Compensation by the E138K Mutation in HIV-1 Reverse Transcriptase for Deficits in Viral Replication Capacity and Enzyme Processivity Associated with the M184I/V Mutations

0022-538X/11/$12.00

doi:10.1128/JVI.05584-11

Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Compensation by the E138K Mutation in HIV-1 Reverse Transcriptase

for Deficits in Viral Replication Capacity and Enzyme Processivity

Associated with the M184I/V Mutations

䌤

Hong-Tao Xu,

Eugene L. Asahchop,

Maureen Oliveira,

Peter K. Quashie,

Yudong Quan,

Bluma G. Brenner,

and Mark A. Wainberg

*

McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada,

and Departments of Medicine

and Microbiology and Immunology,

McGill University, Montreal, Quebec, Canada

Received 1 July 2011/Accepted 8 August 2011

Recently, several phase 3 clinical trials (ECHO and THRIVE) showed that E138K and M184I were the most

frequent mutations to emerge in patients who failed therapy with rilpivirine (RPV) together with two

nucle-os(t)ide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TDF). To investigate the basis for

the copresence of E138K and M184I, we generated recombinant mutated and wild-type (WT) reverse

trans-criptase (RT) enzymes and HIV-1

infectious clones. Drug susceptibilities were determined in cord blood

mononuclear cells (CBMCs). Structural modeling was performed to analyze any impact on deoxynucleoside

triphosphate (dNTP) binding. The results of phenotyping showed that viruses containing both the E138K and

M184V mutations were more resistant to each of FTC, 3TC, and ETR than viruses containing E138K and

M184I. Viruses with E138K displayed only modest resistance to ETR, little resistance to efavirenz (EFV),

and no resistance to either FTC or 3TC. E138K restored viral replication capacity (RC) in the presence of

M184I/V, and this was confirmed in cell-free RT processivity assays. RT enzymes containing E138K, E138K/

184I, or E138K/184V exhibited higher processivity than WT RT at low dNTP concentrations. Steady-state

kinetic analysis demonstrated that the E138K mutation resulted in decreased

K

s for dNTPs. In contrast,

M184I/V resulted in an increased

K

for dNTPs compared to those for WT RT. These results indicate that the

E138K mutation compensates for both the deficit in dNTP usage and impairment in replication capacity by

M184I/V. Structural modeling shows that the addition of E138K to M184I/V promotes tighter dNTP binding.

The reverse transcriptase (RT) of human immunodeficiency

virus type 1 (HIV-1) is a multifunctional enzyme possessing

both RNA- and DNA-dependent DNA polymerase (RDDP

and DDDP, respectively) activities, as well as an RNase H

activity (12). Due to its crucial role in viral replication, HIV-1

RT is an important target for anti-HIV drugs that currently

include nucleoside reverse transcriptase inhibitors (NRTIs),

i.e., zidovudine (AZT or ZDV), didanosine (ddI), stavudine

(d4T), zalcitabine(ddC), lamivudine(3TC), emtricitabine (FTC),

and abacavir (ABC) and a nucleotide reverse transcriptase

inhibitor, tenofovir disoproxil fumarate (TDF), as well as the

nonnucleoside reverse transcriptase inhibitors (NNRTIs)

nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), and

etravirine (ETR). Both NRTIs and NNRTIs are key

compo-nents of highly active antiretroviral therapy (HAART), which

has led to significant declines in HIV-associated morbidity and

mortality (23, 33). However, the development of HIV drug

resistance is a formidable obstacle to the long-term success of

antiretroviral treatment (36, 40), and resistance mutations

have been described for all antiretroviral drugs currently in use

(21).

The rapid replication rate of HIV-1 and the error-prone

nature of its RT drive the development of drug resistance (38).

Resistance mutations arise prior to therapy due to errors in

HIV-1 replication and also can be selected during viral

repli-cation in the presence of incompletely suppressive drug

regi-mens. In the case of NRTIs and NNRTIs, drug resistance can

be due to either single mutations or the accumulation of

mu-tations specific for each individual drug in the HIV-1 RT

genes. First-generation NNRTIs have a low genetic barrier for

resistance, as only a single mutation, such as K103N, is

suffi-cient to confer diminished susceptibility. The same is true for

certain NRTIs, since high-level resistance to FTC and 3TC can

be conferred by the M184V and M184I mutations (39).

Etravirine (ETR) and rilpivirine (RPV) are

second-genera-tion NNRTIs. ETR is a component of therapy for

treatment-experienced patients, while RPV was recently approved by the

Food and Drug Administration for use in drug-naive patients.

ETR is active against HIV-1 containing RT mutations that

confer resistance to the first-generation NNRTIs (8). ETR also

has a high genetic barrier for resistance, requiring the

accu-mulation of several NNRTI-associated mutations for

high-level resistance to become manifest (4). The DUET clinical

trials identified 17 resistance-associated mutations (RAMs),

including V90I, A98G, L100I, K101E/H/P, V106I, E138A,

V179D/F/T, Y181C/I/V, G190A/S, and M230L, that are

asso-ciated with diminished susceptibility to ETR (16, 27, 37). Cell

culture selection experiments with ETR showed that E138K

was the first mutation to emerge, and it conferred low-level

resistance to ETR; E138K also was found to result in lower

viral replication capacity (3). The International AIDS

Society-* Corresponding author. Mailing address: McGill AIDS Centre,

Lady Davis Institute for Medical Research, Jewish General Hospital,

3755 Co

ˆte-Ste-Catherine Rd., Montreal, Quebec H3T 1E2, Canada.

