Non-cleavage site gag mutations in amprenavir-resistant human immunodeficiency virus type 1 (HIV-1) predispose HIV-1 to rapid acquisition of amprenavir resistance but delay development of resistance to other protease inhibitors.
ABSTRACT In an attempt to determine whether mutations in Gag in human immunodeficiency virus type 1 (HIV-1) variants selected with a protease inhibitor (PI) affect the development of resistance to the same or a different PI(s), we generated multiple infectious HIV-1 clones carrying mutated Gag and/or mutated protease proteins that were identified in amprenavir (APV)-selected HIV-1 variants and examined their virological characteristics. In an HIV-1 preparation selected with APV (33 passages, yielding HIV(APVp33)), we identified six mutations in protease and six apparently critical mutations at cleavage and non-cleavage sites in Gag. An infectious recombinant clone carrying the six protease mutations but no Gag mutations failed to replicate, indicating that the Gag mutations were required for the replication of HIV(APVp33). An infectious recombinant clone that carried wild-type protease and a set of five Gag mutations (rHIV(WTpro)(12/75/219/390/409gag)) replicated comparably to wild-type HIV-1; however, when exposed to APV, rHIV(WTpro)(12/75/219/390/409gag) rapidly acquired APV resistance. In contrast, the five Gag mutations significantly delayed the acquisition of HIV-1 resistance to ritonavir and nelfinavir (NFV). Recombinant HIV-1 clones containing NFV resistance-associated mutations, such as D30N and N88S, had increased susceptibilities to APV, suggesting that antiretroviral regimens including both APV and NFV may bring about favorable antiviral efficacy. The present data suggest that the preexistence of certain Gag mutations related to PI resistance can accelerate the emergence of resistance to the PI and delay the acquisition of HIV resistance to other PIs, and these findings should have clinical relevance in the therapy of HIV-1 infection with PI-including regimens.
- SourceAvailable from: pu.ru
Article: HIV drug resistance.New England Journal of Medicine 04/2004; 350(10):1023-35. · 51.66 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Two different responses to the therapy were observed in a group of patients receiving the protease inhibitor indinavir. In one, suppression of virus replication occurred and has persisted for 90 weeks (bDNA, < 500 human immunodeficiency virus type 1 [HIV-1] RNA copies/ml). In the second group, a rebound in virus levels in plasma followed the initial sharp decline observed at the start of therapy. This was associated with the emergence of drug-resistant variants. Sequence analysis of the protease gene during the course of therapy revealed that in this second group there was a sequential acquisition of protease mutations at amino acids 46, 82, 54, 71, 89, and 90. In the six patients in this group, there was also an identical mutation in the gag p7/p1 gag protease cleavage site. In three of the patients, this change was seen as early as 6 to 10 weeks after the start of therapy. In one patient, a second mutation occurred at the gag p1/p6 cleavage site, but it appeared 18 weeks after the time of appearance of the p7/p1 mutation. Recombinant HIV-1 variants containing two or three mutations in the protease gene were constructed either with mutations at the p7/p1 cleavage site or with wild-type (WT) gag sequences. When recombinant HIV-1-containing protease mutations at 46 and 82 was grown in MT2 cells, there was a 68% reduction in its rate of replication compared to the WT virus. Introduction of an additional mutation at the gag p7/p1 protease cleavage site compensated for the partially defective protease gene. Similarly, rates of replication of viruses with mutations M46L/I, I54V, and V82A in protease were enhanced both in the presence and in the absence of Indinavir when combined with mutations in the gag p7/p1 and the gag p1/p6 cleavage sites. Optimal rates of virus replication require protease cleavage of precursor polyproteins. A mutation in the cleavage site that enhanced the availability of a protein that was rate limiting for virus maturation would confer on that virus a significant growth advantage and may explain the uniform emergence of viruses with alterations at the p7/p1 cleavage site. This is the first report of the emergence of mutations in the gag p7/p1 protease cleavage sites in patients receiving protease therapy and identifies this change as an important determinant of HIV-1 resistance to protease inhibitors in patient populations.Journal of Virology 09/1997; 71(9):6662-70. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Mortality among human immunodeficiency virus (HIV)-infected individuals has decreased dramatically in countries with good access to treatment and may now be close to mortality in the general uninfected population. To evaluate changes in the mortality gap between HIV-infected individuals and the general uninfected population. Mortality following HIV seroconversion in a large multinational collaboration of HIV seroconverter cohorts (CASCADE) was compared with expected mortality, calculated by applying general population death rates matched on demographic factors. A Poisson-based model adjusted for duration of infection was constructed to assess changes over calendar time in the excess mortality among HIV-infected individuals. Data pooled in September 2007 were analyzed in March 2008, covering years at risk 1981-2006. Excess mortality among HIV-infected individuals compared with that of the general uninfected population. Of 16,534 individuals with median duration of follow-up of 6.3 years (range, 1 day to 23.8 years), 2571 died, compared with 235 deaths expected in an equivalent general population cohort. The excess mortality rate (per 1000 person-years) decreased from 40.8 (95% confidence interval [CI], 38.5-43.0; 1275.9 excess deaths in 31,302 person-years) before the introduction of highly active antiretroviral therapy (pre-1996) to 6.1 (95% CI, 4.8-7.4; 89.6 excess deaths in 14,703 person-years) in 2004-2006 (adjusted excess hazard ratio, 0.05 [95% CI, 0.03-0.09] for 2004-2006 vs pre-1996). By 2004-2006, no excess mortality was observed in the first 5 years following HIV seroconversion among those infected sexually, though a cumulative excess probability of death remained over the longer term (4.8% [95% CI, 2.5%-8.6%] in the first 10 years among those aged 15-24 years). Mortality rates for HIV-infected persons have become much closer to general mortality rates since the introduction of highly active antiretroviral therapy. In industrialized countries, persons infected sexually with HIV now appear to experience mortality rates similar to those of the general population in the first 5 years following infection, though a mortality excess remains as duration of HIV infection lengthens.JAMA The Journal of the American Medical Association 08/2008; 300(1):51-9. · 29.98 Impact Factor
JOURNAL OF VIROLOGY, Apr. 2009, p. 3059–3068
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 7
Non-Cleavage Site Gag Mutations in Amprenavir-Resistant Human
Immunodeficiency Virus Type 1 (HIV-1) Predispose HIV-1 to Rapid
Acquisition of Amprenavir Resistance but Delay Development of
Resistance to Other Protease Inhibitors?
Manabu Aoki,1,2David J. Venzon,3Yasuhiro Koh,1Hiromi Aoki-Ogata,1Toshikazu Miyakawa,1
Kazuhisa Yoshimura,1Kenji Maeda,1,4and Hiroaki Mitsuya1,4*
Departments of Hematology and Infectious Diseases, Kumamoto University Graduate School of Medical and Pharmaceutical Sciences,
Kumamoto 860-8556, Japan1; Institute of Health Sciences, Kumamoto Health Science University, Kumamoto 861-5598, Japan2; and
Biostatistics and Data Management Section3and Experimental Retrovirology Section, HIV and AIDS Malignancy Branch,4
Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Received 10 December 2008/Accepted 20 January 2009
In an attempt to determine whether mutations in Gag in human immunodeficiency virus type 1 (HIV-1)
variants selected with a protease inhibitor (PI) affect the development of resistance to the same or a
different PI(s), we generated multiple infectious HIV-1 clones carrying mutated Gag and/or mutated
protease proteins that were identified in amprenavir (APV)-selected HIV-1 variants and examined their
virological characteristics. In an HIV-1 preparation selected with APV (33 passages, yielding HIVAPVp33),
we identified six mutations in protease and six apparently critical mutations at cleavage and non-cleavage
sites in Gag. An infectious recombinant clone carrying the six protease mutations but no Gag mutations
failed to replicate, indicating that the Gag mutations were required for the replication of HIVAPVp33. An
infectious recombinant clone that carried wild-type protease and a set of five Gag mutations
cantly delayed the acquisition of HIV-1 resistance to ritonavir and nelfinavir (NFV). Recombinant HIV-1
clones containing NFV resistance-associated mutations, such as D30N and N88S, had increased suscep-
tibilities to APV, suggesting that antiretroviral regimens including both APV and NFV may bring about
favorable antiviral efficacy. The present data suggest that the preexistence of certain Gag mutations
related to PI resistance can accelerate the emergence of resistance to the PI and delay the acquisition of
HIV resistance to other PIs, and these findings should have clinical relevance in the therapy of HIV-1
infection with PI-including regimens.
12/75/219/390/409gag) replicated comparably to wild-type HIV-1; however, when exposed to APV,
12/75/219/390/409gagrapidly acquired APV resistance. In contrast, the five Gag mutations signifi-
Combination antiretroviral therapy using reverse transcrip-
tase inhibitors and protease inhibitors (PIs) produces substan-
tial suppression of viral replication in human immunodefi-
ciency virus type 1 (HIV-1)-infected patients (3, 27, 28, 42).
However, the emergence of drug-resistant HIV-1 variants in
such patients has limited the efficacy of combination chemo-
therapy. HIV-1 variants resistant to all of the currently avail-
able antiretroviral therapeutics have emerged both in vitro and
in vivo (6, 16, 27, 30). Of note, a number of PI resistance-
associated amino acid substitutions in the active site of pro-
tease have been identified, and such substitutions have consid-
erable impact on the catalytic activity of protease. This impact
is reflected by impaired processing of Gag precursors in mu-
tated-protease-carrying virions and by decreased catalytic effi-
ciency of the protease toward peptides with natural cleavage
sites (7, 29, 31, 43).
However, the highly PI-resistant viruses frequently have
amino acid substitutions at the p7-p1 and p1-p6 cleavage
sites in Gag. These mutations have been identified in PI-
resistant HIV-1 variants selected in vitro (2, 5, 8, 29) and in
HIV-1 isolated from patients with AIDS for whom chemo-
therapy including PIs was failing (26, 40, 47, 48). These
mutations are known to compensate for the enzymatic im-
pairment of protease, per se, resulting from the acquisition
of PI resistance-conferring mutations within the protease-
encoding region. Moreover, certain mutations at non-cleav-
age sites in Gag have been shown previously to be essential
for the replication of HIV-1 variants in the presence of PIs
(14, 15). Although a few amino acid substitutions at cleav-
age and non-cleavage sites in Gag have been shown to be
associated with resistance to PIs, the roles and impact of
amino acid substitutions in Gag for the HIV-1 acquisition of
PI resistance remain to be elucidated.
In the present study, we identified novel Gag non-cleavage
site mutations in addition to multiple mutations in the protease
gene during in vitro selection of HIV-1 variants highly resistant
to amprenavir (APV). We show that the non-cleavage site
mutations were important for not only the replication of the
mutated-protease-carrying HIV-1 but also the accelerated ac-
quisition of HIV-1 resistance to APV and an unrelated PI,
nelfinavir (NFV). We also show that recombinant HIV-1
clones containing NFV resistance-associated mutations, such
* Corresponding author. Mailing address: Department of Hematology,
Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-
8556, Japan. Phone: (81) 96-373-5156. Fax: (81) 96-363-5265. E-mail:
?Published ahead of print on 28 January 2009.
as D30N and N88S, had increased susceptibility to APV, sug-
gesting that antiretroviral regimens including both APV and
NFV may bring about favorable antiviral efficacy.
MATERIALS AND METHODS
Cells and antiviral agents. MT-2 and MT-4 cells were grown in RPMI 1640-
based culture medium, and 293T cells were propagated in Dulbecco’s modified
Eagle’s medium. These media were supplemented with 10% fetal calf serum
(HyClone, Logan, UT), 50 U/ml penicillin, and 50 ?g/ml streptomycin. APV was
kindly provided by GlaxoSmithKline, Research Triangle Park, NC. Saquinavir
(SQV) and ritonavir (RTV) were provided by Roche Products Ltd. (Welwyn
Garden City, United Kingdom) and Abbott Laboratories (Abbott Park, IL),
respectively. NFV and indinavir (IDV) were kindly provided by Japan Energy
Generation of PI-resistant HIV-1 in vitro. For the generation of PI-resistant
HIV-1, various PI-resistant HIV-1 strains were propagated in the presence of
increasing concentrations of a drug in a cell-free fashion as described previously
(44, 45). In brief, on the first passage, MT-2 or MT-4 cells (5 ? 105) were exposed
to 500 50% tissue culture infective doses (TCID50) of each infectious molecular
HIV-1 clone and cultured in the presence of various PIs at initial concentrations
of 0.01 to 0.06 ?M. On the last day of each passage (approximately day 7), 1 ml
of the cell-free supernatant was harvested and transferred to a culture of fresh
uninfected cells in the presence of increased concentrations of the drug for the
following round of culture. In this round of culture, three drug concentrations
(increased by one-, two-, and threefold compared to the previous concentration)
were employed. When the replication of HIV-1 in the culture was confirmed by
substantial Gag protein production (greater than 200 ng/ml), the highest drug
concentration among the three concentrations was used to continue the selection
(for the next round of culture). This protocol was repetitively used until the drug
concentration reached the targeted concentration. Proviral DNA from the ly-
sates of infected cells at various passages was subjected to nucleotide sequencing.
Determination of nucleotide sequences. Molecular cloning and the determi-
nation of nucleotide sequences of HIV-1 passaged in the presence of each PI
were performed as described previously (44, 45). In brief, high-molecular-weight
DNA was extracted from HIV-1-infected MT-2 and MT-4 cells by using the
InstaGene matrix (Bio-Rad Laboratories, Hercules, CA) and was subjected to
molecular cloning, followed by sequence determination. The primers used for
the first-round PCR amplification of the entire Gag- and protease-encoding
regions of the HIV-1 genome were LTR F1 (5?-GAT GCT ACA TAT AAG
CAG CTG C-3?) and PR12 (5?-CTC GTG ACA AAT TTC TAC TAA TGC-3?).
The first-round PCR mixture consisted of 5 ?l of proviral DNA solution, 2.0 U
of premix Taq (Ex Taq version; Takara Bio Inc., Otsu, Japan), and 12.5 pmol of
each of the first-round PCR primers in a total volume of 50 ?l. The PCR
conditions used were an initial 2-min step at 94°C, followed by 30 cycles of 30 s
at 94°C, 30 s at 58°C, and 3 min at 72°C, with a final 8 min of extension at 72°C.
The first-round PCR products (1 ?l) were used directly in the second round of
PCR with primers LTR F2 (5?-GAG ACT CTG GTA ACT AGA GAT C-3?) and
Ksma2.1 (5?-CCA TCC CGG GCT TTA ATT TTA CTG GTA C-3?) under the
same PCR conditions described above. The second-round PCR products were
purified with spin columns (MicroSpin S-400 HR; Amersham Biosciences Corp.,
Piscataway, NJ), cloned directly, and subjected to sequencing with a model 377
automated DNA sequencer (Applied Biosystems, Foster City, CA).
Generation of recombinant HIV-1 clones. The PCR products obtained as
described above were digested with two of the three enzymes BssHII, ApaI, and
SmaI, and the obtained fragments were introduced into pHIV-1NLSma, designed
to have a SmaI site by changing two nucleotides (2590 and 2593) of pHIV-1NL4-3
(15, 19). To generate HIV-1 clones carrying the mutations, site-directed mu-
tagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La
Jolla, CA) was performed, and the mutation-containing genomic fragments were
introduced into pHIV-1NLSma. Determination of the nucleotide sequences of
plasmids confirmed that each clone had the desired mutations but no unintended
mutations. 293T cells were transfected with each recombinant plasmid by using
Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA), and the thus-obtained
infectious virions were harvested 48 h after transfection and stored at ?80°C
Drug sensitivity assays. Assays for HIV-1 p24 Gag protein production were
performed with MT-4 cells as described previously (1, 20, 24). In brief, MT-4
cells (105/ml) were exposed to 100 TCID50of infectious molecular HIV-1 clones
in the presence or absence of various concentrations of drugs and were incubated
at 37°C. On day 7 of culture, the supernatant was harvested and the amounts of
p24 Gag protein were determined by using a fully automated chemiluminescent
enzyme immunoassay system (Lumipulse F; Fujirebio Inc., Tokyo). The drug
concentrations that suppressed the production of p24 Gag protein by 50% (50%
inhibitory concentrations [IC50]) were determined by comparing the levels of p24
production with that in a drug-free control cell culture. All assays were per-
formed in triplicate.
