ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2005, p. 3930–3932
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 9
In Vitro Selection and Analysis of Human Immunodeficiency Virus
Type 1 Resistant to Derivatives of
Jennifer L. Hammond,1Urvi M. Parikh,1Dianna L. Koontz,1Susan Schlueter-Wirtz,2
Chung K. Chu,3Hengameh Z. Bazmi,1Raymond F. Schinazi,2
and John W. Mellors1*
Department of Medicine, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
152611; Department of Pediatrics, Emory University School of Medicine, and the Georgia VA Research Center on AIDS
and HIV Infection, Veterans Affair Medical Center, Decatur, Georgia 300332; and College of Pharmacy,
The University of Georgia, Athens, Georgia 306023
Received 19 January 2005/Returned for modification 10 March 2005/Accepted 15 June 2005
Serial passage of human immunodeficiency virus type 1 in MT-2 cells in increasing concentrations of the D-
and L-enantiomers of ?-2?,3?-didehydro-2?,3?-dideoxy-5-fluorocytidine (d4FC) resulted in the selection of viral
variants with reverse transcriptase substitutions M184I or M184V for L-d4FC and I63L, K65R, K70N, K70E,
or R172K for D-d4FC. Phenotypic analysis of site-directed mutants defined the role of these mutations in
reducing susceptibility to L- or D-d4FC.
Derivatives of ?-2?,3?-didehydro-2?,3?-dideoxy-5-fluorocyti-
dine (d4FC) (Fig. 1) are potent and selective inhibitors of both
wild-type and nucleoside reverse transcriptase inhibitor-resis-
tant human immunodeficiency virus type 1 (HIV-1) in clinical
development (2, 6, 10, 12, 15). A phase I-II study with HIV-
infected patients having virus with the M184V resistance mu-
tation showed that the substitution of L-d4FC (elvucitabine or
ACH-126,443) for lamivudine produced a 0.67 to 0.78 log10
decline in plasma HIV RNA (copies/ml) over a 28-day period,
although dose-dependent hematological toxicity was evident
(L. M. Dunkle, J. C. Gathe, D. E. Pedevillano, H. G. Robison,
W. G. Rice, J. C. Pottage, Jr., and the ACH-006 Study Team,
Abstr. XII Int. HIV Drug Resist. Workshop: Basic Princ. Clin.
Implic., abstr. 2, 2003). A phase I-II study in which D-d4FC was
given once daily at 50 mg, 100 mg, or 200 mg for 10 days to 30
antiretroviral naı ¨ve, HIV-infected subjects showed marked re-
ductions in plasma HIV-1 RNA averaging 1.7 log10copies/ml
without discernible toxicity (R. L. Murphy, D. Schu ¨rmann, A.
Beard, L. Cartee, R. F. Schinazi, and M. J. Otto, Abstr. 11th
Conf. Retrovir. Opport. Infect., abstr. 137, 2004). The potency
and favorable safety profile of D-d4FC have led to its acceler-
ated clinical development. Because of the interest in both
enantiomers of d4FC, the goal of the current study was to
evaluate the influence of stereochemistry on resistance to
d4FC resistance by performing in vitro selections.
Selection experiments were conducted as previously de-
scribed (1). A total of 106MT-2 cells (AIDS Research and
Reference Reagent Program, National Institute of Allergy and
Infectious Diseases, National Institutes of Health) were pre-
treated for 2 h with L-d4FC or D-d4FC (synthesized by R.
