M+rtM204V	15.8 ±5.3	1.3	>100	>30
wt-rtA194T	9.5 ±1.4	0.8	4.0 ±1.4	1.2
wt-rtL180M+rtA194Trt+M204V	14.9 ±4.2	1.5	>100	>25
PC	10.4 ±2.4	1.0	1.9 ±0.7	1.0
PC-rtL180M+rtM204V	14.1 ±3.6	1.4	>100	>50
PC-rtA194T	7.2 ±1.2	0.7	2.7 ±0.6	1.4
PC-rtL180M+rtA194T+rtM204V	8.7 ±3.4	0.8	>100	>37
BCP	8.9 ±3.7	1.0	2.6 ±1.0	1.0
BCP-rtL180M+rtM204V	13.9 ±9.2	1.6	>100	>38
BCP-rtA194T	11.5 ±4.0	1.3	2.6 ±1.0	1.0
BCP-rtL180M+rtA194T+rtM204V	16.2 ±8.7	1.8	>100	>38

BCP, basal core promoter; EC₅₀, 50% effective concentration; ND, not done; PC, precore; wt, wild-type.

The constructs were also tested against LAM; rtA194T alone, in wt, PC or BCP genome backbones were susceptible to LAM, while rtL180M+rtM204V and rtL180M+rtA194T+rtM204V were resistant to LAM (Table 1).

Discussion

In this study, we aimed to analyse the effect of the PC (G1896A) or BCP (A1762T/G1764A) mutations alone and in combination with the rtL180M+rtM204V or rtM204I LAM resistance mutations, or the rtA194T with or without rtL180M+rtM204V mutations on HBV DNA replication capacity as well as their sensitivities to TFV and LAM. Repeated testing by our laboratory showed that rtA194T mutation (with or without rtL180M+rtM204V), regardless of wt, PC or BCP genome background remained susceptible to TFV (Table 1), and that rtA194T alone is susceptible to LAM (Table 1). This is in contrast to the previous study that demonstrated 5- to 7-fold resistance to TFV with these constructs [20]. It is possible that differences in methodologies or the difference in HBV genotype used could account for these differences. In our assays, we used a wide range of drug concentrations and twofold serial dilutions so that a finer difference in drug susceptibility could be captured. In addition, we used a sensitive real-time PCR assay that is able to quantify HBV DNA with a linear range between 200 and 10⁸ copies [27]. By contrast, previous results were generated using larger serial dilutions and detection was by a less sensitive dot blot assay. Given that the RT mutations evaluated herein resulted in a significantly reduced replication signal, a less sensitive assay is likely to lead to greater variability. For the confounding factor of difference in viral genotype used in the assays, the current results were obtained using an HBV genotype D laboratory strain, and could not be extended to genotype A strain used in the previous study [20]. The effect of rtA194T, on wt, PC or BCP mutation background, on susceptibility to TFV in vitro in genotype A context remains to be further examined. However, the rtA194T mutation created on a wt genotype A genome background, the same genotype as mentioned above [18,20], was also shown to be susceptible to TFV by our laboratory using Southern blot analysis [19]. Furthermore, our in vitro results are consistent with the clinical results which demonstrated full virological responses to tenofovir disoproxil fumarate among a group of LAM-experienced patients who harboured the rtA194T mutation [28]. rtA194T appears to be a naturally occurring polymorphic substitution that is rarely seen. A recent study using pyrosequencing technology revealed a low level (<1%) prevalence of rtA194T in both treatment-naive and patients previously treated with nucleoside analogues [29]. Our results may also explain why the rtA194T mutation was not observed to develop in various tenofovir disoproxil fumarate clinical trials after >3 years of continuous treatment [30,31].

The results from this study also demonstrated the enhanced HBV DNA replication induced by BCP or PC mutations as well as the decreased replication caused by the rtL180M+rtM204V and rtM204I LAM resistance mutations are consistent with several previous reports [9 ,,–15,32,33]. Furthermore, the co-existence of PC or BCP mutations enhanced the replication levels of the tested LAM resistance mutations, with or without the rtA194T, and are consistent with previously published data [12,13,20]. However, in contrast to a previous study [20], even with the enhancement effect of PC or BCP mutations, the rtL180M+rtM204V, rtL180M+rtA194T+rtM204V and rtM204I mutants had significantly lower replication rates as compared with wt. Of note, PC mutations exist frequently among patients infected with genotype D HBV but rarely occur in genotype A [7,8]. As such, in vitro results of PC mutations on genotype A [20] would have little clinical relevance.

Our results also indicated, when each of the RT mutations was compared with their genome background respectively, the extent of the effect by each RT mutations on virus DNA replication was remarkably similar. For example, rtA194T consistently led to ∼40% reduction in virus DNA replication from each respective wt, PC or BCP mutation genome background, while rtL180M+rtA194T+rtM204V further reduced replication levels. The mechanism by which PC and BCP mutations impact HBV DNA replication remains to be understood. Our results showed that PC mutation abrogated HBeAg expression, while BCP mutations significantly reduced HBeAg levels in culture supernatant, consistent with previous reports [32,33]. The PC mutation also resides within the ε structure [6], causing a T1858:A1896 Watson-Crick base pairing, possibly leading to a more stable ε structure and increased pregenomic RNA packaging and DNA replication [5]. However, the T1858C mutation, which would also stabilize the ε stem-loop replicated at the same level as wt did not appear to enhance HBV DNA replication. The BCP mutations may affect virus DNA replication through other mechanisms, such as altering transcription factor binding to the BCP promoter, mutations in the overlapping HBx protein, or changes in the ratio of PC to pregenomic RNA [32 –35]. Further research is needed to reveal the mechanism of the observed PC or BCP effects. The extent to which these in vitro findings have any clinical impact needs further evaluation. Interestingly, while PC or BCP mutations lead to increased viral replication in vitro, patients with HBeAg-negative disease who presumably harbour these mutations tend to have a lower viral load than HBeAg-positive patients [2].

In summary, we found that PC or BCP mutations each led to enhanced HBV DNA replication in vitro. This enhancement can partly offset the decreased replication effect by various RT mutations including the LAM resistance mutations rtL180M+rtM204V, rtM204I, as well as rtA194T with or without rtL180M+rtM204V. All these RT mutations remained susceptible to TFV regardless of wt, PC or BCP mutation genome background.

Footnotes

Acknowledgements

We thank Dr Michael Miller for reviewing this manuscript and our colleagues at Department of Clinical Virology of Gilead Sciences, Inc. for their scientific discussion and critique of this work.

YZ, MC and KB-E were all employees of Gilead Sciences, Inc. at the time this work was conducted.

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