Phone: (514) 340-8260. Fax: (514) 340-7537. E-mail: mark.wainberg

@mcgill.ca.

䌤

Published ahead of print on 17 August 2011.

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USA (IAS-USA) drug resistance mutation list includes both

E138K and E138G for ETR resistance (17). Recently, two

phase 3 clinical trials (ECHO and THRIVE) on the use of

RPV/TDF/FTC in drug-naïve patients showed that E138K

confers cross-resistance to RPV and ETR and that the

com-bination of the E138K/M184I mutations was common in

pa-tients at treatment failure (20). These results indicate that

E138K is a signature mutation of relevance for the

second-generation NNRTIs ETR and RPV.

In this study, we employed both enzymatic and cell-based

assays to assess the impact of the E138K mutation in

combi-nation with M184I or M184V on enzyme processivity, viral

replication capacity, and phenotypic drug susceptibility. We

also have assessed why E138K/M184I was favored over E138K/

M184V in clinical trials with RPV/FTC/TDF.

MATERIALS AND METHODS

Chemicals, cells, and nucleic acids.Etravirine (ETR) was a gift of Tibotec (Titusville, NJ). Emtricitabine (FTC) was kindly provided by Gilead Sciences (Foster City, California). Lamivudine (3TC) was a gift of Glaxo-SmithKline (Greenford, United Kingdom). Efavirenz (EFV) was obtained from Bristol-Myers Squibb (Princeton, NJ).

Cord blood mononuclear cells (CBMCs) were obtained through the Depart-ment of Obstetrics, Jewish General Hospital, Montreal, Canada. The HEK293T cell line was obtained from the American Type Culture Collection. The following reagents and cells were obtained through the NIH AIDS Research and Refer-ence Reagent Program: the infectious molecular clone pNL4-3 was from Mal-colm Martin, and the TZM-bl (JC53-bl) cells were from John C. Kappes, Xiaoyun Wu, and Tranzyme Inc.

The pNL4.3PFB proviral DNA is a generous gift from Tomozumi Imamichi, National Institutes of Health, Bethesda, MD. pRT6H-PROT is a generous gift from Stuart F. J. Le Grice, National Institutes of Health, Bethesda, MD.

A 497-nucleotide (nt) HIV-1 PBS RNA template spanning the 5⬘untranslated

region (UTR) to the primer binding site (PBS) wasin vitrotranscribed from ACC

I-linearized pHIV-PBS DNA (2) by using a T7-MEGA shortscript kit (Ambion, Austin, TX) as described previously (42). The oligonucleotides used in this study were synthesized by Integrated DNA Technologies Inc. (Coralville, IA) and

purified by 6% polyacrylamide–7 M urea gel electrophoresis. For 5⬘-end labeling

of oligonucleotides with [␥-32

P]ATP, the Ambion KinaseMax kit was used, fol-lowed by purification through Ambion NucAway spin columns according to protocols provided by the supplier (Applied Biosystems, Streetsville, Canada). The names and sequences of the oligonucleotides used in this study are the

following: NLBalF, 5⬘-TGGCCATTGACAGAAGAAAAAATAAAAGCA-3⬘;

NLPFLMR, 5⬘-ATAATACACTCCATGTACtGGTTC-3⬘; and D25, 5⬘-GGAT

TAACTGCGAATCGTTCTAGCT-3⬘.

Site-directed mutagenesis and preparation of virus stocks. To construct

HIV-1 RT expression plasmids and HIV-1NL4-3variants harboring desired

mu-tations in the RT gene, site-directed mutagenesis reactions were carried out using a QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) on HIV-1 RT expression plasmid pbRT6H-PROT (42), which contains the

RT (p66) coding region of HIV-1NL4-3(GenBank accession number AF324493)

with a C-terminal His tag and was constructed from pRT6H-PROT (32). To

make recombinant HIV-1NL4-3viruses containing the desired RT mutations, we

first amplified fragments spanning RT amino acids (aa) 25 to 314 from the pbRT6H-PROT variants by PCR using primers NLBalF and NLPFLMR. After digestion with restriction enzymes MscI and PflMI, the resultant 871-bp mutant DNAs were used to replace the corresponding 871-bp fragment of pNL4.3PFB proviral DNA. DNA sequencing was performed to verify the absence of spurious

mutations and the presence of any desired mutation. Recombinant HIV-1NL4-3

wild-type and mutant viruses were generated by the transfection of the corre-sponding proviral plasmid DNAs into HEK293T cells using Lipofectamine 2000 (Invitrogen, Burlington, Canada) according to the manufacturer’s instructions. Viral supernatants were harvested at 48 h posttransfection, centrifuged for 5 min

at 800⫻gto remove cellular debris, filtered through a 0.45-␮m-pore size filter,

aliquoted, and stored at⫺80°C. Levels of p24 in viral supernatant were measured

by a Perkin-Elmer HIV-1 p24 antigen enzyme-linked immunosorbent assay (ELISA) kit. Virion-associated RT activity was measured as described previously

(12) with 50␮l of RT reaction mixture containing 10␮l of culture supernatants;

0.5 U/ml of poly(rA)/p(dT)12-18template/primer (T/P) (Midland Certified

Re-agent Company, Midland, TX) in 50 mM Tris-HCl, pH 7.8; 75 mMKCl; 5 mM

dithiothreitol; 5 mM MgCl2; 0.05% Triton X-100; 2% ethylene glycol; 0.3 mM

reduced glutathione; and 5␮Ci of [3_{H]dTTP (70 to 80 Ci/mmol; 2.5 mCi/ml).}

Following a 240-min incubation at 37°C, the reaction mixture was quenched by adding 0.2 ml of 10% cold trichloroacetic acid (TCA) and 20 mM sodium pyrophosphate and incubated for at least 30 min on ice. The precipitated prod-ucts were filtered onto Millipore 96-well MutiScreen HTS FC filter plates

(MSFCN6B) and sequentially washed with 200␮l of 10% TCA and 150␮l of

95% ethanol. The radioactivity of incorporated products was analyzed by liquid scintillation spectrometry using a Perkin-Elmer 1450 MicroBetaTriLux micro-plate scintillation and luminescence counter.