Replication kinetic assay. MT-2 or MT-4 cells (105) were exposed to each
infectious HIV-1 clone (5 ng of p24 Gag protein/ml) for 3 h, washed twice with
phosphate-buffered saline, and cultured in 10 ml of complete medium as de-
scribed previously (1, 14). Culture supernatants (50 ?l) were harvested every
other day, and the p24 Gag amounts were determined as described above.
CHRA. Two titrated infectious clones to be compared for their replicative
capabilities or fitness in the competitive HIV-1 replication assay (CHRA)
were combined and added to freshly prepared MT-4 cells (2 ? 105) in the
presence or absence of various concentrations of PIs as described previously
(21, 36). Briefly, a fixed amount (200 TCID50) of one infectious clone was
combined with three different amounts (100, 200, and 300 TCID50) of the
other infectious clone, and the mixture was added to the culture of MT-4
cells. On the following day, one-third of infected MT-4 cells were harvested
and washed twice with phosphate-buffered saline, and cellular DNA was
extracted and subjected to nested PCR and sequencing as described above.
The HIV-1 coculture that best approximated a 50:50 mixture on day 1 was
further propagated, and the remaining cultures were discarded. Every 7 days,
the cell-free supernatant of the virus coculture was transmitted to fresh
uninfected MT-4 cells. The cells harvested at the end of each passage were
subjected to direct DNA sequencing, and viral population changes were
determined. The persistence of the original amino acid substitutions was
confirmed for all infectious clones used in this assay.
Statistical analysis of selection profiles of infectious HIV-1 clones. The selec-
tion profiles of various infectious HIV-1 clones were compared as follows.
The logarithms of the concentrations were modeled as normally distributed
variables with possible left censoring. The mean was assumed to be a qua-
dratic function of the passage number. The difference between two curves was
assessed by combining the estimated covariance-weighted differences of the
linear and quadratic coefficients and comparing the result to computer sim-
ulations for the same quantity generated under the specific null hypothesis for
that difference. SAS 9.1.3 (SAS Institute, Cary, NC) was used for all the
computations. All P values are two tailed, and for figures with more than two
curves, the values were corrected by the Hochberg method for multiple
Amino acid sequences of Gag and protease of HIV-1 pas-
saged in the presence of APV. A wild-type HIV-1 strain
(HIVWT) was propagated in MT-2 cells in the presence of
increasing concentrations of APV, and the proviral DNA se-
quences in those MT-2 cells were determined at passages 3, 12,
and 33 (Fig. 1). By passage 3, when HIV-1 was propagating in
the presence of 0.04 ?M APV (yielding HIVAPVp3), no amino
acid substitutions in protease were identified but 5 of 10 clones
had acquired the substitution of arginine for leucine at position
75 (L75R) in Gag. By passage 12 (at 0.18 ?M APV), two
APV-related resistance mutations (L10F and M46L) in pro-
tease had emerged and one mutation (H219Q) in Gag had
been added. By passage 33 (at 10 ?M; yielding HIVAPVp33), six
APV-related amino acid substitutions, one primary mutation
(I84V) and five secondary mutations (L10F, V32I, M46I,
I54M, and A71V), in protease had emerged (Fig. 1A). In
addition, a p1-p6 cleavage site mutation in Gag (L449F) was
identified in all 10 HIV-1 clones of HIVAPVp33examined, and
five non-cleavage site mutations (E12K, L75R, H219Q,
V390D, and R409K) were seen in Gag of HIVAPVp33(Fig. 1B).
Cleavage site mutations have been known to emerge when
amino acid substitutions in protease are accumulated and
HIV-1 develops resistance to PIs both in vitro and in vivo (5,
8). Intriguingly, the present data suggest that certain amino
acid substitutions in non-cleavage sites of Gag (i.e., L75R and
3060AOKI ET AL.J. VIROL.
H219Q) may emerge earlier and in greater numbers than
amino acid substitutions in protease, at least in the case of
HIV-1 selection with APV. The amino acid substitutions that
emerged in the virus and the pattern and order of such sub-
stitutions were largely in agreement with the data in the pre-
vious report by Gatanaga et al. (15). The present results sug-
gested that the non-cleavage site mutations observed may play
a key role in the development of HIV-1 resistance against PIs
and that especially the two Gag mutations H219Q and R409K
may be required for the development of PI resistance.
Mutations in Gag are required for the replication of
HIVAPVp33. In order to examine the effects of the mutations
identified in Gag as described above on the replication pro-
file of HIV-1, we generated infectious recombinant HIV-1
clones containing the six mutations (L10F, V32I, M46I,
I54V, A71V, and I84V) in protease seen in HIVAPVp33. A
recombinant HIV-1 clone containing the protease of
HIVAPVp33plus a wild-type Gag (rHIVAPVp33pro
the L449Fcleavage site
the 7-day period of culture (Fig. 2A), indicating that these
Gag species do not support the growth of HIVAPVp33.
Therefore, we next generated a recombinant HIV-1 clone con-
taining the protease of HIVAPVp33and the Gag protein with
the five non-cleavage site mutations (E12K, L75R, H219Q,
V390D, and R409K; rHIVAPVp33pro
replicated moderately under the same conditions (Fig. 2A).
The addition of the cleavage site mutation L449F, generating
lication of the virus. In MT-4 cells, in which HIV-1 generally
replicates more quickly and efficiently than in MT-2 cells,
449gag) failed to replicate in MT-2 cells over
12/75/219/390/409/449gag, further improved the rep-
erately; however, both rHIVAPVp33pro
HIVWT(Fig. 2B), due presumably to the greater replication of
HIV-1 in MT-4 cells, making the difference relatively indis-
tinct. These data clearly indicate that both non-cleavage site
and cleavage site mutations in Gag contribute to the robust
fitness of HIVAPVp33. We also attempted to examine the effects
of combined Gag mutations on the replication of HIV-1 con-
taining wild-type protease and generated three recombinant
HIV clones, rHIVWTpro
three recombinant clones turned out to be comparable to that
of HIVWTwhen examined in MT-2 and MT-4 cells (Fig. 2C
and D), unlike the finding by Doyon and his colleagues that the
cleavage site mutation L449F compromised the replication of
HIV-1 containing wild-type protease (8).
Gag mutations predispose HIV-1 to rapidly acquire APV
resistance. The appearance of two non-cleavage site mutations
(L75R and H219Q) in Gag prior to the emergence of muta-
tions in protease (Fig. 1) prompted us to examine whether
these two Gag mutations predisposed the virus to the acquisi-
tion of APV resistance-associated mutations in protease. We
thus attempted to select APV-resistant HIV-1 by propagating
HIVNL4-3(HIVWT) and rHIVWTpro
increasing concentrations of APV (Fig. 3). When we compared
the selection curves of these two viruses, there was no signifi-
cant difference (P, 0.53 and 0.65 for propagation in MT-2 and
MT-4 cells, respectively). We then examined the effects of two
mutated Gag species containing two and five mutations
(H219Q and R409K and E12K, L75R, H219Q, V390D, and
12/75/219/390/409/449gagreplicated comparably to
12/75/219/390/409gag. The replication rates of these
75/219gagin the presence of
FIG. 1. Amino acid sequences deduced from the nucleotide sequences of protease (A)- and Gag (B)-encoding regions of proviral DNA isolated
at the indicated passages (p3, p12, and p33) from HIV-1NL4-3variants selected in the presence of APV. The amino acid sequences of the protease
and Gag proteins of wild-type HIV-1NL4-3are shown at the top as a reference. Identity to the sequence at individual amino acid positions is
indicated by dots. The numbers of clones with the given amino acid substitutions among a total of 10 clones are listed.
VOL. 83, 2009NON-CLEAVAGE SITE Gag MUTATIONS AND RESISTANCE TO PIs3061
R409K [yielding mGag12/75/219/390/409gag], respectively) on the
selection curves. The selection profile of a newly generated
recombinant HIV clone (rHIVWTpro
ent from that of HIVWTin MT-2 cells (P ? 0.22); however,
219/409gag) was not differ-
than HIVWTwhen propagated in MT-4 cells (P ? 0.0001). The
recombinant clone with five non-cleavage site mutations
lines in the presence of APV significantly earlier than HIVWT,
with P values of 0.0080 and ?0.0001 for MT-2 and MT-4 cells,
respectively (Fig. 3).
We then asked whether additional amino acid substitutions
occurred and accelerated the acquisition of APV resistance by
the virus when the Gag mutations were present. To investigate
this issue, we determined the nucleotide sequence of the pro-
tease-encoding gene of each virus. Only one protease mutation
(L10F) wasseen by passage
ence of APV (Fig. 4A and B). In contrast, two mutations
(M46L and I84V) had been acquired by rHIVWTpro
passage 20. Of note, when rHIVWTpro
propagated in MT-2 cells in the presence of APV, a mutation
(L10F) had occurred by an early passage (passage 5) and four
mutations (L10F, V32I, M46I, and I84V) had emerged by
passage 17 (Fig. 4D). When examined in MT-4 cells, HIVWT
I84V and M46L and I84V, respectively) by passage 10, al-
had acquired three and four mutations (L10F, M46I, and I84V
and L10F, V32I, M46I, and I84V, respectively) by the same
passage (Fig. 4E to H). These data, taken together, indicate
that the two sets of Gag mutations (H219Q and R409K and
E12K, L75R, H219Q, V390D, and R409K) clearly predisposed
the virus to rapidly acquire APV resistance-associated muta-
tions in protease and begin to propagate in the presence
Gag mutations in HIVAPVp33delay viral acquisition of re-
sistance to other PIs. We next asked whether the presence of
219/409gagacquired resistance to APV much earlier
12/75/219/390/409gag) started to propagate in both cell
75/219gagwere propagated in MT-2 cells in the pres-
75/219gaghad acquired two mutations (L10F and
FIG. 2. Replication kinetics of Gag mutant clones with or without protease mutations. MT-2 cells (A and C) and MT-4 cells (B and D) were
exposed to Gag mutant clones with (A and B) or without (C and D) protease mutations. Virus replication was monitored by the amounts of p24
Gag produced in the culture supernatants. The results shown are representative of results from three independent experiments. HIVAPVp33variants
had six mutations (L10F, V32I, M46I, I54M, A71V, and I84V) in the viral protease.
FIG. 3. In vitro selection of APV-resistant variants using HIV-1 carrying
increasing concentrations of APV (starting at 0.03 ?M) in MT-2 cells
(A) or MT-4 cells (B). The selection was carried out in a cell-free
manner for a total of 14 to 29 passages. The results of statistical
evaluation of the selection profiles are as follows: panel A,
12/75/219/390/409gag(E), were propagated in the presence of
12/75/219/390/409gag, P ? 0.0065; rHIVWTpro
219/409gag, P ? 0.15; and rHIVWTpro
12/75/219/390/409gag, P ? 0.0001; rHIVWTpro
219/409gag, P ? 0.0001; and rHIVWTpro
219/409gag, P ? 0.088.
3062AOKI ET AL.J. VIROL.
the five Gag mutations (E12K, L75R, H219Q, V390D, and
R409K) accelerated the viral acquisition of resistance to
other currently available PIs (SQV, IDV, RTV, and NFV)
(Fig. 5). To this end, we propagated two HIV-1 strains
the presence of increasing concentrations of each PI and
compared the replication profiles. The initial drug concen-
trations used were 0.01 ?M for SQV, 0.03 ?M for IDV and
NFV, and 0.06 ?M for RTV, and each virus was selected by a
concentration of up to 5 ?M. The selection was carried out in
a cell-free manner for a total of 13 to 32 passages as described
previously (44, 45). There was no significant difference in the
selection profiles of the two strains when they were passaged in
the presence of SQV (P ? 0.8) or IDV (P ? 0.22) (Fig. 5A and
B). However, rHIVWTpro
nificantly later in the presence of RTV (P ? 0.0001 (Fig. 5C). The
selection profiles of HIVWTand rHIVWTpro
the presence of NFV were examined in two independent ex-
periments. Both curves in the first and second sets depicted in
Fig. 5D showed statistically significant difference, with P values
of ?0.0001 and 0.0016, respectively. These data strongly sug-
gest that the Gag mutations seen in HIVAPVp33predispose
HIV-1 to the rapid acquisition of APV resistance; however,
such Gag mutations delay the viral acquisition of resistance to
Gag mutations seen in HIVAPVp33do not affect viral suscep-
tibilities to PIs. Since the Gag mutations seen in HIVAPVp33
were found to contribute to the rapid acquisition of viral
resistance to APV but they delayed the emergence of viral
resistance to other PIs, we examined whether such Gag
mutations affected the susceptibilities of HIV-1 to various
PIs in the HIV-1 drug susceptibility assay. As shown in
Table 1, none of three sets of Gag mutations, as examined in
the context of rHIVWTpro
12/75/219/390/409gag) in MT-4 cells in
12/75/219/390/409gagstarted to replicate sig-
FIG. 4. Number of amino acid substitutions corresponding to the protease-encoding region of each infectious HIV-1 clone selected in the
presence of APV. Nucleotide sequences of proviral DNA of HIVWT(A and E) and three infectious HIV-1 clones, rHIVWTpro
and MT-4 cells (E to H) at the termination of each passage and compared to the nucleotide sequence of HIV-1NL4-3. The number within each
symbol represents the number of mutations identified in the protease when each infectious HIV-1 clone was selected in the presence of APV.
75/219gag(B and F),
219/409gag(C and G), and rHIVWTpro
12/75/219/390/409gag(D and H), were determined using lysates of HIV-1-infected MT-2 cells (A to D)
FIG. 5. In vitro selection of PI-resistant variants using HIV-1 carrying
‚) were propagated in MT-4 cells in the presence of increasing concen-
trations of SQV (A), IDV (B), RTV (C), or NFV (D). The initial drug
concentrations used were 0.01 ?M for SQV, 0.03 ?M for IDV and NFV,
and 0.06 ?M for RTV, and each virus was selected by up to a 5 ?M
concentration of each PI. The selection was carried out in a cell-free
manner for a total of 13 to 32 passages. NFV selection was performed
twice. Data from the first selection are shown with a solid line; the second
(with NFV at 0.7 ?M), and the data are shown with a dashed line. The
results of statistical evaluation of the selection profiles are as follows:
panel A, P ? 0.80; panel B, P ? 0.22; panel C, P ? 0.0001; and panel D,
first selection, P ? 0.0001, and second selection, P ? 0.0016.
VOL. 83, 2009NON-CLEAVAGE SITE Gag MUTATIONS AND RESISTANCE TO PIs 3063
HIV-1 to any of five PIs (APV, SQV, IDV, RTV, and NFV).
Indeed, the IC50s for HIVWTwere highly comparable to
those for any of the three recombinant clones carrying com-
bined Gag mutations.
Replication rate difference is not the cause of the contrast-
ing resistance acquisition patterns. Our observations of the
more rapidly than HIVWTwhen selected with APV (Fig. 3)
quisition of resistance to other PIs compared to HIVWT
(Fig. 5), prompted us to ask whether the replication rates of
affected by the presence of PIs. We therefore compared the
replication rates of rHIVWTpro
12/75/219/390/409gag, affected the susceptibility of
12/75/219/390/409gagacquired resistance to APV
12/75/219/390/409gagsignificantly delayed the ac-
12/75/219/390/409gagand HIVWTwere differentially
in the presence or absence of APV, SQV, IDV, RTV, or
NFV by using the CHRA (21). As shown in Fig. 6,
the absence or presence of PIs. Comparing the divergence
patterns of the curves for rHIVWTpro
HIVWTin the absence and presence of APV (Fig. 6A and B)
revealed that those for growth in the presence of APV
diverged more quickly than those for growth in the absence
of APV (Fig. 6B). However, similar divergence patterns
were seen with SQV, IDV, RTV, and NFV (Fig. 6C, D, E,
and F), suggesting that the replication advantage of
cause for the observed contrasting resistance acquisition
NFV resistance-conferring protease mutations increase
HIV-1 susceptibility to APV. There have been reports that an
12/75/219/390/409gagoutgrew HIVWTregardless of
12/75/219/390/409gagseen in the CHRA was not the
FIG. 6. Results from the CHRA for HIVWTand rHIVWTpro
HIVWT(f) and rHIVWTpro
?M RTV (E), or 0.03 ?M NFV (F) were examined by the CHRA. The cell-free supernatant was transferred to fresh MT-4 cells every 7 days.
High-molecular-weight DNA extracted from infected cells at the end of each passage was subjected to nucleotide sequencing, and the proportions
of Arg and Lys at position 409 in Gag were determined.
12/75/219/390/409gagin the absence or presence of each drug. Replication profiles of
12/75/219/390/409gag(E) in the absence (A) or presence of 0.03 ?M APV (B), 0.02 ?M SQV (C), 0.03 ?M IDV (D), 0.03
TABLE 1. Sensitivities of infectious HIV-1 clones with Gag mutations to various PIs
Infectious HIV-1 clone
0.031 ? 0.0008
0.031 ? 0.003
0.029 ? 0.003
0.032 ? 0.0001
0.021 ? 0.002
0.017 ? 0.003
0.020 ? 0.01
0.023 ? 0.005
0.032 ? 0.002
0.032 ? 0.003
0.032 ? 0.001
0.032 ? 0.003
0.032 ? 0.0005
0.031 ? 0.0007
0.031 ? 0.004
0.032 ? 0.0001
0.028 ? 0.002
0.029 ? 0.003
0.028 ? 0.002
0.028 ? 0.002
aData shown are mean values (with 1 standard deviation) derived from the results of three independent experiments conducted in triplicate. The IC50s were
determined by employing MT-4 cells exposed to each infectious HIV-1 clone (50 TCID50) in the presence of each PI, with the inhibition of p24 Gag protein production
as an end point.
3064 AOKI ET AL.J. VIROL.
NFV-related resistance mutation, N88S, renders HIV-1 sus-
ceptible to APV (33, 49). Since the acquisition of viral resis-
tance to PIs such as NFV was significantly delayed when HIV-1
had the Gag mutations seen in HIVAPVp33, we asked if another
NFV-related resistance mutation (D30N) would render HIV-1
more susceptible to APV. We also asked whether the presence
of multiple NFV resistance-associated mutations (D30N,
M46I, and V77I) would make HIV-1 susceptible to APV.
Moreover, we examined the effects of the Gag mutations seen
in HIVAPVp33on HIV-1 susceptibilities to APV and NFV.
was more susceptible to APV than
HIVWTby a factor of 20, in agreement with the reports by
Ziermann et al. and Resch et al. (33, 49). We found that the
D30N mutation in rHIVD30Npro
more susceptible to APV, by a factor of 10. Interestingly,
D30N, K45I, and A71V, was more resistant to NFV than
HIVWTby a factor of 9; however, the recombinant virus
remained more susceptible to APV than HIVWT(Table 2).
The introduction of the five Gag mutations (E12K, L75R,
H219Q, V390D, and R409K) into rHIV10/30/45/71pro
change the susceptibility profile (Table 2). Another recom-
binant HIV-1 clone with three protease mutations (D30N,
M46I, and V77I), rHIV30/46/77pro
tant to NFV (by a factor of 9) and more susceptible to APV
than HIVWT. The introduction of the five Gag mutations,
the susceptibility of rHIV30/46/77pro
Taken together, the data suggest that, as seen in the case
of the lamivudine (3TC) resistance-associated mutation
M184V that restores zidovudine (ZDV) sensitivity (37), NFV
resistance-associated mutations paradoxically render HIV-1
more susceptible to APV.
WTgagalso made HIV-1
WTgag, with the four mutations L10F,
WTgag, was also more resis-
12/75/219/390/409gag, did not affect
WTgagto APV or NFV
Certain amino acid substitutions in Gag are known to occur
in common with resistance to PIs (11, 15, 32, 36); however, no
salient features such as patterns and orders of the occurrence
have been identified for a number of amino acid substitutions
seen in Gag in PI-resistant HIV-1 variants. The roles and
impact of such amino acid substitutions in Gag for the repli-
cation of HIV-1 have not been delineated, either. These lim-
itations have been worsened since the functions and tertiary
structures of entire HIV-1 Gag proteins remain to be deter-
mined, although some structures of certain parts of Gag pro-
teins have been lately elucidated (13, 34, 41).
In the present study, we attempted to determine the effects
of non-cleavage site mutations in Gag which emerged during
the in vitro selection of HIV-1 in the presence of APV on the
viral acquisition of resistance to APV and other currently ex-
isting PIs. When we selected HIV-1 in vitro in the presence of
increasing concentrations of APV, six amino acid substitutions
apparently critical for the development of APV resistance
emerged. Such substitutions included five non-cleavage site
mutations (E12K, L75R, H219Q, V390D, and R409K) and one
cleavage site mutation, L449F (Fig. 1B).
HIV-1 variants containing PI resistance-conferring amino
acid substitutions in protease plus wild-type Gag often have
highly limited replicative abilities (7, 31). Indeed, in the
present study, the recombinant HIV-1 clone containing
containing Gag (rHIVAPVp33pro
MT-2 cells (Fig. 2A), indicating that neither of the two Gag
species supported the growth of HIVAPVp33. However, a re-
combinant HIV-1 clone containing the protease of HIVAPVp33
and the five Gag non-cleavage site mutations,
the same conditions (Fig. 2A), an observation in agreement
with reports by others that some PI resistance-associated mu-
tations compromise the catalytic activity of protease and/or
alter polyprotein processing, often leading to slower viral rep-
lication (29, 36, 43). Since some of the five non-cleavage site
mutations emerged before mutations in protease developed,
we examined the effects of three sets of non-cleavage site
amino acid mutations upon the emergence of APV resistance.
Interestingly, HIV-1 with either of two sets of Gag mutations
quired APV resistance significantly faster than HIVWT(Fig. 3),
while such mutations alone did not alter the susceptibilities of
HIV to the PIs examined (Table 1), a finding providing the first
report that Gag mutations expedite the emergence of PI-resis-
tant HIV-1 variants. At this time, it is apparently unknown
whether certain Gag mutations associated with viral resistance
to PIs persist when highly active antiretroviral therapy
(HAART) regimens including a PI(s) are interrupted or
changed to regimens containing no PIs. However, the non-
cleavage site mutations in Gag examined in this study did not
reduce the viral fitness (Fig. 2 and 6), suggesting that Gag
mutations may persist longer in circulation and/or in the HIV-1
reservoir in the body than mutations in protease when antiret-
roviral therapy including a PI(s) is interrupted. Such persisting
Gag mutations may enable HIV-1 to rapidly acquire resistance
WTgag) or the L449F cleavage site mutation-
449gag) failed to replicate in
12/75/219/390/409gag, replicated moderately under
TABLE 2. Phenotypic sensitivities of recombinant HIV-1 clones
passaged with NFVa
Infectious HIV-1 clone
IC50(?M) ? SD (change, n-fold) of:
0.031 ? 0.0008 (1)
0.0015 ? 0.0007 (0.05)
0.0031 ? 0.0001 (0.1)
0.014 ? 0.0021 (0.45)
0.020 ? 0.002 (0.64)
0.0069 ? 0.0024 (0.22)
0.0046 ? 0.0019 (0.15)
0.028 ? 0.002 (1)
0.028 ? 0.001 (1)
0.045 ? 0.001 (1.6)
0.26 ? 0.03 (9)
0.32 ? 0.03 (11)
0.25 ? 0.04 (9)
0.21 ? 0.06 (8)
rHIV10/30/45/71pro12/75/219/390/409gagwere generated to have a set of four pro-
tease mutations (L10F, D30N, K45I, and A71V) and wild-type Gag or Gag
withfive mutations, whileother
rHIV30/46/77pro12/75/219/390/409gag, were generated with three protease muta-
tions (D30N, M46I, and V77I) and wild-type Gag or Gag with five mutations.
Both sets of protease mutations were seen when HIV-1 was propagated in the
presence of NFV. The IC50s were determined by employing MT-4 cells
exposed to each recombinant HIV-1 clone (50 TCID50) in the presence of
each PI, with the inhibition of p24 Gag protein production as an end point.
All values were determined in triplicate, and the data are shown as mean
values ?1 standard deviation of results from two or three independent ex-
periments. The numbers in parentheses are changes (n-fold) compared to the
IC50of each PI for HIVWT.
HIV clones rHIV10/30/45/71proWTgag
VOL. 83, 2009 NON-CLEAVAGE SITE Gag MUTATIONS AND RESISTANCE TO PIs 3065
to that very PI when treatment with the PI is resumed. It is of
note that on the other hand, two sets of Gag non-cleavage site
mutations seen in HIVAPVp33(H219Q and R409K and E12K,
L75R, H219Q, V390D, and R409K) significantly delayed the
emergence of resistance to other PIs such as RTV and NFV
(Fig. 5). These data suggest that if a HAART regimen includ-
ing APV is changed to an alternative regimen, the inclusion of
a different PI in the alternative regimen is likely to delay the
emergence of resistance to the different PI.
It is known that the L449F cleavage site mutation renders
recombinant HIV-1 carrying a protease mutation (I50V)
more resistant to APV (25). In the present study, a recom-
binant HIV-1 clone containing the protease of HIVAPVp33
plus the L449F cleavage site mutation-containing Gag
data strongly suggest that the L449F mutation alone pre-
vents HIVAPVp33from replicating, although HIVAPVp33did
not contain the I50V mutation. The observation in the
present study that the addition of five non-cleavage site
ability of the virus indicates that the presence of non-cleav-
age site Gag mutations plays an important role in the rep-
lication of APV-resistant HIV-1 variants.
sition of resistance to other PIs (Fig. 5), we examined
whether the replication rates of rHIVWTpro
and HIVWTwere associated with the observed contrasting
resistance acquisition patterns by using the CHRA (21). We
found that rHIVWTpro
gardless of the presence or absence of PIs (Fig. 6), suggest-
ing that thedifference in
for the contrasting resistance acquisition patterns. As for
HIVWT, it is well explained by the presence of the H219Q
mutation. His-219 is located within the cyclophilin A
(CypA) binding loop of p24 Gag protein. It is thought that
CypA plays an essential role in the HIV-1 replication cycle
(4, 35) by destabilizing the capsid (p24 Gag protein) shell
during viral entry and uncoating (12) and/or by performing
an additional chaperone function, thus facilitating correct
capsid condensation during viral maturation (17, 39). CypA
is also known to support the replication of HIV-1 by binding
to the Ref-1 restriction factor and/or TRIM5?, the human
cellular inhibitors that impart resistance to retroviral infec-
tion (18, 38). It has also been demonstrated previously that
the effect of CypA on HIV-1 replicative ability is bimodal:
both high and low CypA contents limit HIV-1 replication
(14). We have demonstrated previously that certain human
cells, such as MT-2 and H9 cells, contain large amounts of
CypA (14). We have determined more recently that MT-2
cells contain more CypA by about fivefold and that MT-4
cells contain about three times more than peripheral blood
mononuclear cells (PBMCs) (unpublished data). In fact,
HIV-1 produced in MT-4 cells contains large amounts of
CypA, presumably resulting in compromised replication of
the HIV-1. However, the H219Q mutation apparently re-
449gag) failed to replicate (Fig. 2A). These
12/75/219/390/409/449gag, restored the replicative
12/75/219/390/409gagacquired resistance to
12/75/219/390/409gagsignificantly delayed the acqui-
the replication ratesof
12/75/219/390/409gagand HIVWTwas not the cause
duces the incorporation of CypA into the virions through
significantly distorting the CypA binding loop and restores
the replicative ability of virions produced in MT-4 cells (14).
Therefore, H219Q should contribute at least in part to the
replication advantage of rHIVWTpro
noteworthy that of 156 different HIV-1 strains whose se-
quences were compiled in the HIV Sequence Compendium
2008 (22), 95 and 45 strains had histidine and glutamine,
respectively, at position 219. Hence, position 219 is a poly-
morphic amino acid site, and it is thought that this polymor-
phic position is associated with the acquisition of resistance
to certain PIs. Indeed,
using fresh phytohemagglutinin-stimulated PBMCs (14).
Since H219Q confers a replication advantage on HIV-1 in
PBMCs, it is likely that HIV-1 with H219Q may acquire
resistance more rapidly than HIV-1 without H219Q.
Two groups, Ziermann et al. and Resch et al., have re-
ported that an NFV-related resistance mutation, N88S, ren-
ders HIV-1 susceptible to APV (33, 49), and indeed, Za-
chary et al. have reported an anecdotal finding that the
infection of an individual with HIV-1 containing N88S was
successfully managed with an ensuing APV-based regimen
(46). Therefore, we examined the effect of another NFV
resistance-associated mutation, D30N, in addition to that of
the N88S mutation on HIV-1 susceptibility to APV. It was
found that the mutations (D30N and N88S) clearly in-
creased the susceptibility of HIV-1 to APV by 10- and 20-
fold, respectively. These data are reminiscent of the obser-
vation that the 3TC resistance-associated mutation M184V
in a background of mutations conferring resistance to ZDV
restores ZDV sensitivity (37) and that ZDV-3TC combina-
tion therapy has proven to be more beneficial than ZDV
monotherapy in patients harboring HIV-1 with the M184V
mutation (9, 23), although the structural mechanism of the
restoration of ZDV sensitivity by M184V is not clear. When
a set of four protease mutations (L10F, D30N, K45I, and
A71V), which had emerged by passage 10 when HIVWTwas
selected with NFV, were introduced into HIVWT, generat-
was more resistant to NFV than HIVWTby a factor of 9
while the clone was slightly more sensitive to APV (Table 2).
When we introduced mGag12/75/219/390/409gaginto HIV-1
containing a set of three NFV resistance-associated pro-
tease mutations (D30N, M46I, and V77I), generating
more resistant to NFV by a factor of 8 but more sensitive to
APV by a factor of 6.7 (Table 2).
There has been a report that dual PI therapy with APV plus
NFV is generally safe and well tolerated and that the combi-
nation of APV with NFV may have the most beneficial phar-
macokinetic interactions, based on the results of a phase II
clinical trial of dual PI therapies, APV in combination with
IDV, NFV, or SQV, although this phase II trial was handi-
capped by the presence of substantial PI resistance at the
baseline and the small number of patients in the study, pre-
cluding conclusions about the relative activities or toxicities of
the dual PI combinations (10). The hypothesis that a HAART
regimen combining APV with NFV may bring about more
12/75/219/390/409gag. It is
WTgagin the CHRA
WTgag, the recombinant HIV-1 clone
12/75/219/390/409gag, the recombinant clone was
3066AOKI ET AL. J. VIROL.
favorable antiviral efficacy for HIV-1-infected individuals
should merit further study.
This work was supported in part by a grant-in-aid for scientific
research (priority areas) from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (Monbu-Kagakusho); a
grant to the Cooperative Research Project on Clinical and Epidemio-
logical Studies of Emerging and Reemerging Infectious Diseases
(Renkei Jigyo; no. 78, Kumamoto University) of Monbu-Kagakusho
(H.M.); a grant for the promotion of AIDS research from the Ministry
of Health, Welfare, and Labor of Japan (Kosei-Rohdosho; H15-AIDS-
001); a grant-in-aid from the Institute of Health Sciences, Kumamoto
Health Science University (M.A.); and in part by the intramural re-
search program of the Center for Cancer Research, National Cancer
Institute, National Institutes of Health.
1. Amano, M., Y. Koh, D. Das, J. Li, S. Leschenko, Y. F. Wang, P. I. Boross,
I. T. Weber, A. K. Ghosh, and H. Mitsuya. 2007. A novel bis-tetrahydrofura-
nylurethane-containing nonpeptidic protease inhibitor (PI), GRL-98065, is
potent against multiple-PI-resistant human immunodeficiency virus in vitro.
Antimicrob. Agents Chemother. 51:2143–2155.
2. Bally, F., R. Martinez, S. Peters, P. Sudre, and A. Telenti. 2000. Polymor-
phism of HIV type 1 gag p7/p1 and p1/p6 cleavage sites: clinical significance
and implications for resistance to protease inhibitors. AIDS Res. Hum.
3. Bhaskaran, K., O. Hamouda, M. Sannes, F. Boufassa, A. M. Johnson, P. C.
Lambert, and K. Porter. 2008. Changes in the risk of death after HIV
seroconversion compared with mortality in the general population. JAMA
4. Braaten, D., and J. Luban. 2001. Cyclophilin A regulates HIV-1 infectivity,
as demonstrated by gene targeting in human T cells. EMBO J. 20:1300–1309.
5. Carrillo, A., K. D. Stewart, H. L. Sham, D. W. Norbeck, W. E. Kohlbrenner,
J. M. Leonard, D. J. Kempf, and A. Molla. 1998. In vitro selection and
characterization of human immunodeficiency virus type 1 variants with in-
creased resistance to ABT-378, a novel protease inhibitor. J. Virol. 72:7532–
6. Clavel, F., and A. J. Hance. 2004. HIV drug resistance. N. Engl. J. Med.
7. Colonno, R., R. Rose, C. McLaren, A. Thiry, N. Parkin, and J. Friborg. 2004.
Identification of I50L as the signature atazanavir (ATV)-resistance mutation
in treatment-naive HIV-1-infected patients receiving ATV-containing regi-
mens. J. Infect. Dis. 189:1802–1810.
8. Doyon, L., G. Croteau, D. Thibeault, F. Poulin, L. Pilote, and D. Lamarre.
1996. Second locus involved in human immunodeficiency virus type 1 resis-
tance to protease inhibitors. J. Virol. 70:3763–3769.
9. Eron, J. J., S. L. Benoit, J. Jemsek, R. D. MacArthur, J. Santana, J. B.
Quinn, D. R. Kuritzkes, M. A. Fallon, and M. Rubin for the North American
HIV Working Party. 1995. Treatment with lamivudine, zidovudine, or both in
HIV-positive patients with 200 to 500 CD4? cells per cubic millimeter.
N. Engl. J. Med. 333:1662–1669.
10. Eron, J. J., R. Haubrich, W. Lang, G. Pagano, J. Millard, J. Wolfram, W.
Snowden, L. Pedneault, and M. Tisdale. 2001. A phase II trial of dual
protease inhibitor therapy: amprenavir in combination with indinavir, nelfi-
navir, or saquinavir. J. Acquir. Immune Defic. Syndr. 26:458–461.
11. Gallego, O., C. de Mendoza, A. Corral, and V. Soriano. 2003. Changes in the
human immunodeficiency virus p7-p1-p6 gag gene in drug-naive and pre-
treated patients. J. Clin. Microbiol. 41:1245–1247.
12. Gamble, T. R., F. F. Vajdos, S. Yoo, D. K. Worthylake, M. Houseweart, W. I.
Sundquist, and C. P. Hill. 1996. Crystal structure of human cyclophilin A
bound to the amino-terminal domain of HIV-1 capsid. Cell 87:1285–1294.
13. Ganser-Pornillos, B. K., A. Cheng, and M. Yeager. 2007. Structure of full-
length HIV-1 CA: a model for the mature capsid lattice. Cell 131:70–79.
14. Gatanaga, H., D. Das, Y. Suzuki, D. D. Yeh, K. A. Hussain, A. K. Ghosh, and
H. Mitsuya. 2006. Altered HIV-1 Gag protein interactions with cyclophilin A
(CypA) on the acquisition of H219Q and H219P substitutions in the CypA
binding loop. J. Biol. Chem. 281:1241–1250.
15. Gatanaga, H., Y. Suzuki, H. Tsang, K. Yoshimura, M. F. Kavlick, K. Na-
gashima, R. J. Gorelick, S. Mardy, C. Tang, M. F. Summers, and H. Mit-
suya. 2002. Amino acid substitutions in Gag protein at non-cleavage sites are
indispensable for the development of a high multitude of HIV-1 resistance
against protease inhibitors. J. Biol. Chem. 277:5952–5961.
16. Grabar, S., C. Pradier, E. Le Corfec, R. Lancar, C. Allavena, M. Bentata, P.
Berlureau, C. Dupont, P. Fabbro-Peray, I. Poizot-Martin, and D. Costagli-
ola. 2000. Factors associated with clinical and virological failure in patients
receiving a triple therapy including a protease inhibitor. AIDS 14:141–149.
17. Gross, I., H. Hohenberg, C. Huckhagel, and H. G. Krausslich. 1998. N-
terminal extension of human immunodeficiency virus capsid protein converts
the in vitro assembly phenotype from tubular to spherical particles. J. Virol.
18. Hatziioannou, T., D. Perez-Caballero, S. Cowan, and P. D. Bieniasz. 2005.
Cyclophilin interactions with incoming human immunodeficiency virus type
1 capsids with opposing effects on infectivity in human cells. J. Virol. 79:
19. Koh, Y., S. Matsumi, D. Das, M. Amano, D. A. Davis, J. Li, S. Leschenko, A.
Baldridge, T. Shioda, R. Yarchoan, A. K. Ghosh, and H. Mitsuya. 2007.
Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule
inhibitors of protease dimerization. J. Biol. Chem. 282:28709–28720.
20. Koh, Y., H. Nakata, K. Maeda, H. Ogata, G. Bilcer, T. Devasamudram, J. F.
Kincaid, P. Boross, Y. F. Wang, Y. Tie, P. Volarath, L. Gaddis, R. W.
Harrison, I. T. Weber, A. K. Ghosh, and H. Mitsuya. 2003. Novel bis-
tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI)
UIC-94017 (TMC114) with potent activity against multi-PI-resistant human
immunodeficiency virus in vitro. Antimicrob. Agents Chemother. 47:3123–
21. Kosalaraksa, P., M. F. Kavlick, V. Maroun, R. Le, and H. Mitsuya. 1999.
Comparative fitness of multi-dideoxynucleoside-resistant human immunode-
ficiency virus type 1 (HIV-1) in an in vitro competitive HIV-1 replication
assay. J. Virol. 73:5356–5363.
22. Kuiken, C., B. Foley, P. Marx, S. Wolinsky, T. Leitner, B. Hahn, F. Mc-
Cuthan, and B. Korber. 2008. HIV sequence compendium 2008. LA-UR
08-03719. Theoretical Biology and Biophysics Group, Los Alamos National
Laboratory, Los Alamos, NM.
23. Larder, B. A., S. D. Kemp, and P. R. Harrigan. 1995. Potential mechanism
for sustained antiretroviral efficacy of AZT-3TC combination therapy. Sci-
24. Maeda, K., K. Yoshimura, S. Shibayama, H. Habashita, H. Tada, K. Sagawa,
T. Miyakawa, M. Aoki, D. Fukushima, and H. Mitsuya. 2001. Novel low
molecular weight spirodiketopiperazine derivatives potently inhibit R5
HIV-1 infection through their antagonistic effects on CCR5. J. Biol. Chem.
25. Maguire, M. F., R. Guinea, P. Griffin, S. Macmanus, R. C. Elston, J. Wol-
fram, N. Richards, M. H. Hanlon, D. J. Porter, T. Wrin, N. Parkin, M.
Tisdale, E. Furfine, C. Petropoulos, B. W. Snowden, and J. P. Kleim. 2002.
Changes in human immunodeficiency virus type 1 Gag at positions L449 and
P453 are linked to I50V protease mutants in vivo and cause reduction of
sensitivity to amprenavir and improved viral fitness in vitro. J. Virol. 76:
26. Mammano, F., C. Petit, and F. Clavel. 1998. Resistance-associated loss of
viral fitness in human immunodeficiency virus type 1: phenotypic analysis of
protease and gag coevolution in protease inhibitor-treated patients. J. Virol.