Schinazi) (12) and then infected with HIV-1LAIat a multiplic-
ity of infection of 0.1 50% tissue culture infective doses/cell. A
100-?l aliquot was used to initiate a new cycle of infection and
the selective pressure (i.e., drug concentration) was generally
doubled every two to three passages. The passaged virus was
regularly monitored for a reduction in susceptibility to the
compounds by determining the 50% inhibitory concentration
(IC50) of the passaged virus relative to that of the unpassaged
HIV-1LAI(7). Student’s t test was used to determine the sta-
tistical significance of differences between log-transformed
Amino acid changes in passaged virus were identified by
extracting RNA (TRIZOL reagent; Gibco BRL, Grand Island,
NY) from pelleted virions (25,000 ? g for 1 h at 4°C). The near
full-length coding region of reverse transcriptase (RT) (amino
acids [aa] 1 to 511) was amplified by RT-PCR (1), purified
(Wizard PCR purification system; Promega, Madison, WI),
and sequenced (aa 1 to 350) by using an ABI 377 sequencer
(Applied Biosystems, Foster City, CA). To confirm the role of
these changes, recombinant mutants were made by using oli-
gonucleotide-directed mutagenesis (Altered Sites II; Promega)
or by cassette cloning of the XmaI-to-XbaI RT fragment from
passaged virus into the pxxHIV-1LAIvector (14). Infectious
recombinant HIV-1 was generated as previously described
In two independent experiments, virus resistant to L-d4FC
was isolated after 4 (selection no. 1) or 10 (selection no. 2)
passages. Both selections were initiated at an L-d4FC concen-
tration of 0.5 ?M (?5 times the IC50of the wild type). The
selective pressure could not be increased above 1.1 ?M be-
cause of cytotoxicity for MT-2 cells. Phenotypic analyses of
viruses from passages 4 and 10 showed ?20-fold (selection no.
1) and ?11-fold (selection no. 2) levels of resistance to L-d4FC,
respectively; and genotype analyses of these viruses identified
mutations at codon 184 (M to I or V) (Table 1). A recombinant
HIV-1 encoding the M184V mutation showed ?46-fold resis-
tance to L-d4FC. The M184V mutation was also selected in
* Corresponding author. Mailing address: 818 Scaife Hall, Univer-
sity of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh,
PA 15261. Phone: (412) 624-8512. Fax: (412) 383-7982. E-mail: mellors
HIV-1LAIpassaged with L-d4FC in human peripheral blood
mononuclear cells (13).
Viruses resistant to D-d4FC were isolated in three indepen-
dent passage experiments. The first selection was initiated at a
concentration of 0.75 ?M (?6 times the IC50of D-d4FC) and
increased to a final concentration of 4.0 ?M over the course of
37 passages. Phenotypic analysis of the virus from passage 37
showed a 14-fold decrease in susceptibility to D-d4FC (Table
1). Genotype analysis of virus from this passage identified
mutations at codons 70 (K to N), 90 (V to I), and 172 (R to K).
All three mutations are novel and, with the exception of
R172K, are in highly conserved regions of HIV-1 RT. Site-
directed mutants encoding K70N or V90I/R172K mutations
showed a 2.1-fold reduction in susceptibility. Both the site-
directed mutant encoding K70N/V90I/R172K mutations and
the p37 RT recombinant virus also showed significant reduc-
tions in susceptibility to D-d4FC, 3.9- and 2.4-fold, respectively
The second and third selections for D-d4FC-resistant virus
were initiated at a lower concentration, 0.20 ?M (?1.5 times
the IC50). The selective pressure was increased to a concen-
tration of 4.8 ?M over the course of 20 passages (selection no.
2) and to 1.6 ?M over the course of 16 passages (selection no.
3). Passage 20 virus from selection no. 2 showed an 8.1-fold
decrease in susceptibility to D-d4FC, and genotypic analysis
identified mutations at codons 65 (K to R) and 179 (V to D) in
RT (Table 1). In site-directed mutants, the K65R mutation
alone decreased D-d4FC susceptibility 6.2-fold; the addition of
the V179D mutation did not increase the level of resistance
(5.5-fold) (Table 2), nor did it affect the replicative fitness of
virus with the K65R mutation as determined in competition
experiments in MT-2 cells (not shown). These results are con-
sistent with findings from Geleziunas et al., who recently re-
ported that K65R is the sole mutation after in vitro selection
with D-d4FC (2).
In the third selection, virus with the I63L mutation was
identified at passage 12 (13-fold resistance) and further selec-
FIG. 1. Structures
TABLE 1. Selection of HIV-1 resistant to D- and L-d4FC
?11 ATG3ATA M184I
D-d4FC1 0.75–4.037 14 AAA3AAT
2 0.2–4.820 8.1AAA3AGA
3 0.2–1.6 12
aResistance relative to wild-type virus (HIV-1LAIpassage 0).
bAmino acid change relative to wild-type virus (HIV-1LAIpassage 0).
TABLE 2. D-d4FC Susceptibility of recombinant viruses encoding
mutations selected by serial passage in D-d4FC
0.19 ? 0.13
0.40 ? 0.28
0.39 ? 0.26
0.72 ? 0.63
0.45 ? 0.17
1.15 ? 0.43
1.03 ? 0.28
0.27 ? 0.13
0.49 ? 0.22
aHIV-1LAIencoding the indicated resistance mutations.
bIC50(means ? standard deviations from at least three independent experi-
ments) determined by inhibition of p24 antigen production in MT-2 cells.
cResistance relative to wild-type virus.
dFor the comparison of log-transformed mean IC50s by using Student’s t test.
eMutant derived by cassette cloning of the RT gene from D-d4FC p37 virus
into the xxHIVLAIvector (see Materials and Methods).
VOL. 49, 2005NOTES 3931
tion resulted in virus having greater resistance (16-fold) and Download full-text
the K70E mutation (Table 1). A site-directed mutant with the
I63L mutation was not significantly resistant to D-d4FC (1.5-
fold; P ? 0.25), but the K70E mutation significantly decreased
susceptibility to D-d4FC by 2.6-fold (Table 2). We were unable
to select D-d4FC-resistant viruses in human peripheral blood
mononuclear cells after more than 20 passages (data not
The site-directed mutant encoding the M184V substitution
showed no reduction in susceptibility to D-d4FC (0.9-fold)
(data not shown). Conversely, the K65R mutant showed 41-
fold resistance to L-d4FC (not shown).
Selection of the M184I/V mutation by L-d4FC is consistent
with previous studies of lamivudine. The crystal structure of
HIV-1 RT with a bound deoxynucleotide triphosphate predicts
that replacing M184 with a ?-branched amino acid, such as I or
V, will create a “steric clash” between residue 184 and the
pseudosugar ring of nucleoside analogs in the L configuration,
including L-d4FC (3, 11).
D-d4FC selected several different mutation patterns: (i) a
combination of three novel mutations (K70N, V90I, and
R172K); (ii) a mutation known to confer broad nucleoside
reverse transcriptase inhibitor resistance (K65R) (1, 9); and
(iii) two other mutations in the fingers subdomain of RT (I63L
and K70E). Although the contribution of the I63L mutation to
D-d4FC resistance is uncertain, the K70E mutation, which re-
placed the I63L mutation at higher drug concentrations, is
located near the incoming deoxynucleotide triphosphate and
could affect the position of the bound analog triphosphate (3).
It is likely that the mutation confers some level of discrimina-
tion against incorporation of D-d4FC triphosphate, although
the specific molecular interactions that underlie this are not yet
The selection of several different genotypes by D-d4FC in-
dicates that there is more than one genetic route to D-d4FC
resistance. It is notable, however, that each genotype conferred
only low-level resistance (?6.2-fold) to D-D4FC in site-di-
rected mutants. The reason different genotypes were selected
may have been related to the variation in starting drug con-
centration (4, 5) or the stochastic nature of in vitro resistance
selection (i.e., chance). Although our results suggest multiple
genetic mechanisms of viral escape from D-d4FC, clinical stud-
ies are needed to determine which, if any, of these occur in
Our finding of multiple mutations in the fingers subdomain
of RT suggests that resistance to D-d4FC is due to discrimina-
tion rather than excision. This concept is supported by the
observation that D-d4FC retains potency against virus contain-
ing the zidovudine resistance mutations D67N, K70R, T215Y,
and K219Q, which confer excision competence (8, 12). Thus,
D-d4FC may be a potent inhibitor for patients with virus having
zidovudine resistance mutations, although clinical studies are
needed to confirm this.
In summary, the results of this study should prove useful in
directing resistance testing of viruses from patients treated
with L- or D-d4FC and in choosing combinations of nucleoside
analogs for treatment and prevention of drug-resistant HIV-1.
This work was supported by grants from the National Cancer Insti-
tute (SAIC contract 20XS190A) and from the National Institute of
Allergy and Infectious Diseases, Division of AIDS (AACTG Central
Group grant U01AI38858). R.F.S. is supported by National Institutes
of Health grant R37-AI-41980 and the Department of Veterans Af-
fairs. R.F.S. is the principal founder and shareholder in Pharmasset,
Inc., and will receive future royalties from sales of D-d4FC (Reverset).
J.W.M. is a consultant to and holds share options in Pharmasset, Inc.
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3932NOTESANTIMICROB. AGENTS CHEMOTHER.