Replication capacity in TZM-bl cells.The relative replicative capacities of

recombinant wild-type HIV-1NL4-3and E138K, M184I, M184V, E138K/M184I,

and E138K/M184V mutants were evaluated in a noncompetitive short-term infectivity assay using TZM-bl cells as previously described (3, 43). Twenty thousand cells per well were added in triplicate into a 96-well culture plate in 100

␮l of Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) supplemented

with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin, and 1%L

-glu-tamine (Invitrogen). Viral stocks for both wild-type and mutant viruses were normalized by p24, and recombinant viruses were serially diluted 2-fold from

viral stock suspensions. After 4 h, 50␮l of DMEM was removed from the wells

and replaced by 50␮l of virus dilution; a control well did not contain virus. Virus

and cells were cocultured for 48 h, after which 100␮l of Bright-Glo reagent was

added and luciferase activity measured in a luminometer as described in the manufacturer’s instructions (Promega). The viral replication level was expressed as a percentage of relative light units (RLU) with reference to wild-type virus.

Phenotypic drug susceptibility assays.Phenotypic susceptibility analysis of the RT inhibitors ETR, FTC, EFV, and 3TC was performed with recombinant

HIV-1NL4-3clones in a cell-basedin vitroassay as described previously (3).

Briefly, CBMCs were infected for 2 h with either wild-type recombinant virus or mutants, washed with cold Dulbecco’s phosphate-buffered saline to remove un-bound virus, and plated onto 96-well plates in the presence of each RT inhibitor. After 3 days in culture, the culture wells were refreshed with media containing the corresponding drug dilutions. After 7 days, the culture supernatants were collected and analyzed for RT activity to determine the dose-response curve. The

EC50(50% effective concentration) was calculated using the program GraphPad

Prism (GraphPad Software, San Diego, CA).

Recombinant reverse transcriptase expression and purification.Recombinant RTs in heterodimeric forms were expressed from plasmid pbRT6H-PROT and purified as described previously, with minor modifications (42). In brief, RT

expression inEscherichia coliM15 (pREP4) (Qiagen, Mississauga, Canada) was

induced with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) at room

tem-perature. The pelleted bacteria were lysed under native conditions with Bug-Buster protein extraction reagent containing benzonase (Novagen, Madison, WI) according to the manufacturer’s instructions. After clarification by high-speed centrifugation, the clear supernatant was subjected to the batch method of nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography (QIAexpres-sionist) (Qiagen). All buffers contained Complete protease inhibitor cocktail (Roche). Histidine-tagged RT was eluted with an imidazole gradient. RT-con-taining fractions were pooled, passed through DEAE-Sepharose (GE Health-care), and further purified using SP-Sepharose (GE Healthcare, Mississauga, Canada). Fractions containing purified RT were pooled, dialyzed against storage buffer (50 mM Tris-HCl [pH 7.8], 50 mM NaCl, and 50% glycerol), and con-centrated to 4 to 8 mg/ml with Centricon Plus-20 MWCO30 kDa (Millipore).

Aliquots of proteins were stored at⫺80°C. Protein concentration was measured

by a Bradford protein assay kit (Bio-Rad Laboratories), and the purity of the recombinant RT preparations was verified by SDS-PAGE. The polymerase ac-tivity of each recombinant RT preparation was evaluated in duplicate as de-scribed previously (29) using various amounts of RTs and a synthetic

homopoly-meric poly(rA)/(dT)12-18T/P (Midland Certified Reagent Company).

Processivity assays.The processivity of recombinant RT proteins was analyzed

under various dNTP concentrations (0.5 and 200␮M) using the heteropolymeric

RNA template in the presence of a heparin enzyme trap to ensure a single processive cycle, i.e., a single round of binding and of primer extension and dissociation. The T/P was prepared by annealing the 497-nt HIV PBS RNA with

the32_{P-5⬘end-labeled 25-nt DNA primer D25 at a molar ratio of 1:1, denatured}

at 85°C for 5 min, and then slowly cooled to 55°C for 8 min and 37°C for 5 min to allow for the specific annealing of primer to the template. Reactions were performed essentially as described previously (42). RT enzymes with equal amounts of activity and 40 nM T/P were preincubated for 5 min at 37°C in a

buffer containing 50 mMTris-HCl (pH 7.8), 50 mM NaCl, and 6 mM MgCl2.

Reactions were initiated by the addition of dNTPs and heparin trap (final concentration, 3.2 mg/ml) and were incubated at 37°C for 30 min. Two volumes

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of stop solution (90% formamide, 10 mM EDTA, and 0.1% each of xylene cyanol and bromophenol blue) were added to stop the reaction. Each reaction was run in duplicate. Reaction products were denatured by heating at 95°C and analyzed using 6% denaturing polyacrylamide gel electrophoresis and phosphorimaging. The effectiveness of the heparin trap to limit polymerization on the RNA tem-plate was verified in control reactions in which the heparin trap was preincubated with substrate before the addition of RT enzymes and dTTP.

RNA-dependent DNA polymerase activity assay.The 497-nt RNA and 5⬘-end

32

P-labeled D25 primers described above were used to assess the polymerization rate of recombinant RT enzymes in time course experiments. The final reaction mixtures contained 20 nM T/P, 400 nM RT enzyme, 50 mM Tris-HCl (pH 7.8),

and 50 mM NaCl. Reactions were initiated by adding 6 mM MgCl2and dNTPs

at various concentrations as described above and sampled at variable time points, i.e., 30 s, 60 s, 5 min, and 20 min, respectively, and mixed with two volumes of stop solution. Reaction products were separated by 6% denaturing polyacryl-amide gel electrophoresis and analyzed as described above.

Steady-state kinetic analysis.Kinetic studies were carried out as described

previously (30) using homopolymeric poly(rA)/p(dT)12-18and complementary

dTTP as the nucleotide substrate, with modifications. The reaction mixture (10

␮l) contained 50 mM Tris-HCl (pH 7.8), 60 mM KCl, 6 mM MgCl2, 5 mM DTT,

50␮g/ml poly(rA)/p(dT)12-18, 8.5 nM RT enzymes, and various concentrations of

tracer [3_{H]dTTP and cold dTTP (0.2 to 200␮M). Reactions were run at 37°C and}

quenched by adding 0.2 ml of 10% cold TCA and 20 mM sodium pyrophosphate; products were filtered onto Millipore 96-well MutiScreen HTS FC filter plates

(MSFCN6B) and sequentially washed with 200␮l of 10% TCA and 150␮l of

95% ethanol. The radioactivity of incorporated products was analyzed by liquid scintillation spectrometry using a Perkin-Elmer 1450 MicroBetaTriLux micro-plate scintillation and luminescence counter. The steady-state kinetic parameter

(Km) for nucleotide substrates was determined by the nonlinear regression

anal-ysis of the substrate concentration and initial velocity data using the Michaelis-Menten equation with the program GraphPad Prism according to the manufac-turer’s instructions.

Structural modeling of E138K/M184V and E138K/M184I double mutants.

Homology structural models of E138K/M184V and E184K/M184I double

mu-tants were obtained by means of multiple template modeling (MTM) andab

initiosimulations from the I-TASSER 3D protein prediction server (31) using the lead PDB template of 3KK2 (19), a crystal structure of wild-type HIV-1 RT in complex with template DNA, and dNTP bound in the dNTP binding pocket. Initial model assessment was based on root mean square deviations (RMSDs) of the global monomeric homology structure from the monomeric lead template, and the resulting structures were aligned to appropriate crystal structural files using the RCSB PDB protein comparison tool (28) to verify structural homology as well as to ensure that all structural files used have similar orientations. To mimic the binding of dNTPs into ternary RT complexes, dNTP was removed from the 3KK2 structure and docked using high stringency into the dNTP binding pocket of the various structures using AUTODOCK Tools and AUTODOCK Vina. (34) PyMol (available at http//www.pymol.org; accessed 20 March 2011) was used for structural visualization, alignment, and image pro-cessing.

RT-catalyzed RNase H activity.RNase H activity was assayed using a 41-mer

5⬘-32

P heteropolymeric RNA template, kim40R, that was annealed to a comple-mentary 32-nucleotide DNA oligomer, termed kim32D, at a 1:4 molar ratio as described previously (18). Reactions were conducted at 37°C in mixtures con-taining an RNA-DNA duplex substrate with equal activities of RT enzymes in

assay buffer, 50 mM Tris-HCl, pH 7.8, 60 mM KCl, and 5 mM MgCl2in the

presence of a heparin trap (2 mg/ml). Aliquots were removed at different time points after the initiation of reactions and quenched by using an equal volume of formamide loading dye. The samples were heated to 90°C for 3 min, cooled on ice, and electrophoresed through 6% polyacrylamide–7 M urea gels. The gels were analyzed by phosphorimaging. The efficacy of the heparin trap was verified by preincubation experiments done by 10-min preincubation of enzymes with substrate and various concentrations of heparin trap, followed by the initiation of RNase H activities with magnesium and dNTPs.

RESULTS

The E138K mutation compensates for the impaired viral

replication capacity (RC) of HIV containing either M184I or

M184V.

We previously showed that the replication capacity of

HIV-1 containing the E138K mutation was impaired by 2- to

3-fold compared to that of wild-type virus (3). Here, we wanted

to investigate the impact of the E138K, M184I, M184V,

E138K/M184I, and E138K/M184V mutations on RC by

infect-ing TZM-bl cells with serially diluted viral stocks normalized

by p24 antigen. The infectivities of the WT and mutant

vi-ruses were determined by measuring luciferase activity at 48 h

postinfection. The relative RC of viruses containing M184V

was decreased by

⬃

2-fold compared to that of the WT, while

the replication capacity of viruses containing either E138K or

M184I were decreased by

⬃

3-fold (Fig. 1). Interestingly, the

replication capacity of the E138K/M184I and E138K/M184V

double mutant viruses was equal to that of the WT virus. Thus,

the addition of E138K to either M184I or M184V compensates

for the impaired RC observed with each of the singly mutated

viruses. The order of replication was the following: WT

⫽

E138K/M184I

⫽

E138K/M184V

⬎

M184V

⬎

E138K

⫽

M184I. We did not observe an advantage of E138K/M184V in

RC over E138K/M184I.

Drug susceptibilities monitored in cell culture phenotyping

assay.

Previous cell culture assays showed that HIV-1 clones

containing E138K displayed phenotypic resistance to ETR

(3.8-fold) (3). In this study, we determined the impact of the

interactions of E138K and the M184I/V mutations in HIV-1 on

susceptibility to each of the RT inhibitors FTC, 3TC, ETR, and

EFV. The results are summarized in Table 1. E138K conferred

no resistance on its own to the NRTIs FTC and 3TC and had

only minor impact on sensitivity to EFV but conferred

low-level resistance to ETR (3.2-fold-change in EC

), which is

consistent with our previous report (3). For FTC and 3TC, all

FIG. 1. E138K mutation in HIV-1 reverse transcriptase

compen-sates for the impaired viral replication capacity of both M184I and

M184V. Viral stocks of wild-type virus and viruses containing E138K,

M184I, M184V, E138K/M184I, and E138K/M184V were normalized

for p24 and used to infect TZM-bl cells. Luciferase activity was

mea-sured at 48 h postinfection as an indication of viral replication. The

relative infectivity of WT compared to mutant viruses is shown on the

y

axis, while the

x

axis denotes the input of p24. Each experiment was

performed in triplicate. The figure is representative of two

indepen-dent experiments. These values translate to a 2-fold virus replication

disadvantage for M184V virus and a 3-fold disadvantage for the M184I

and E138K viruses compared to the replication of the WT virus.

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of the viruses containing the M184I or M184V mutation, either

alone or in combination with E138K, exhibited high-level

re-sistance. We also observed that the M184V-containing viruses

showed a higher level of resistance to FTC and 3TC than the

M184I-containing viruses. Neither M184I nor M184V

dramat-ically altered susceptibility to the NNRTIs ETR and EFV. For

ETR, the E138K/M184I double mutation resulted in a 2.3-fold

change in EC

and a 2.7-fold change for the E138K/M184V

virus. These results indicate that the high prevalence of E138K/

M184I rather than E138K/M184V in clinical samples cannot

be explained by levels of drug resistance.

Purification of recombinant HIV-1 RT enzymes.

Recombi-nant WT heterodimeric (p66/p51) RT and RT enzymes

con-taining each of E138K, E138K/M184I, M184I, E138K/M184V,

and M184V substitutions were purified to

⬎

95% homogeneity;

the RT p66 and p51 subunits were processed to similar molar

ratios based on SDS-PAGE analysis (data not shown). The

mutations introduced into the recombinant HIV-1 RT did not

interfere with either heterodimer formation or enzyme

purifi-cation. Consistently with previously published data, the E138K

mutation did not have an effect on RT dimerization (1, 25).

E138K restores the enzyme processivity of RT containing

either M184I or M184V at low dNTP concentrations.

The

pro-cessivity of a polymerase is defined as the number of

nucleo-tides incorporated in a single round of binding, elongation, and

dissociation. Earlier studies showed that diminished HIV-1 RT

processivity is the major determinant of impaired viral

repli-cation capacity associated with M184I/V mutations, especially

at low dNTP concentrations (5, 24). We wished to determine

whether the compensatory effect of E138K on the viral

repli-cation capacity of M184I/V viruses was due to a restoration of

enzyme processivity. To this end, we performed single-cycle

processivity assays with recombinant RT enzymes at both low

and high dNTP concentrations (Fig. 2). Assays performed with

TABLE 1. Drug susceptibilities for recombinant HIV-1

WT and site-directed mutant viruses assayed in CBMC cultures

Virus EC50⫾SD (␮M) (fold change in resistance) EC50⫾SD (nM) (fold change in resistance)

FTC 3TC ETV EFV

WT

0.008

⫾

0.005

0.041

⫾

0.029

1.0

⫾

0.3

1.2

⫾

0.6

E138K

0.018

⫾

0.006 (2.2)

0.024

⫾

0.025 (0.6)

3.3

⫾

0.4 (3.2)

2.2

⫾

0.4 (1.8)

E138K/M184I

2.1

⫾

0.9 (250)

26.5

⫾

14.0 (

⬎

500)

2.4

⫾

0.1 (2.3)

2.0

⫾

0.9 (1.6)

E138K/M184V

⬎

50 (

⬎

1,000)

⬎

100 (

⬎

1,000)

2.8

⫾

0.9 (2.7)

2.5

⫾

0.9 (2.1)

M184I

1.5

⫾

0.4 (185)

14.0

⫾

2.7 (350)

1.2

⫾

0.5 (1.2)

1.2

⫾

0.5 (1.0)

M184V

⬎

50 (

⬎

1,000)

⬎

100 (

⬎

1,000)

1.2

⫾

0.5 (1.2)

1.4

⫾

1.0 (1.2)

a_EC

50s (50% drug effective concentrations) were determined by RT assays using culture fluids of CBMCs. Data represent the averages from two to three

representative experiments performed in duplicate. Values in parentheses represent the fold change in EC50for mutated viruses compared to that of the WT virus.

FIG. 2. E138K mutation in HIV-1 RT restores enzyme processivity of both the M184I- and M184V-containing enzymes at low dNTP

concentrations. The processivity of purified recombinant RT enzymes was analyzed using 5

⬘

-end-labeled DNA primer (D25) annealed to a 471-nt

RNA template as the substrate; the resulting full-length DNA (FL DNA) is 471 nt in size. Processivities were determined by the size distribution

of DNA products in fixed-time experiments at two different concentrations of dNTPs in the presence of heparin trap, a low dNTP concentration

(0.5

␮

M) (A), and a high dNTP concentration (200

␮

M) (B). Each reaction was performed in duplicate and terminated at 30 min. The sizes of

some fragments of the

P-labeled 25-bp DNA ladder (Invitrogen) in nucleotide bases are indicated on the left side of the panel. All reaction

products were resolved by denaturing 6% polyacrylamide gel electrophoresis and visualized by phosphorimaging. Positions of

P-labeled D25

primer (

P-D25) and the 471-nt full-length extension DNA (FL DNA) product are indicated on the right.

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0.5

␮

M each dNTP showed that both M184I and M184V RTs

have lower processivity than wild-type RT, whereas the three

E138K-containing mutant enzymes had higher processivity,

in-dicating that E138K compensates for the diminished enzyme

processivity associated with M184I/V (Fig. 3A). We did not

observe a processivity advantage for E138K/M184I over

E138K/M184V. At 200

␮

M each dNTP, all enzymes displayed

similar processivity based on the production of full-length

products (Fig. 3B); the distortion of running samples in each

lane was from blotting the gel onto Whatman paper. This is in

agreement with previous studies showing that the processivity

defect of the M184I/V RT mutants is minimized at high dNTP

concentrations (5, 11) and implies that the restoration of

pro-cessivity by E138K is due the compensation of dNTP usage

associated with M184I/V.

Effects of dNTP concentrations on polymerization rates of

recombinant RT enzymes.

To measure the rate of DNA

polymerization at specific dNTP concentrations, we performed

RNA-dependent DNA polymerase reactions in time course

experiments for 30 s, 60 s, 5 min, and 20 min using a 497-nt

HIV-1 PBS RNA template (Fig. 3). RT molecules were used at

an

⬃

20-fold excess above the substrate, so that any RTs that

dissociated from the primer terminus during synthesis would

be rapidly replaced. In this case, the rate-limiting step would be

nucleotide addition (11). Polymerase reactions were carried

out at two different dNTP concentrations, 0.5 and 200

␮

M. The

rate of polymerization was calculated as the number of

nucle-otide additions divided by reaction time. The longest extension

products made after 60 s were used to calculate the

polymer-ization rate. At 0.5

␮

M each dNTP, the rate of synthesis of

reaction product for WT, E138K, E138K/M184I, and E138K/

M184V RTs was

⬃

0.9 nt/s. However, for M184I and M184V,

the

P-labeled primer D25 was hardly extended (Fig. 4B).

When reactions were performed at 200

␮

M dNTPs, all of the

RT variant enzymes except E138K showed similar

polymeriza-tion rates of

⬃

3.2 nt/s, while the rate for E138K was

⬃

2.0 nt/s

(Fig. 4B), which is consistent with previously reported

maxi-mum polymerization rates for WT HIV-1 RT on RNA

tem-plates (10, 11). These data confirm that the M184I/V mutants

have defects in dNTP usage (5, 11), and that the E138K

mu-tation can compensate for this deficit, even though the E138K

enzyme itself has lower catalytic efficiency at high dNTP

con-centrations.

Steady-state kinetic analyses of WT and mutant RTs.

To

assess the impact of the E138K mutation in the context of

M184I/V on dNTP binding affinity, steady-state kinetic

analy-ses were carried out using a homopolymeric poly(rA)/

p(dT)

template/primer to determine the steady-state

ki-netic constants

K

and

V

for WT and mutant HIV-1

recombinant RTs; the results are summarized in Table 2. In

the case of RTs containing M184V or M184I compared to WT

RT, increased

K

values for dTTP of 1.4-fold and 2.4-fold,

respectively, were observed. For E138K RT, the value was

0.46-fold of that of the WT. For the double mutants E138K/

M184I and E138K/M184V, the fold change in

K

value was

1.25 and 1.1, respectively. Although

K

is not a direct measure

of dNTP binding, changes in

K

values suggest that the

mu-tations affect dNTP binding affinity. These results indicate that

the deficit in dNTP binding affinity associated with the HIV-1

RT M184I/V mutations was compensated by the copresence of

E138K; this is further demonstrated in structural modeling as

described below.

Homology modeling shows that the E138K/M184V and

E138K/M184I double mutants promote tighter dNTP binding.

Individual models of RT molecules containing E138K/M184I

that were generated from either WT, E138K, or M184V RT

FIG. 3. Time course experiments showing the effects of dNTP concentrations on rates of DNA synthesis by recombinant HIV-1 RT enzymes.

The

P-labeled D25 primer (

P-D25) was annealed to the 497-nt RNA template, and extension assays were performed at an excess of

recombinant RT enzymes at dNTP concentrations of 0.5

␮

M (A) and 200

␮

M (B). The reactions were stopped at 30 s (30

⬙

), 60 s (60

⬙

), 5 min (5

⬘

),

and 20 min (20

⬘

), respectively. The sizes of some fragments of the

P-labeled 25-bp DNA ladder (Invitrogen) in nucleotide bases are indicated

on the left side of the panel. The longest extension products generated at 60 s were identified by arrows and indicate differences in rates of

polymerization.

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did not differ significantly from WT RT in global structure.

This attests to the accuracy of the modeling method and the

reliability of the models produced. E138K/M184I and E138K/

M184V homology structures that were formed using either

WT, M184I, or E138K as the lead template were in agreement

with wild-type and single mutant structures with mean RMSDs

in the range of 0.3 to 2.3 Å (Fig. 4A).There did not appear

to be large variations between the structures of the E138K/

M184V and E138K/M184I double mutants (RMSD,

⬍

0.5 Å).

In the WT RT, the finger subdomain aids in binding the

dNTP in the right conformation (Fig. 4B), while mutations at

M184 move this subdomain away and open up the dNTP

bind-ing pocket, thus causbind-ing dNTP to bind loosely (Fig. 4C). The

presence of the E138K mutation in association with the

M184I/V mutations (Fig. 4D and E) caused a series of steric

interferences with several p66 residues (Y181 and Y183) at the

interface between p66 and p51. These changes translate into a

FIG. 4. Modeling and docking studies showing the concerted effect of the E138K and M184I/V double mutations. (A) The overlay of wild-type

(blue) and M184I (yellow) crystal structures with homology models E138K/M184I (pink) and E138K/M184V (green) shows minimal differences

in global structure. (B to E) dNTP docked into identical binding sites/coordinates in all structures with residue 184 is indicated by a black arrow,

and template DNA is indicated in white (T). (B) The finger subdomain which assists in binding and orienting bound dNTP in the wild-type RT

is altered when the M184I mutation is present. (C) M184I also opens up the dNTP binding pocket and prevents the tight association of dNTP with

RT. (D) The E138K/M184V double mutant has a less open dNTP binding site than M184I, thus contributing to tighter dNTP binding. (E) The

E138K/M184I double mutant appears to display the partial recovery of the finger subdomain and shows tight binding similar to that of

E138K/M184V RT. (F) Ribbon overlay of the M184I crystal structure with the E138K/M184I homology model. The blue arrows indicate the

downward movement of residues and the subsequent closing up of the dNTP binding site in the p66 unit induced by the repositioning of the side

chain containing residue 138 in the p51 subunit.

TABLE 2. Kinetic parameters of recombinant RT enzymes as

determined by steady-state kinetic analysis

Virus

Parametera

Vmax(pM␮g⫺ 1

min⫺1

) Km(␮M) (FC

b

)

WT

139.3

⫾

5.1

5.63

⫾

0.1 (1)

E138K

112.9

⫾

3.2

2.59

⫾

0.1 (0.46)

E138K/M184I

177.9

⫾

4.3

7.0

⫾

0.15 (1.25)

M184I

193.2

⫾

7.1

14.6

⫾

0.3 (2.6)

E138K/M184V

221.3

⫾

6.4

6.2

⫾

0.3 (1.1)

M184V

192.2

⫾

5.6

7.88

⫾

0.4 (1.4)

a

The steady-state kinetic parametersVmaxandKmfor dTTP of WT HIV-1 RT

and its mutant derivatives were measured using poly(rA)/p(dT)12-18template/

primer. The recombinant RT enzymes were purified in heterodimeric forms, and

the mutations were introduced into both subunits. Values are averages⫾

stan-dard deviations from representative experiments performed in triplicate.

b

Fold change (FC) ofKmof mutant RT variants compared to that of the WT.

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downward shift of residue 184 (Fig. 4F) and partially close up

the dNTP binding site (Fig. 4D and E). Thus, the E138K/

M184I and E138K/M184V double mutants appear to have

tighter dNTP binding than the M184I/V single mutants.

RNase H activity.

We previously showed that M230L in HIV

RT impairs RNase H activity and contributes to reductions in

viral replication capacity (43). We wished to test the impact of

the E138K mutation alone and in combination with M184I/V

on intrinsic RNase H activity. Therefore, we performed an

RNase H time course study in the presence of a heparin trap

by using a recessed 32-mer DNA primer hybridized to a 5

⬘

-end-labeled 40-mer RNA to monitor 3

⬘

-DNA-directed RNase

H activity (Fig. 5.). The presence of the heparin trap permits

the analysis of cleaved products from a single binding event of

RT to the substrate. The results show that the RNase H activity

of the E138K mutant RT was lower than that of the WT RT,

and that the M184I/V mutations can restore the RNase H

activity of E138K, albeit not to the WT level (Fig. 5). Thus, the

E138K mutation also impairs RNase H activity despite the fact

that it also results in a defect in regard to initial rates of

polymerization.

DISCUSSION

The diarylpyrimidine (DAPY) compounds etravirine (ETR;

TMC125) and rilpivirine (RPV; TMC278) are representative

potent second-generation NNRTIs. ETR has been licensed by

the FDA and is recommended for use in salvage therapy in

combination with other active antiretrovirals (ARVs). Most

clinical data sets regarding ETR resistance are based on the

DUET studies (16, 37), and little information is available on

the roles of individual resistance mutations on RT enzyme

properties, viral replication, and diminished sensitivity to ETV.

Even less information is available for RPV in this regard (4).

We previously reported that E138K in RT is usually the first

resistance mutation to emerge in tissue culture with ETR

se-lection. Recently, the ECHO and THRIVE phase 3 clinical

trials showed that E138K/M184I was the most frequent

mu-tation combination in patients who failed regimens of RPV

combined with FTC/TDF. We believe that E138K possesses

clinical relevance to ETR and RPV. Our current findings

demonstrate that the E138K mutation can restore the enzyme

processivity of the RT and viral replication capacity of viruses

containing the M184I/V mutation. This renders the doubly

mutated viruses containing E138K/M184I and E138K/M184V

highly competent in replication capacity and resistant to each

of FTC, 3TC, and ETR.

Our cell culture-based phenotypic data show that

recombi-nant HIV-1

viruses harboring E138K and M184I/V

dou-ble mutations possessed high-level resistance to FTC and 3TC,

as does the M184I/V single mutation, and possessed modest

resistance to ETR, as does the E138K single mutation. None of

the mutations tested in this study significantly affected EFV

susceptibility. We did not observe that the E138K/M184I

combination conferred higher levels of resistance to ETR

and 3TC compared to those of an E138K/M184V double

mutant. Therefore, the high prevalence of E138K/M184I

in-stead of E138K/M184V among patients who failed

RPV/FTC-containing therapy in the ECHO and THRIVE clinical trials

cannot be explained by differences in levels of drug resistance

(20). Although we did not test FTC and ETR in combination,

it is unlikely that significant differences would have been

ob-tained. Nonetheless, this experiment is being planned. It is

possible, of course, that M184I has an advantage over M184V

when both ETR and FTC are present, since both the M184I

and E138K mutations were selected in the ECHO and

THRIVE clinical trials that employed these two drugs.

Of note, we observed that recombinant HIV-1 virus

harbor-ing E138K confers 3.2-fold resistance to ETR, which is

consis-tent with our previous report (3), while the combination of

E138K and either M184I or M184V conferred 2.4- or 2.8-fold

resistance to ETR, respectively. The published lower clinical

cutoff (CCO) for ETR is 3.0 (37); it is conceivable that

addi-tional clinical data will be necessary to reevaluate the role of

ETR mutations such as E138K and G190A in regard to the

durability of ETR-based therapy (41). Although we did not

have access to rilpivirine (RPV) for these studies, we believe

that findings similar to those obtained with ETR would have

been obtained with this compound.

Our replication capacity studies carried out in TZM-bl cells

show that recombinant viruses harboring E138K, M184I, or

M184V alone exhibited 2- to 3-fold lower replication capacity

compared to that of WT virus and that the copresence of

E138K with either M184I or M184V restored viral replication

capacity to WT levels. Previously, we observed that the HIV-1

E138K mutation results in lower replication capacity in several

HIV-1 subtypes, and M184I/V viruses also have impaired

rep-lication capacity, especially in cells with low dNTP

concentra-tions (15, 39). Although some compensatory mutaconcentra-tions in RT

might augment viral resistance levels and fitness (7, 22, 26), this

is the first report on the compensatory effects of E138K on the

enzymatic activity of RT containing M184V or M184I. Our

biochemical assays provide the first evidence that E138K RT

has a lower rate of polymerization and lower RNase H activity

than WT RT, which helps to explain the lower replication

capacity of the E138K virus. In our studies, the combination of

E138K/M184I did not confer a replication advantage over

FIG. 5. RNase H activity of WT and E138K-containing

recombi-nant RTs. (A) Graphic representation of the RNA/DNA (kim40R/

kim32D) duplex substrate used to monitor the cleavage efficiency of

recombinant RT. The 40-mer RNA kim40R was labeled at its 5

⬘

terminus with

P and annealed to the 32-mer DNA oligonucleotide

kim32D. (B) RNase H activity was analyzed by monitoring substrate

cleavage in time course experiments in the presence (right) of a

hep-arin trap. The positions of cleaved products are indicated on the left.

The uncleaved substrate indicates variations in RNase H activity

among different RT enzymes. All reactions were resolved by

denatur-ing 6% polyacrylamide gel electrophoresis.

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E138K/M184V in the absence of drugs, which means that the

high prevalence of E138K/M184I instead of E138K/M184V in

the ECHO and THRIVE clinical trials cannot be explained by

differences in viral replication capacity. Others have shown

that the combination of E138K/M184I was more fit than

E138K/M184V in a sensitive growth competition assay (14,

14a). It is possible that the different cell types and assay

sys-tems used have affected results obtained.

Our enzyme assays with purified recombinant RT proteins

provide evidence at a molecular level for the compensatory

effects between E138K and M184I/V. M184V RT is known to

have lower enzyme processivity than WT RT, especially at low

dNTP concentrations (5, 9, 35), and M184I is even more

im-paired than M184V in terms of processivity (11, 15). Deficits in

M184I/V RT primer elongation rate, processivity, and strand

transfer may all be attributable to defective dNTP utilization

(9, 11, 15, 35, 39). Here, we have shown that E138K

compen-sates for the defect in dNTP usage associated with M184I/V,

thus restoring enzyme processivity and viral replication

capac-ity. Indeed, RT enzymes containing E138K alone, E138K/

M184I, or E138K/M184V exhibited higher processivity than

WT RT at low dNTP concentrations. Steady-state kinetic

anal-ysis also demonstrated that the E138K mutation resulted in

decreased

K

values. This was further supported by structural

modeling showing that the addition of E138K to M184I/V

promotes tighter dNTP binding.

Under selection pressure with 3TC or FTC, either

in vitro

or

in vivo

, M184I usually emerges first as a resistance mutation

and is rapidly replaced by M184V because of its fitness

advan-tage over M184I (6, 9, 13, 18, 35). The reason that M184I

(ATG

3

ATA) appears before M184V (ATG

3

GTG) is that

G

3

A is the most frequent mutation to occur during HIV-1

replication (6, 13).The fact that M184I is more impaired than

M184V RT in dNTP usage and processive DNA synthesis

contributes to the

in vivo

instability of the M184I mutation and

its supplementation by M184V (5, 11, 15, 39). What has

prob-ably happened in the ECHO and THRIVE clinical trials is that

E138K stabilized M184I as a result of the compensatory effects

described here. In clinical studies on the use of ETR or RPV

and FTC or 3TC, it is not known whether E138K or M184I

emerges first. The clonal analysis of clinical samples from

pa-tients undergoing treatment failure should be able to answer

this question.

ACKNOWLEDGMENTS

We thank Stuart Le Grice for providing the pRT6H-PROT DNA,

Tomozumi Imamichi for the pNL4.3PFB plasmid DNA, and Daniela

Moisi for technical assistance in DNA sequencing reactions.

This work was supported by research grants from the Canadian

Institutes of Health Research (CIHR) and by the International

Part-nership on Microbicides.

We have no conflicts of interest to declare.

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