27. Mitsuya, H., and J. W. Erickson. 1999. Discovery and development of anti-
retroviral therapeutics for HIV infection, p. 751–780. In T. C. Merigan, J. G.
Bartlet, and D. Bolognes (ed.), Textbook of AIDS medicine. The Williams &
Wilkins Co., Baltimore, MD.
28. Murphy, E. L., A. C. Collier, L. A. Kalish, S. F. Assmann, M. F. Para, T. P.
Flanigan, P. N. Kumar, L. Mintz, F. R. Wallach, and G. J. Nemo. 2001.
Highly active antiretroviral therapy decreases mortality and morbidity in
patients with advanced HIV disease. Ann. Intern. Med. 135:17–26.
29. Nijhuis, M., N. M. van Maarseveen, S. Lastere, P. Schipper, E. Coakley, B.
Glass, M. Rovenska, D. de Jong, C. Chappey, I. W. Goedegebuure, G.
Heilek-Snyder, D. Dulude, N. Cammack, L. Brakier-Gingras, J. Konvalinka,
N. Parkin, H. G. Krausslich, F. Brun-Vezinet, and C. A. Boucher. 2007. A
novel substrate-based HIV-1 protease inhibitor drug resistance mechanism.
PLoS Med. 4:e36.
30. Paredes, R., A. Mocroft, O. Kirk, A. Lazzarin, S. E. Barton, J. van Lunzen,
T. L. Katzenstein, F. Antunes, J. D. Lundgren, and B. Clotet. 2000. Predic-
tors of virological success and ensuing failure in HIV-positive patients start-
ing highly active antiretroviral therapy in Europe: results from the Euro-
SIDA study. Arch. Intern. Med. 160:1123–1132.
31. Perrin, V., and F. Mammano. 2003. Parameters driving the selection of
nelfinavir-resistant human immunodeficiency virus type 1 variants. J. Virol.
32. Peters, S., M. Munoz, S. Yerly, V. Sanchez-Merino, C. Lopez-Galindez, L.
Perrin, B. Larder, D. Cmarko, S. Fakan, P. Meylan, and A. Telenti. 2001.
Resistance to nucleoside analog reverse transcriptase inhibitors mediated by
human immunodeficiency virus type 1 p6 protein. J. Virol. 75:9644–9653.
33. Resch, W., R. Ziermann, N. Parkin, A. Gamarnik, and R. Swanstrom. 2002.
Nelfinavir-resistant, amprenavir-hypersusceptible strains of human immuno-
deficiency virus type 1 carrying an N88S mutation in protease have reduced
infectivity, reduced replication capacity, and reduced fitness and process the
Gag polyprotein precursor aberrantly. J. Virol. 76:8659–8666.
34. Saad, J. S., S. D. Ablan, R. H. Ghanam, A. Kim, K. Andrews, K. Nagashima,
F. Soheilian, E. O. Freed, and M. F. Summers. 2008. Structure of the
myristylated human immunodeficiency virus type 2 matrix protein and the
role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting. J.
Mol. Biol. 382:434–447.
35. Steinkasserer, A., R. Harrison, A. Billich, F. Hammerschmid, G. Werner, B.
VOL. 83, 2009NON-CLEAVAGE SITE Gag MUTATIONS AND RESISTANCE TO PIs 3067
Wolff, P. Peichl, G. Palfi, W. Schnitzel, E. Mlynar, et al. 1995. Mode of action
of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with ac-
tivity against human immunodeficiency virus type 1 (HIV-1): interference
with early and late events in HIV-1 replication. J. Virol. 69:814–824.
36. Tamiya, S., S. Mardy, M. F. Kavlick, K. Yoshimura, and H. Mistuya. 2004.
Amino acid insertions near Gag cleavage sites restore the otherwise com-
promised replication of human immunodeficiency virus type 1 variants re-
sistant to protease inhibitors. J. Virol. 78:12030–12040.
37. Tisdale, M., S. D. Kemp, N. R. Parry, and B. A. Larder. 1993. Rapid in vitro
selection of human immunodeficiency virus type 1 resistant to 3?-thiacytidine
inhibitors due to a mutation in the YMDD region of reverse transcriptase.
Proc. Natl. Acad. Sci. USA 90:5653–5656.
38. Towers, G. J., T. Hatziioannou, S. Cowan, S. P. Goff, J. Luban, and P. D.
Bieniasz. 2003. Cyclophilin A modulates the sensitivity of HIV-1 to host
restriction factors. Nat. Med. 9:1138–1143.
39. Turner, B. G., and M. F. Summers. 1999. Structural biology of HIV. J. Mol.
40. Verheyen, J., E. Litau, T. Sing, M. Daumer, M. Balduin, M. Oette, G.
Fatkenheuer, J. K. Rockstroh, U. Schuldenzucker, D. Hoffmann, H. Pfister,
and R. Kaiser. 2006. Compensatory mutations at the HIV cleavage sites
p7/p1 and p1/p6-gag in therapy-naive and therapy-experienced patients. An-
tivir. Ther. 11:879–887.
41. Verli, H., A. Calazans, R. Brindeiro, A. Tanuri, and J. A. Guimaraes. 2007.
Molecular dynamics analysis of HIV-1 matrix protein: clarifying differences
between crystallographic and solution structures. J. Mol. Graph. Model.
42. Walensky, R. P., A. D. Paltiel, E. Losina, L. M. Mercincavage, B. R. Schack-
man, P. E. Sax, M. C. Weinstein, and K. A. Freedberg. 2006. The survival
benefits of AIDS treatment in the United States. J. Infect. Dis. 194:11–19.
43. Watkins, T., W. Resch, D. Irlbeck, and R. Swanstrom. 2003. Selection of
high-level resistance to human immunodeficiency virus type 1 protease in-
hibitors. Antimicrob. Agents Chemother. 47:759–769.
44. Yoshimura, K., R. Kato, M. F. Kavlick, A. Nguyen, V. Maroun, K. Maeda,
K. A. Hussain, A. K. Ghosh, S. V. Gulnik, J. W. Erickson, and H. Mitsuya.
2002. A potent human immunodeficiency virus type 1 protease inhibitor,
UIC-94003 (TMC-126), and selection of a novel (A28S) mutation in the
protease active site. J. Virol. 76:1349–1358.
45. Yoshimura, K., R. Kato, K. Yusa, M. F. Kavlick, V. Maroun, A. Nguyen, T.
Mimoto, T. Ueno, M. Shintani, J. Falloon, H. Masur, H. Hayashi, J. Erick-
son, and H. Mitsuya. 1999. JE-2147: a dipeptide protease inhibitor (PI) that
potently inhibits multi-PI-resistant HIV-1. Proc. Natl. Acad. Sci. USA 96:
46. Zachary, K. C., G. J. Hanna, and R. T. D’Aquila. 2001. Human immunode-
ficiency virus type 1 hypersusceptibility to amprenavir in vitro can be asso-
ciated with virus load response to treatment in vivo. Clin. Infect. Dis. 33:
47. Zennou, V., F. Mammano, S. Paulous, D. Mathez, and F. Clavel. 1998. Loss
of viral fitness associated with multiple Gag and Gag-Pol processing defects
in human immunodeficiency virus type 1 variants selected for resistance to
protease inhibitors in vivo. J. Virol. 72:3300–3306.
48. Zhang, Y. M., H. Imamichi, T. Imamichi, H. C. Lane, J. Falloon, M. B.
Vasudevachari, and N. P. Salzman. 1997. Drug resistance during indinavir
therapy is caused by mutations in the protease gene and in its Gag substrate
cleavage sites. J. Virol. 71:6662–6670.
49. Ziermann, R., K. Limoli, K. Das, E. Arnold, C. J. Petropoulos, and N. T.
Parkin. 2000. A mutation in human immunodeficiency virus type 1 protease,
N88S, that causes in vitro hypersensitivity to amprenavir. J. Virol. 74:4414–
3068AOKI ET AL.J. VIROL.