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Journal of Virology, November 2001, p. 11227-11233, Vol. 75, No. 22
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.22.11227-11233.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Functional Correlates of Insertion Mutations in the Protease Gene
of Human Immunodeficiency Virus Type 1 Isolates from Patients
Eun-Young
Kim,1,*
Mark A.
Winters,1
Ron M.
Kagan,2 and
Thomas C.
Merigan1
Center for AIDS Research, Stanford
University, Stanford,1 and Quest
Diagnostics, San Juan Capistrano,2
California
Received 23 March 2001/Accepted 18 August 2001
 |
ABSTRACT |
Twenty-four of over 24,000 patients genotyped over the past 3 years
were found to have human immunodeficiency virus (HIV) isolates that
possess an insert in the protease gene. In this report, we evaluated
the spectrum of protease gene insertion mutations in patient
isolates and analyzed the effect of these various insertion mutations
on viral phenotypes. The inserts were composed of 1, 2, 5, or 6 amino acids that mapped at or between codons 35 and 38, 17 and 18, 21 and 25, or 95 and 96. Reduced susceptibility to protease
inhibitors was found in isolates which possess previously reported drug
resistance mutations. Fitness assays, including replication and
competition experiments, showed that most of the isolates with inserts
grew somewhat better than their counterparts with a deletion of the
insert. These experiments demonstrate that, rarely, insertion mutations
can develop in the HIV type 1 protease gene, are no more resistant than
any other sequences which have similar associated resistance mutations,
and can provide a borderline advantage in replication.
| |
TEXT |
The low fidelity of human
immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT),
combined with the lack of an associated proofreading function
(20), results in high levels of mutation and increasing
genetic variation that produces quasispecies of HIV. These mechanisms
make it possible for a number of mutations in the protease and RT genes
of HIV to emerge and to be selected as the predominant isolates during
drug treatment (3, 15, 23, 32). The resulting amino acid
substitutions affect the structure of the viral enzyme, which can alter
the kinetics of enzyme function or change the ability of inhibitors to
access the active site (16, 18), thus providing the mutant
virus a competitive advantage under drug pressure (13).
Recently it has been shown that resistance to multiple nucleoside
analogs can result from several insertion mutations near codon 69 of
the RT gene (2, 14, 28, 31). However, all previously
described mutations associated with resistance to protease inhibitors
have been single codon substitutions that have resulted from 1- or 2-base point mutations in the protease gene (1, 16, 30). In this study, we characterized HIV-1 isolates possessing various amino
acid insertions in the protease gene using several different methodological approaches, including drug susceptibility assays, kinetics of viral antigen production, and competitive replication assays.
Genotypes of insert-containing isolates and patterns of insertion
mutations.
The patient plasma or serum specimens studied here were
submitted for HIV-1 protease genotyping to Quest Diagnostics Inc., San
Juan Capistrano, California; Stanford University Hospital, Stanford,
Calif.; and the University of Oregon, Portland. To make an RT-PCR
artifact less likely, genotyping was performed independently by
population-based sequencing of plasma-derived HIV RNA at both Stanford
University and Quest Diagnostics as previously described (30). In addition, genotyping was repeated over time and
the presence of an insertion was confirmed in the four instances where additional patients' specimens were available. Both laboratories demonstrated the same sequence result in all cases. The patterns of
insertion mutations in the protease gene showed that the nucleic acid
compositions of the inserts were typically duplications of neighboring
sequences (Table 1). Most of the inserts
(in 19 out of 24 isolates) were composed of 1 to 5 amino acids that
mapped between codons 35 and 38. Two were found to be a
single-amino-acid insertion between codons 21 and 25, one was a
single-amino-acid insertion positioned between codons 17 and 18, and 5- and 6-amino-acid insertions were observed at positions 95 to 96 (Table
1). Ten isolates had at least two major resistance-associated protease gene mutations (G48V, I54V, V82A, I84V, and/or L90M), with an average
of 10 other amino acid changes from consensus B. The eight other
isolates did not possess any major protease gene mutations but did
posses an average of seven other changes compared to consensus B. Nineteen insertions appeared preferentially in the flap region (Table
1), which encompasses amino acids 33 to 62 (4, 5, 21, 29).
Furthermore, the database available to us showed that 10 of 11 simian
immunodeficiency virus-African green monkey isolates had two amino acid
insertions in the protease gene at codons 34 to 37 (25;
http://hivdb.stanford.edu/hiv/). Molecular modeling
experiments have shown that the insertions cause conformational changes in the geometry of the flap region and contribute to structural alterations in more distant region of the molecule (M. A. Winters, E.-Y. Kim, S. Chou, A. Warford, R. Kagan, R. Fenwick, L. Kovari, and
T. C. Merigan, Abstr. 7th Conf. Retrovir. Opportun. Infect., abstr. 723, 2000; L. Kovari, personal communication, 2000).
Because the flap region does not contribute strongly to the enzyme's
stability (29) and the flap region overlies the catalytic
aspartate residues located in the substrate binding site (4,
5), mutation of flap residues might provide an effective means
for the virus to block protease inhibitor access.
Recombinant viruses and drug susceptibility.
Twelve
recombinant viruses of patient-derived HIV isolates were constructed by
previously described homologous recombination methods
(19). In brief, the purified PCR product of the protease gene was cotransfected into C8166 cells with HXB2 lacking the protease
gene (pHXB2-
PR). Additional recombinants of the five representative
isolates in which each insertion mutation was deleted by PCR-based
site-directed mutagenesis were constructed (10). Primers
used to remove the protease gene insert were designed after the
sequences were analyzed with sequence analysis programs at the Stanford
HIV database (25; http://hivdb.stanford.edu/hiv/), which
is based on optimal sequence alignment of the amino acids between
codons of the protease (26) and manually corrected. Recombinants were constructed for 12 of the insertion-containing isolates. In addition, five representative viral constructs lacking the
insertion were created. These insertion strains were able to function
as infectious clones. These results demonstrate that all the
insertion-containing proteases have intact biological activities
(33), a result which is not expected with a PCR artifact. In vitro susceptibility to indinavir (IDV), saquinavir (SQV), or
nelfinavir (NFV) was measured for each of the 12 isolates containing the insertion and for the 5 corresponding isolates lacking the insertion using a previously described method (11).
Results are expressed as mean 50% inhibitory concentrations
(IC50s) of four to eight values obtained from two to four
different experiments per isolate (Table
2). When we compared the susceptibilities of insertion and deletion pairs, the IC50s for three
of the five insertion-containing isolates (Q781, Q822, and Q058) were
similar to or higher than those for corresponding insertion-lacking
isolates. The insertion in Q781 conferred reduced susceptibility to all three protease inhibitors, as demonstrated by three- to fivefold-higher IC50s than those for the corresponding constructs with the
insertion deleted. The assays showed that there was no substantial
difference in drug susceptibility in the insertion-containing isolates
that lacked protease inhibitor resistance mutations in the protease gene (Q781, Q822, and U099) compared to that of the wild-type virus.
Isolates that had major protease inhibitor resistance mutations showed
a 4- to 45-fold decrease in susceptibility to protease inhibitors
compared to that of the wild type. Phenotypic results suggest that
previously reported drug resistance mutations seem to be primarily
responsible for protease inhibitor resistance even in the
presence of the insertions and that insertion mutations may not
contribute directly to drug resistance.
Replication kinetics and competition studies.
A replication
kinetics assay was carried out by modification of previously described
methods (27). Five thousand 50% tissue culture infective
doses (TCID50) (9) of each virus was used to
infect 5 × 106 phytohemagglutinin (PHA)-stimulated
peripheral blood mononuclear cells (PBMCs) (multiplicity of infection,
0.001) in both replication and competition experiments. p24 antigen
production was measured for 7 days, and at least three independent
assays were performed with five different isolates (Fig.
1). The Q058 insertion-deletion pair was
selected to serve as a related protease inhibitor-resistant control. In
the absence of drug, all isolates showed lower replication rates than
that of wild-type NL4-3. Although three of five isolates lacking the
insertion (Q781, Q822, and U099) do not have major protease inhibitor
resistance mutations, there were several differences in their
genotypes. Previous studies showed that several protease mutations
confer reduced enzyme activity due to either an inability to refold and
autoprocess or an intrinsic lack of protease activity (18, 22,
24, 29). It is possible that point mutations or insertions in
the protease gene might have caused an impairment of protease function,
and the insertion and point mutations were likely selected for in order
to restore protease activity and viral replicative fitness. Four of the
five isolates tested (Q781, Q822, Q164, and Q058) showed that the
insertion-containing isolates grew better than their corresponding
insertion/lacking isolates (Fig. 1). Tests in the presence of the
IC50s of IDV, SQV, and NFV revealed that Q058 isolates
exhibited markedly different replication profiles (Fig. 1). To evaluate
the fitness of an isolate containing the insert relative to that of its
counterpart lacking the insert, we tested Q058 and Q781 isolate pairs
as the most protease inhibitor resistant and a protease inhibitor
sensitive insertion isolates, respectively (Fig.
2). In competition studies, the relative
fitness of an insertion/insertion-less pair has been evaluated by
allowing the two virus populations to compete with each other until one isolate becomes dominant (7, 13). To ensure that an
increase in the proportion of one isolate suggests a relatively better replicative capacity than that of its counterpart, the isolate pairs
were administered at three different ratios: 80 and 20%, 50 and 50%,
and 20 and 80%, based on TCID50. The cells were fed with
medium containing 1% interleukin-2 twice per week and with PHA-stimulated PBMCs 7 and 14 days after infection. RNAs were extracted
from each of the mixtures of virus on day 0, and RT-PCR and sequencing
verified the insertion-containing/insertion-lacking isolate
ratio of the mixture. At days 1, 7, 14, and 21, chromosomal DNA from
infected cells was purified and the HIV-1 protease coding region was
amplified as described above. The proportion of insertion-containing and insertion-lacking isolates was determined with relative peak heights in electropherograms. In order to determine the impact of an
insertion mutation on fitness under protease inhibitor pressure, two
different concentrations of drugs were used as a selective condition
based on the minimum and maximum IC50s. In the absence of
protease inhibitors, insertion-containing isolates outgrew their
corresponding insertion-lacking isolates by day 7, regardless of
starting concentrations of viruses (Fig. 2). The Q781 pair showed that
the insertion-containing isolate outgrew the insertion-lacking isolate
regardless of the presence of drugs (Fig. 2a). In the presence of a
protease inhibitor, the domination occurred earlier, indicating that
the replication rates of the insertion isolates are more affected by
drug pressure. In the presence of both concentrations of SQV, the Q058
insertion-containing isolate outgrew its insertion-lacking control
(Fig. 2b). When competition cultures were done with IDV present, Q058
failed to outgrow its insertless partner at low drug concentrations but
grew quickly at high concentrations. This result for low
concentrations contrasts with the drug susceptibility results presented
above.
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FIG. 1.
Replication kinetics of HIV-1 recombinant isolates. One
thousand TCID50 of each virus was used per 106
PHA-stimulated PBMCs. Virus production was monitored every day by p24
antigen assay. Culture supernatants were collected every day until day
7, and p24 antigen production was monitored by enzyme-linked
immunosorbent assay. Data are the means of results of three different
tests. To serve as a related protease inhibitor-resistant control, Q058
insertion (Ins) and Q058 deletion (Del) isolates were cultured with the
IC50 of each protease inhibitor (PI) and p24 values were
measured daily for 7 days.
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FIG. 2.
Competitive HIV-1 replication assay of insertion (INS)
and deletion (DEL) isolate pairs of Q781 with and without the insertion
(a) and Q058 with and without the insertion (b). Data were generated
based on relative peak heights in electropherograms produced from DNA
sequencing of the HIV-1 genome. In the absence of protease inhibitors,
insertion and deletion pairs were combined at three different ratios,
80:20, 50:50, and 20:80 based on TCID50. In the presence of
protease inhibitions, insertion and deletion isolates were coinfected
at the same ratios and cultured in the presence of two different
concentrations of three protease inhibitors (IDV, SQV, and NFV).
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|
Of more than 24,000 patients genotyped by Quest Diagnostics over the
past 3 years, 22 individuals (0.09%) were found to have
HIV isolates
that possess an insertion in the protease gene. The
prevalence of
isolates containing protease insertion mutations
is substantially lower
than the occurrence of other types of protease
mutations and 10-fold
lower than the occurrence of RT insertions
in the same group of
patients (
31). The lower prevalence of
insert-containing
isolates suggests that a unique set of host
conditions and virus
characteristics may be required for the insert-containing
isolates to
occur and/or emerge under drug selection pressure.
The inserts may have
been selected during protease inhibitor therapy,
as available
pretherapy samples did not show evidence of the insert
in one case
(U099). The patient had been treated with stavudine,
lamivudine, and
IDV for 8 months and then treated with zidovudine,
lamivudine, and NFV.
The codon 35TD insertion was absent until
2 years of treatment had
passed (Winters et al., Abstr. 7th Conf.
Retrovir. Opportun.
Infect.; S. