The non-nucleoside reverse transcriptase inhibitor efavirenz stimulates replication of human immunodeficiency virus type 1 harboring certain non-nucleoside resistance mutations
We measured the effects of non-nucleoside reverse transcriptase (RT) inhibitor-resistant mutations K101E+G190S, on replication fitness and EFV-resistance of HIVNL4-3. K101E+G190S reduced fitness in the absence of EFV and increased EFV resistance, compared to either single mutant. Unexpectedly, K101E+G190S also replicated more efficiently in the presence of EFV than in its absence. Addition of the nucleoside resistance mutations L74V or M41L+T215Y to K101E+G190S improved fitness and abolished EFV-dependent stimulation of replication. D10, a clinical RT backbone containing M41L+T215Y and K101E+G190S, also demonstrated EFV-dependent stimulation that was dependent on the presence of K101E. These studies demonstrate that non-nucleoside reverse transcriptase inhibitors can stimulate replication of NNRTI-resistant HIV-1 and that nucleoside-resistant mutants can abolish this stimulation. The ability of EFV to stimulate NNRTI-resistant mutants may contribute to the selection of HIV-1 mutants in vivo. These studies have important implications regarding the treatment of HIV-1 with combination nucleoside and non-nucleoside therapies.
The non-nucleoside reverse transcriptase inhibitor efavirenz stimulates replication
of human immunodeﬁciency virus type 1 harboring certain non-nucleoside
, H. Liang
, L. Bacheler
, K. Deriziotis
, L.M. Demeter
, C. Dykes
Department of Medicine, 601 Elmwood Ave., University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 USA
Department of Biostatistics and Computational Biology, 601 Elmwood Ave., University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 USA
Department of Microbiology & Immunology, 601 Elmwood Ave., University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 USA
Virco Labs, Inc. Chapel Hill, NC USA
Received 15 January 2010
Returned to author for revision
20 February 2010
Accepted 11 March 2010
Available online 18 April 2010
Drug-dependent stimulation of replication
Non-nucleoside reverse transcriptase
Nucleoside reverse transcriptase inhibitors
We measured the effects of non-nucleoside reverse transcriptase (RT) inhibitor-resistant mutations K101E+
G190S, on replication ﬁtness and EFV-resistance of HIV
. K101E+G190S reduced ﬁtness in the absence of
EFV and increased EFV resistance, compared to either single mutant. Unexpectedly, K101E+G190S also
replicated more efﬁciently in the presence of EFV than in its absence. Addition of the nucleoside resistance
mutations L74V or M41L+T215Y to K101E+G190S improved ﬁtness and abolished EFV-dependent
stimulation of replication. D10, a clinical RT backbone containing M41L+T215Y and K101E+G190S, also
demonstrated EFV-dependent stimulation that was dependent on the presence of K101E. These studies
demonstrate that non-nucleoside reverse transcriptase inhibitors can stimulate replication of NNRTI-
resistant HIV-1 and that nucleoside-resistant mutants can abolish this stimulation. The ability of EFV to
stimulate NNRTI-resistant mutants may contribute to the selection of HIV-1 mutants in vivo. These studies
have important implications regarding the treatment of HIV-1 with combination nucleoside and non-
© 2010 Elsevier Inc. All rights reserved.
Human immunodeﬁciency virus type 1 (HIV-1) reverse transcrip-
tase (RT) is an important target of antiretroviral therapy. Non-
nucleoside reverse transcriptase inhibitors (NNRTIs) selectively bind
to HIV-1 RT in a hydrophobic binding pocket adjacent to the po-
lymerase active site, which is located in the palm subdomain of the
p66 subunit (Kohlstaedt et al., 1992). NNRTI binding causes an
allosteric change in RT that leads to non-productive binding of the
incoming nucleotide during DNA polymerization (Spence et al., 1995).
HIV-1 resistance to NNRTIs is caused by mutations in the NNRTI
binding pocket, which interfere with drug binding (reviewed in
Domaoal and Demeter, 2004).
Efavirenz (EFV) is the most commonly used NNRTI clinically,
because of its demonstrated potent antiviral activity and clinical
efﬁcacy when combined as ﬁrst-line therapy with two nucleoside
analogs (Gulick et al., 2006, 2004; Riddler et al., 2008; Robbins et al.,
2003; Staszewski et al., 1999). K103N is the most frequently observed
NNRTI resistance mutation in patients failing EFV-containing regi-
mens (Bacheler et al., 2000; Riddler et al., 2008). Other NNRTI
resistance mutations, such as G190S, confer similar or greater degrees
of EFV resistance compared to K103N but develop uncommonly in
patient isolates. Uncommonly occurring NNRTI resistance mutations
introduced into a laboratory strain cause substantially greater re-
ductions in replication ﬁtness than K103N, as measured in cell culture
in the absence and presence of drug, suggesting that replication
ﬁtness inﬂuences the likelihood of a mutant emerging during treat-
ment failure of an NNRTI-containing regimen (Archer et al., 2000;
Gerondelis et al., 1999; Koval et al., 2006; Wang et al., 2006).
An interesting question is why mutations that confer reductions in
HIV-1 replication ﬁtness in cell culture ever appear in clinical samples,
if ﬁtness signiﬁcantly impacts mutant selection in patients. One
hypothesis is that second-site mutations, either within or outside of
RT, could compensate for the replication deﬁcits conferred by these
drug-resistant mutations. Another possibility is that second-site
mutations could augment HIV-1 drug resistance sufﬁciently to favor
selection of the less-ﬁt mutant in the presence of drug. One example
of this type of mutation is L74V, which confers resistance to the
nucleoside analogs abacavir and didanosine and improves the rep-
lication ﬁtness of the NNRTI-resistant mutants G190E and K103N+
L100I (Kleim et al., 1996; Koval et al., 2006).
Virology 402 (2010) 228–237
⁎Corresponding author. Infectious Diseases Division, University of Rochester
School of Medicine and Dentistry, 601 Elmwood Avenue, Box 689, Rochester, NY
14642 USA. Fax: +1 585 442 9328.
E-mail address: Carrie_Dykes@urmc.rochester.edu (C. Dykes).
0042-6822/$ –see front matter © 2010 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/yviro
K101E has been observed in patients failing NNRTIs, including EFV,
and most often occurs in combination with other known NNRTI
resistance mutations (Bacheler et al., 2001, 2000). K101E in
combination with either K103N or G190S increases EFV resistance
more than 10-fold (Bacheler et al., 2001; Petropoulos et al., 2000).
K101E and G190S are also of interest because they are associated with
HIV-1 resistance to the next-generation NNRTI, etravirine (Llibre et
al., 2008) (Picchio, G., Vingerhoets, J., Staes, M., Tambuyzer, L.,
Bacheler, L., Pattery, T., de Bethune, M. P. . 15th Conference on
Retroviral and Opportunistic Infections, Boston, MA., Abstract #866). We
further explored the effects of K101E on HIV-1 replication ﬁtness and
EFV resistance alone and in combination with G190S and evaluated
the ability of other RT mutations to modulate these effects.
K101E reduces the replication ﬁtness of G190S, except in the presence of
high EFV concentrations
We have previously shown that the relative ﬁtness of G190S is 40%
reduced relative to wild type (Wang et al., 2006). Since K101E has been
observed in combination with G190S in patients failed EFV, we wanted
to determine the effect of the addition of K101E on the replication of
G190S. We compared the relative ﬁtness of recombinant viruses
carrying K101E or G190S alone and in combination, in an NL4-3
backbone, using multiple-cyclegrowth competition assays in PM1 cells.
The K101E mutation alone-reduced HIV-1 replication ﬁtness by 20%
compared to wild type (PRR= 0.81± 0.05) and had slightly increased
ﬁtness relative to G190S (PRR= 1.07± 0.01).The combination of K101E
and G190S reduced replication ﬁtness in the absence of EFV to a much
greater extent than either mutation alone (PRR= 0.57± 0.06 versus
G190S), but the ﬁtness deﬁcit of the double mutant relative to G190S
was reversed at concentrations of EFV exceeding 200 nM (Fig. 1).
The K101E+G190S mutant in an NL4-3 backbone demonstrates
EFV-dependent stimulation of virus replication
In order to better understand how K101E augments the relative
ﬁtness of G190S in the presence of EFV, we performed drug resistance
assays, in which PM1 cells were infected separately by each mutant,
and supernatant p24 antigen concentration was measured after 6
days of growth in different concentrations of EFV. The IC
G190S was substantially higher than either single mutant, as expected
from the results of growth competition assays in the presence of EFV
(Table 1). Surprisingly, when reviewing the drug resistance data, we
found that the K101E+G190S double mutant's replication was 2.0- to
2.5-fold higher in 200–800 nM of EFV than in the absence of drug
(Fig. 2A, pb0.001, comparing p24 concentration in 400 nM EFV vs. no
drug). This property of EFV-dependent growth stimulation was not
observed with either single mutant (Fig. 2B and C). We also measured
replication of the G190S and K101E+G190S mutants using a pre-
viously published replication assay in which a reporter gene product
expressed by HIV-infected cells is detected by ﬂow cytometry (Dykes
et al., 2006). Using this assay, concentrations of EFV that stimulated
replication of K101E+G190S, as deﬁned by p24 antigen production,
also increased the number of infected cells compared to the no-drug
control (data not shown). We also observed the phenomenon of EFV-
dependent stimulation in both primary PBMCs and lymphoid cell lines
and at different virus inocula (data not shown), suggesting that this
property is not dependent on the experimental conditions used.
There are no published reports of NNRTIs stimulating HIV-1
replication, although the M230L mutant was reported to display this
property in presented but unpublished work (Huang W., Parkin N.T.,
Lie, Y.S., et al. 4th International Workshop on HIV Drug Resistance and
Treatment Strategies, June 2000, Abstract #30; in Antiviral Therapy
volume 5, supplement 3, pp. 24-25). Of interest is that at least one
clinical isolate in that study also contained K101E and G190S. We
conﬁrmed that the M230L mutant in an NL4-3 backbone does
replicate better in the presence of low concentrations of EFV than in
the absence of drug; the magnitude of EFV-dependent stimulation is
similar to that observed with K101E+G190S, although the peak of
growth stimulation occurred at a much lower EFV concentration than
K101E+G190S (10 nM vs. 400 nM, Fig. 2D). The peak p24 concen-
tration for the K101E+G190S double mutant in 400 nM EFV was
almost 10-fold greater than the p24 concentration of G190S in a
similar concentration of EFV (Fig. 2A and B), consistent with the
hypothesis that the property of EFV-dependent growth stimulation
contributes to the improved ﬁtness of K101E+G190S relative to
G190S in 400 and 600 nM EFV (Fig. 1). Studies using PHA- and IL-2-
stimulated primary human PBMCs conﬁrmed that the properties of
the K101E+G190S mutant are also observed in primary cells (data
Fig. 1. Relative replication ﬁtness of K101E+G190S, in an NL4-3 backbone, in the absence and
presence of EFV. The graph represents the relative proportion of the double mutant K101E+
G190S relative to the reference strain G190S NL4-3, as determined by bulk sequence analysis
in growth competition experiments, as outlined in Materials and methods. Growth
competition experiments were performed in the absence of EFV (closed diamonds) or in
the presence of 50 nM (closed squares), 100 nM (closed triangles), 200 nM (closed circles),
400 nM (open squares), or 600 nM (open circles) of EFV. Data represent the mean and
standard deviation of results from a minimum of three independent infections. The average
and standard deviation of the production rate ratio (PRR) at each EFV concentration is shown
to the left of the graph.
pb0.001 compared to PRR in absence of EFV.
EFV susceptibilities of drug resistant mutants.
Drug Resistance Mutations EFV IC
NL4-3 Wild type (IC
G190S 66 1.5 –
K101E 13 1.1 –
K101E+G190S 3,113 37.7 b0.0001
L74V+(K101E+G190S) 2,808 92.4 NS
(M41L+T215Y)+(K101E+G190S) 903 420 b0.0001
D10 (M41L+T215Y)+(K101E+G190S) 5,695 116.8 –
17,110 3,315 b0.01
RT, reverse transcriptase; EFV, efavirenz; IC
, 50% inhibitory concentration; IC
inhibitory concentration; SD, standard deviation.
All viruses tested have NL4-3 sequence other than codons 15–560 of RT.
Values represent the mean of at least 4 replicates done on at least 2 separate days.
previously reported by Bacheler et al. (Bacheler et al., 2001).
Compared to G190S or K101E single mutants in the NL4-3 RT backbone.
Compared to K101E+G190S in the NL4-3 RT backbone.
M41L and T215Y were reverted to wild type; other D10 polymorphisms were
Compared to (M41L+T215Y)+(K101E+G190S) in the D10 RT backbone.
229J. Wang et al. / Virology 402 (2010) 228–237
Identiﬁcation of a clinical RT sequence containing K101E+G190S that
has improved ﬁtness compared to K101E+G190S in an NL4-3 backbone
In order to determine the impact of RT backbone sequences on the
properties of the K101E+G190S double mutant, we constructed a
pNL4-3 clone containing an RT sequence derived from patient plasma
(clone D10), which contained K101E+G190S. This clinical RT se-
quence also contained the nucleoside resistance mutations M41L+
T215Y, in addition to 28 coding changes in RT compared to NL4-3
(Table 2). In the absence of EFV, NL4-3 virus containing the D10 RT
sequence was somewhat more ﬁt than K101E+G190S in an NL4-3 RT
backbone (Fig. 3A) but still remained substantially less ﬁt than G190S
in an NL4-3 backbone (Fig. 3B).
Of note is that NL4-3 virus with the D10 RT sequence demon-
strated marked EFV-dependent stimulation of growth, with peak p24
concentrations 17 times higher than the no-drug control in 800 nM
EFV (Fig. 3C). With this level of stimulation, the p24 values of the D10
clone exceeded those of the G190S mutant beginning at an EFV
concentration of 200 nM (compare Figs. 2B and 3C). Because p24
Fig. 2. EFV susceptibilities of NNRTI-resistant mutants of NL4-3. Graphs represent the results of EFV susceptibility assays of the NNRTI-resistant mutants K101E+G190S (panel A),
G190S (panel B), K101E (panel C), and M230L (panel D). Each mutant was introduced into an NL4-3 RT backbone. x-Axis represents time after infection; y-axis represents virus
replication, expressed as log
p24 antigen concentration in culture supernatant. The peak fold increases in p24 concentration compared to the p24 concentration without drug is
noted on each graph at the appropriate EFV concentration. Note that the scales of the x- and y-axes differ for each mutant. Data points represent the mean and standard deviation of
at least three independent infections. For K101E, no increase in mean p24 antigen concentration relative to the no-drug control was observed at any of the EFV concentrations tested.
For G190S, p24 concentrations of some replicate cultures of the G190S mutant in 20 nM EFV appeared to be higher than the no-drub control; however, this apparent different was
not statistically signiﬁcant (95% conﬁdence intervals cross 1.00).
Codon changes in the D10 RT compared to NL4-3.
Class of Mutation Codon Changes Relative to NL4-3
nRTI resistance M41L T215Y
NNRTI resistance K101E G190S
RT polymorphisms T27S K43N
K102Q I142V C162S Q174K
Q207N R211K Q258L R277K T286A A288S V293I E298K
K358R A371T A376T T386I E399D
A400T T403S I435V D460N R461K V467I P468S Q480E L491S
nRTI, nucleoside re verse transcriptase inhibitor; NN RTI, non-nucleo side reverse
transcriptase inhibitor; RT, reverse transcriptase.
230 J. Wang et al. / Virology 402 (2010) 228–237
concentrations of independent cultures may not be directly compa-
rable, we used growth competition assays to conﬁrm that NL4-3 virus
with the D10 RT sequence was more ﬁt than G190S in the presence of
EFV concentrations associated with drug-dependent stimulation of
replication (Fig. 4).
Effects of the nucleoside resistance mutations M41L+T215Y on the
replication ﬁtness, EFV resistance, and EFV-dependent stimulation of
In order to understand the effects of the nucleoside resistance
mutations M41L+T215Y present in the D10 RT, we introduced these
two mutations into NL4-3 containing K101E+G190S. We observed that
M41L+T215Y improved the replication ﬁtness of the NNRTI-resistant
K101E+G190S NL4-3 mutant, although the quadruple mutant still had
substantially reduced ﬁtness compared to G190S alone (Fig. 5AandB).
However,the ([M41L+T215Y]+[K101E+G190S]) quadruple mutantin
an NL4-3 RT backbone became less ﬁt compared to K101E+G190S at
higher concentrations of EFV (PRR = 0.85 ± 0.03 at 400 nM EFV),
suggesting that (M41L+T215Y) reduces the EFV-dependent stimula-
tion and/or EFV resistance of (K101E+G190S). This hypothesis was
conﬁrmed by drug susceptibility assays showing that (M41L+T215Y)
reduced the EFV IC
of (K101E+G190S) in an NL4-3 RT backbone
(Table 1,pb0.0001) and abolished the EFV-dependent stimulation of
viral growth phenotype of (K101E+G190S) (Fig. 5Cvs.Fig. 2A). Thus,
the presence of (M41L+T215Y) at least partially explains the improved
ﬁtness of NL4-3 containing the D10 RT relative to NL4-3 (K101E+
G190S) in the absence of drug but is not consistent with the observed
stimulation of replication of the D10-NL4-3 virus by EFV.
Effects of other RT polymorphisms on the replication ﬁtness, drug
resistance, and EFV-dependent growth stimulation conferred by the D10
clinical RT sequence
The above studies suggested that RT polymorphisms present in the
D10 clinical RT sequence allowed the persistence of the EFV-
dependent stimulation of virus replication conferred by (K101E+
G190S) despite the presence of (M41L+T215Y). In order to further
study the role of other RT coding sequences on the phenotypes of the
D10 clinical RT sequence, we separately back-mutated the (M41L+
T215Y) and K101E mutations in the D10 RT to the corresponding
Fig. 3. Effects of the D10 RT sequence on HIV-1 replication in the absence and presence
of EFV. (Panel A) Growth competition experiment with NL4-3 virus containing the D10
RT sequence (with the resistance mutations [K101E+G190S]+[M41L+T215Y]), versus
the reference strain (K101E+G190S) in an NL4-3 RT backbone. The average and
standard deviation of the production rate ratio (PRR) is shown on the graph. (Panel B)
Growth competition experiment with NL4-3 virus containing the D10 RT sequence
versus the reference strain, G190S in an NL4-3 RT backbone. The average and standard
deviation of the production rate ratio (PRR) is shown on the graph. (Panel C) Drug
susceptibility assay using virus with the D10 RT grown in the presence of varying
concentrations of EFV. The peak fold increase in p24 concentration compared to the p24
concentration without drug is noted on the graph at 800 nM EFV.
Fig. 4. Relative replication ﬁtness of D10 RT versus G190S in NL4-3 in the absence and
presence of EFV. Growth competition assays of virus with the D10 RT versus G190S in
NL4-3, in the presence of no drug (closed diamonds), or 100 nM (closed squares),
200 nM (closed triangles), or 400 nM (closed circles) of EFV. Bars represent standard
deviations from a total of at least three independent infections. The average and
standard deviation of the production rate ratio (PRR) at each EFV concentration is
shown to the left of the graph.
pb0.001 compared to PRR in absence of
pb0.001 compared to PRR in absence of EFV.
231J. Wang et al. / Virology 402 (2010) 228–237
wild-type sequences. All resultant clones were sequenced on both
strands through all of RT to ensure no spurious mutations were in-
troduced. These studies demonstrated that in the D10 clinical RT
backbone, K101E reduced replication ﬁtness (Fig. 6A) and (M41L+
T215Y) improved replication ﬁtness in the absence of EFV (Fig. 6B),
similar to the effects of these mutations in an NL4-3 RT backbone. The
phenotype of EFV-dependent stimulation of virus replication was
abolished by mutating K101E to wild type (Fig. 6C). Of interest is that
removal of (M41L+T215Y) enhanced the degree of EFV-dependent
stimulation relative to the original D10 RT sequence, indicating that
these nucleoside resistance mutations did have some suppressive
effect on EFV-dependent stimulation when combined with (K101E+
G190S) in the D10 backbone (Fig. 6C). However, the presence of
(M41L+T215Y) in the D10 background did not fully suppress EFV-
dependent growth stimulation of (K101E+G190S) (Fig. 6C), as it did
in the NL4-3 backbone (Fig. 5C). Thus, the polymorphisms in D10
appear to augment the EFV-dependent growth stimulation conferred
by K101E but do not directly contribute to EFV-dependent growth
stimulation in the absence of this mutation. In addition, polymorph-
isms in D10 limit the suppressive effect of (M41L+T215Y) on the EFV-
dependent growth stimulation conferred by (K101E+G190S).
Effects of the nucleoside resistance mutation L74V on the
replication ﬁtness, EFV resistance, and EFV-dependent stimulation
We also evaluated whether another nucleoside resistance muta-
tion, L74V, might affect the replication of (K101E+G190S), since L74V
has been shown to improve the replication ﬁtness of at least two other
NNRTI-resistant mutants, G190E and (K103N+L100I) (Boyer et al.,
1998; Kleim et al., 1996; Koval et al., 2006). In the absence of EFV, L74V
substantially improved the replication ﬁtness of (K101E+G190S) in an
NL4-3 RT backbone in the absence of drug (Fig. 7A). The magnitude of
improvement in relative ﬁtness was greater than conferred by the
addition of (M41L+T215Y), since the L74V+(K101E+G190S) triple
mutant's ﬁtness was as good as, if not better than, G190S alone in the
absence of EFV (Fig. 7B). These data indicate that L74V fully com-
pensates for the replication ﬁtness impairment conferred by K101E
in combination with G190S. No reduction in ﬁtness of the L74V+
(K101E+G190S) triple mutant relative to (K101E+G190S) was
observed in the presence of increasing EFV concentrations, as was
seen with the ([M41L+T215Y]+[K101E+G190S]) mutant (PRR of
triple= 1.40 ± 0.16 at 1200 nM EFV). Drug susceptibility assays
indicated that the L74V mutation abolished the EFV-dependent
growth stimulation of (K101E+G190S), without reducing its degree
of EFV resistance (Fig. 7C, Table 1). Fig. 8 summarizes the effects of
drug resistance mutations and RT backbone on HIV-1 replication
ﬁtness, EFV resistance, and EFV-dependent stimulation of virus
These studies provide evidence that the replication of some drug-
resistant mutants of HIV-1 can be stimulated in the presence of an
antiretroviral drug. We have observed this phenomenon with the
(K101E+G190S) mutant and have conﬁrmed the initial report that
M230L has this property (Huang W., Parkin N.T., Lie, Y.S., et al. 4th
International Workshop on HIV Drug Resistance and Treatment
Strategies, June 2000, Abstract #30; in Antiviral Therapy volume 5,
supplement 3 pp. 24–25). We have also demonstrated that both
nucleoside resistance mutations abolished EFV-dependent stimula-
tion, despite having different effects on EFV IC
. Because an as-
sociation between EFV-dependent stimulation and EFV resistance was
not consistently observed, we believe that it is reasonable to make a
distinction between these two phenotypes, even though they both
represent changes in virus replication rates in the presence of drug.
Fig. 5. Impact of the thymidine analog mutations (M41L+T215Y) on replication of HIV-
1 containing the NNRTI resistance mutations (K101E+G190S) in an NL4-3 backbone.
(Panels A and B) Growth competition experiments of ([K101E+G190S]+[M41L+
T215Y]) in an NL4-3 RT backbone, in the absence of drug. Reference strains are NL4-3
(K101E+G190S) in panel A and NL4-3 (G190S) in panel B. The average and standard
deviation of the production rate ratio (PRR) is shown on each the graph for the
quadruple mutant. (Panel C) EFV susceptibility assay.
232 J. Wang et al. / Virology 402 (2010) 228–237
Although there appeared to be a correlation between reduced
replication ﬁtness and the presence of EFV-dependent stimulation,
this also was not a consistent ﬁnding, since the D10 mutant RT
sequence conferred improved ﬁtness and augmented EFV-dependent
stimulation compared to virus with an NL4-3 RT backbone containing
the same resistance mutations. Moreover, we observed this phenom-
enon in two different RT backbones. Studies in which resistance
mutations in the D10 backbone were reverted to wild type indicate
that some RT polymorphisms can augment the impact of K101E+
G190S on EFV-dependent stimulation of replication and reduce the
ability of M41L+T215Y to reverse the drug-dependent stimulation
conferred by K101E+G190S. The modulating effects of RT poly-
morphisms in the D10 RT backbone are dependent on the presence of
K101E (and also presumably G190S, although this was not directly
tested in our studies).
Another important ﬁnding of these studies is that the NNRTI resistance
mutations K101E and G190S interact to affect a variety of phenotypes,
leading to increased EFV resistance, reduced replication ﬁtness in the
absence of drug, and EFV-dependent stimulation of virus replication. It is
important to note that these phenotypes could not be predicted from
studies of each single mutant. A limited number of previously published
studies have evaluated interactions among NNRTI-resistant variants and
have also found unexpected effects on replication ﬁtness (Collins et al.,
2004; Koval et al., 2006). Understanding interactions among NNRTI
resistance mutations is important, since clinical virus isolates are evolving
more complex patterns of NNRTI resistance mutations, now that next-
generation NNRTIs such as etravirine are being used to treat patients who
have failed ﬁrst-line NNRTIs.
These studies have also demonstrated important interactions
between nucleoside and NNRTI resistance mutations that affect all of
the phenotypes that we evaluated: EFV resistance, replication ﬁtness
in the absence of drug, and EFV-dependent stimulation of virus
replication. L74V, in addition to improving the ﬁtness of G190E and
K103N+L100I (Kleim et al., 1996; Koval et al., 2006), also has the
same effect on (K101E+G190S). This ﬁnding is compatible with
previously published studies of L74V and NNRTI-resistant variants
(Boyer et al., 1998; Koval et al., 2006; Trivedi et al., 2008). Our study,
however, is the ﬁrst demonstration that thymidine analog mutations
(TAMs), such as M41L+T215Y, can also augment the replication ﬁt-
ness of an NNRTI-resistant mutant. The TAM double mutant (M41L+
T215Y) also abolishes EFV-dependent stimulation of growth, similar
to L74V, but unlike L74V, sensitizes the virus to EFV. The ability of
these two TAMs to sensitize HIV to EFV is consistent with a number of
previously published studies demonstrating the inﬂuence of TAMs on
EFV hyper-susceptibility (Clark et al., 2006; Shulman et al., 2004;
Whitcomb et al., 2002). Our studies are shown to be in agreement
with those of Huang et al. (2003), who tested the resistance and
ﬁtness of patient RT sequences with various substitutions at the G190
position. They showed that the ﬁtness of G190 mutations correlated
with their prevalence in patients and that they were primarily
responsible for the NNRTI resistance pattern. They also showed that
the ﬁtness of very poorly replicating mutants was better in the patient
backbone where the mutation occurred and that L74V enhanced the
replication of G190S and other mutants, which is consistent with our
results. They believe the reduced ﬁtness of G190 substitutions is the
result of reduced RT in the virions. They did not see any stimulation of
virus replication by NNRTIs.
Our studies have demonstrated that each of these three pheno-
types can inﬂuence the relative prevalence of two mutants in culture.
The relative importance of EFV-dependent stimulation in a clinical
setting is unclear but could very well contribute to selection for an
otherwise unﬁt mutant. Although the culture conditions studied do
Fig. 6. Impact of drug resistance mutations in the D10 RT backbone on HIV-1 replication
in the absence and presence of EFV. (Panels A and B) Growth competition assays of
virus with the D10 RT in which K101E (panel A) or [M41L+T215Y] (panel B) were
reverted to wild type, competed against NL4-3 with the intact D10 RT as a reference
strain. The average and standard deviation of the production rate ratio (PRR) is shown
on each graph. (Panel C) EFV susceptibility assays with variants of D10 in which
different resistance mutations were reverted to wild type. The peak EFV-dependent
stimulation concentration for D10 with M41L+T215Y back mutated to wild type was
3200 nM EFV and the fold stimulation was 100.73± 28.8; wt, wild type.
233J. Wang et al. / Virology 402 (2010) 228–237
not fully replicate the situation in patients, a number of studies have
found that relative replication in cell culture of drug-resistant mutants
generally correlates with their prevalence in patients (Archer et al.,
2000; Garcia-Lerma et al., 2000; Gerondelis et al., 1999; Harrigan et
al., 1998; Hu et al., 2006; Huang et al., 2003; Kosalaraksa et al., 1999;
Koval et al., 2006; Perrin and Mammano, 2003; Sugiura et al., 2002;
Wang et al., 2006). One concern in extrapolating results from tissue
culture experiments to patients is that the protein concentration in
cell culture is lower than patient serum, and since efavirenz is highly
protein-bound, free drug concentrations are likely quite different in
the experimental and clinical settings. One study, which directly
compared efavirenz IC90s under standard tissue culture conditions to
those obtained in protein concentrations similar to that in serum,
derived a protein-binding factor of 16.5 to allow comparisons of IC90s
with plasma concentrations in patients (Corbett et al., 1999). Using
this factor adjusts mean Cmin and Cmax values of 5.6 μM and 12.9 μM
observed in patients (quoted in the efavirenz package insert) to give
corresponding tissue culture values of 339 nM and 781 nM, respec-
tively, which are in the range of the efavirenz concentrations at which
we observed stimulation of the K101E+G190S mutants. Although
these ﬁgures are estimates, they do suggest that efavirenz-dependent
stimulation of these mutants occurs at clinically relevant efavirenz
concentrations. Our studies also demonstrate that EFV-dependent
stimulation contributes to a selective advantage for some mutants in
certain drug concentrations, and this property may contribute to the
selection of some drug-resistant mutants in clinical infection.
There is no current evidence that explains the mechanism by which
EFV stimulates virus replication. However, we believe that it is through
its interaction with RT and not some other protein. There are several
possible mechanisms for the stimulation. It could enhance RT protein
incorporation into virions, polymerization, dimerization, and/or RNase
H activity. We believe that the latter two mechanisms are most likely
since EFV has already been shown to enhance these activities. EFV
enhances dimerization of HIV-1 reverse transcriptase (Tachedjian et al.,
2001), and the wild-type 101 K residue in the p66 subunit can form a
salt bridge with 138E in the p51 subunit (Ren et al., 2006; Ren and
Stammers, 2008). Thus, it is possible that the K101E mutation in
combination with G190S leads to destabilization of the reverse
transcriptase heterodimer that is partially compensated for by weak
binding of EFV,resulting in EFV-dependent stimulation of HIV-1 reverse
transcription. Unlike 101 K, the wild-type 230 M residue does not
appear todirectly promotedimerization, although it possibly could have
indirect effects on dimer stability. It is unclear at present how both L74V
and TAMs might reduce or eliminate EFV-dependent stimulation; this
effect appears independent of the effects of these nucleoside resistance
mutations on EFV IC
. Our preliminary evidence shows that dimeriza-
tion of K101E+G190S is not stimulated by EFV. However, we plan to
more thoroughly examine the mechanism of dimerization in order to
deﬁnitively determine if it is responsible for stimulation.
An alternative potential explanation for EFV-dependent stimulation
of virus replication is that EFV, which has been found to increase RNase
H cleavage rates (Palaniappan et al., 1995; Radzio and Sluis-Cremer,
2008), could compensate for RNase H defects, which have been
documented for several NNRTI-resistant mutants (Archer et al., 2000;
Gerondelis et al., 1999; Wang et al., 2006). We believe that this
hypothesis is more likely and may depend on theparticular combination
of mutations. Not all NNRTI-resistant mutants that have reduced RNase
H activity are stimulated by EFV, but it will be important to determine
whether certain combinations do, and if nucleoside resistance muta-
tions can affect the stimulation of RNase H activity by EFV.
In summary, these studies demonstrate that the replication of some
NNRTI-resistant mutants is stimulated by EFV and that nucleoside
resistance mutationscan reverse this phenotype. In addition, nucleoside
Fig. 7. Impact of the nucleoside resistance mutation L74V on replication by (K101E+
G190S) NL4-3 in the absence and presence of EFV. (Panels A and B) Growth competition
experiments. The average and standard deviation of the production rate ratio (PRR) is
shown on each graph. (Panel C) EFV susceptibility assay. x-Axis, EFV concentration; y-
p24 concentration in culture supernatant 6 days after infection.
234 J. Wang et al. / Virology 402 (2010) 228–237
and NNRTI resistance mutations can interact in complex ways to affect
replication ﬁtness in the absence of drug, EFV resistance, and EFV-
dependent stimulation of virus replication, and these effects can be
modulated by RT polymorphisms not known to be involved in drug
resistance. The mechanism(s) and clinical signiﬁcance of EFV-depen-
dent stimulation are not understood, but this phenomenon has
implications for the more rational design of effective NNRTI combina-
Materials and methods
Reagents and cells
The following reagents were obtained through the AIDS Research
and Reference Reagent Program, Division of AIDS, National Institute of
Allergy and Infectious Disease: the infectious molecular clone pNL4-3
was obtained from Malcolm Martin, and the PM-1 neoplastic CD4+ T
cell line expressing both the CCR5 and CXCR4 co-receptors was
obtained from Marvin Reitz (Adachi et al., 1986; Lusso et al., 1995).
EFV was obtained from Dupont Pharmaceuticals Company; it was
dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mg/
ml (15.7 mM) and stored at −20 °C. The human primary embryonal
kidney cell line 293 (American Type Culture Collection; Manassas, VA)
was grown in Dulbecco's modiﬁed Eagle's medium (DMEM) supple-
mented with 10% (vol/vol) fetal bovine serum, L-glutamine (2 mM),
penicillin (100 U/ml), and streptomycin (100 U/ml). PM1 cells were
grown in RPMI supplemented with 10% fetal bovine serum, penicillin
(100 U/ml), and streptomycin (100 U/ml). Primary human peripheral
blood mononuclear cells (PBMCs) were isolated by Ficoll gradient
centrifugation from blood that was obtained from HIV-uninfected
volunteer donors, after obtaining written informed consent. PBMCs
were cryopreserved and stored in liquid nitrogen until needed for
studies. PBMCs were stimulated with 5 μg/ml phytohemagglutinin
(PHA) and 20 U/ml interleukin-2 (IL-2) for 2 days before being
infected and were grown in RPMI supplemented with 20% fetal bovine
serum. All cells were propagated in 5% CO
at 37 °C.
Stored plasma samples were obtained from HIV-infected subjects
who received EFV in combination with indinavir, after obtaining
informed consent in the DMP 266-003 study; a subset of patients that
initially received indinavir monotherapy subsequently received EFV
in combination with the nucleoside analogs stavudine (d4T) and
lamivudine (3TC) (Bacheler et al., 2000). One stored sample identiﬁed
as having K101E+G190S on bulk sequence was further evaluated.
These studies are compliant with federal guidelines relating to human
subjects research and were approved by the University of Rochester
Research Subjects Review Board (RSRB).
Subcloning patient RT sequences
Viral RNA was extracted from patient plasma using QIAamp
MinElute Virus Spin Kit (Qiagen, Inc., Valencia, CA). The HIV-1 RT
sequence spanning amino acids 15–560 of RT was ampliﬁed using
primers containing silent XmaI and XbaI restriction enzyme cleavage
Fig. 8. Summary of interactions among resistance mutations and RT polymorphisms that affect HIV-1 resistance to EFV, EFV-dependent stimulation of virus replication, and
replication ﬁtness in the absence of drug. Rectangles represent the RT sequence from codons 15–560. RT backbone polymorphisms are represented by the color of the rectangle:
white, NL4-3, and black, D10. NNRTI resistance mutations are in regular font, nucleoside resistance mutations are in bold. Relative ﬁtness value is the mean PRR ± standard deviation.
The value “maximal stimulation by EFV”is deﬁned as the highest p24 antigen concentration observed in the drug susceptibility assay, divided by the p24 antigen concentration of the
no-drug control. Values represent the mean ± standard deviation of at least three replicates. Viral variants for which the maximal stimulation is “none”had no p24 values in the
presence of EFV that were signiﬁcantly higher than the no-drug control. The concentrations of EFV at which maximal stimulation occurred are listed in the last column. n/a, not
235J. Wang et al. / Virology 402 (2010) 228–237
sites, as described previously (Dykes et al., 2001). PCR products were
cloned into the pCR2.1 TOPO vector (TOPO TA cloning kit, Invitrogen,
Inc.; Valencia, CA). After verifying the RT sequence using forward and
reverse primers, plasmids were digested with XmaI and XbaI, and the
RT insert was cloned into pNL4-3XX as described previously (Dykes
et al., 2001).
The L74V,K101E, and M41L+T215Y mutationswere introducedinto
pNL4-3XX containing wild-type or G190S RT sequences, as previously
described(Wang et al., 2006). Mutagenic primers were as follows:L74V,
5′-CAG TAC TAA ATG GAG AAA AGT AGT AGA TTT CAG AGA AC-3′
(forward) and 5′-GTT CTC TGA AAT CTA CTA CTT TTC TCC ATT TAG TAC
TG-3′(reverse); K101E, 5′-GCA GGG TTA GAA AAG AAA AAA TCA G-3′
(forward) and 5′-CTG ATT TTT TCT TTT CTA ACC CTG C-3′(reverse);
M41L, 5′-GCA TTA GTA GAAATT TGT ACA GAA TTG GAA AAG GAA GG-3′
(forward) and 5′-CC TTC CTT TTC CAA TTC TGT ACA AAT TTC TAC TAA
TGC-3′(reverse); and T215Y, 5′-GTG GGG ATT TTA CAC ACC AGA CAA
AAA AC-3′(forward) and 5′-GT TTT TTG TCT GGT GTG TAA AAT CCC
CAC-3′(reverse). M230L was introduced into wild-type pNL4-3 using
the primers: 5′-CCT TTG GCT GGG TTA TGA ACT CCA TC-3′(forward)
and 5′-GAT GGA GTT CAT AAC CCA GCC AAA GG-3′(reverse).
Generation of virus stocks
The cell line, 293, was transiently transfected with 40 µg of each
plasmid DNA by lipofection (SuperFect, Qiagen, Santa Clarita, CA);
supernatants were harvested after 72 h, and stored at −80 °C. HIV-1
virus capsid protein (p24) quantitation was performed on virus stocks
using an ELISA (Perkin Elmer; Wellesley, MA).
Replication ﬁtness assays
HIV-1 replication ﬁtness was quantiﬁed using a multiple cycle
growth competition assay in the PM1 cell line; a subset was also tested
in the H9 T-cell line and in IL-2 and PHA-stimulated PBMC. The relative
proportions of the test and reference strains were measured using
direct sequence analysis, as previously described (Archer et al., 2000;
Koval et al., 2006; Wang et al., 2006). Virus replication was quantiﬁed
by measuring p24 antigen content in the culture supernatant. We
quantiﬁed relative replication ﬁtness using the production rate ratio
(PRR), which is the ratio of the relative infection rate of the mutant and
reference strains (Wu et al., 2006), using a publicly available web site
Drug susceptibility assays
The effects of EFV on virus replication were measured using a
modiﬁcation of the ACTG/DoD method (Japour et al., 1993) in both
PM1 cells and PHA- and IL2-stimulated PBMC cells, as previously
described (Koval et al., 2006). A virus inoculum of 150 ng p24 was
used to infect 3 × 10
PM1 cells or 4 × 10
stimulated PBMCs in a total
volume of 1 ml in the absence of drug. Cells were then washed and
cultured either in the absence of drug or in the presence of varying
concentrations of EFV, which were determined empirically for each
mutant (maximum concentration was 25.6 μM, with no evidence for
cytotoxicity as measured by trypan blue exclusion). Virus replication
was assayed at day 6 after infection by measuring p24 antigen
concentration in the culture supernatant. At a minimum, all assays
were performed in triplicate.
The K101E+G190S and G190S mutantswere also evaluated for EFV-
dependent stimulation of replication using a modiﬁcation of a pre-
viously published HIV replication ﬁtness assay in which infected cells
are detected by ﬂow cytometry using antibodies directed against a
virus-expressed Thy 1 reporter gene (Dykes et al., 2006). The K101E+
G190S and G190S mutants were subcloned into the pAT2 vector, using
silent XmaIandXbaI sites ﬂanking reverse transcriptase, as previously
described (Dykes et al., 2006). The pAT2 vector was derived from pNL4-
3andcarriestheThy 1.2 gene in place of nef. Virus stocks derived by
transient transfection of 293 cells were used to infect PM1 cells using
inocula designed to give similar levels of replication in the no-drug
control (5 ng p24 per million cells for G190S and 50 ng p24 per million
cells for K101E+G190S). Thy 1.2 expressed on the surface of infected
cells was detected using R-phycoerythrin (PE)-labeled antibody, as
previously described, 6 days after infection (Dykes et al., 2006). The
limit of detection of this assay is approximately 0.05% of cells.
Calculation of IC
Traditionally, the following nonlinear model has been used to
estimate the IC
where F(x) is the proportional reduction in virus replication at a
given drug concentration, relative to a no-drug control, and dis a
shape parameter (Chou, 1976). An important feature of this model is
that the value F(x) increases from 0% to 100% as drug concentration
increases. This model does not account for the possibility of virus
replication in the presence of drug being greater than 100% of the no-
drug control (i.e., drug-dependent stimulation of virus replication).
We therefore applied a more ﬂexible, non-parametric model to ﬁt
the observations when drug-dependent stimulation of virus replica-
tion occurs. This model can be used to describe curves determined by
observations and has been widely used in biological and biomedical
research (Fan and Gijbels, 1996; King and Roth, 2003). Our approach
to estimate IC
for a mutant whose growth is also stimulated at some
drug concentrations is a novel application of this approach. We
assumed that the percentage Yand concentration xare related in the
form Y=m(x), where m(•) is a function in the mathematical sense,
but did not put any restrictions on the form of m(•), i.e. whether m(•)
is linear or nonlinear in x, etc. Hence, it is up to empirical analysis to
use the observed data to ﬁnd out more about m(•). We used the
) to estimate m(•) by adapting
local linear regression techniques (Fan and Gijbels, 1996), referred
to as m
(x). The basic idea of local linear regression is that we estimate
, then we ﬁt locally a straight line using the observations in a
window around x
. After ﬁtting the line, the estimation m
provided by the value of this line at x
. By repeating this procedure for
, one can get the estimation function m
(x). The window width
is constant. Formally, the local linear regression is computed by
solving a weighted least square problem. The IC
may be identiﬁed as
the point x*, which satisﬁes m
(x*) =0.5. Data analysis was conducted
using the statistical software R. For each experiment, we applied our
nonparametric approach to obtain an IC
value. Then we used Wilcox
test for a comparison of IC
values for each pair of mutants to obtain a
This work was supported in part by NIH R01 AI-041387 to L.M.D.,
NIH R01 AI-59773 to H.L, NSF grant DMS-0806097 to H.L., and the
University of Rochester Developmental Center for AIDS Research (D-
CFAR) P30 AI-078498. These funding sources played no role in the
study design, data collection or analysis, or in the manuscript
submission. We thank Kora Fox, Dongge Li, and Sue Liu for the
excellent technical assistance and Robert Bambara for the helpful
comments on the manuscript.
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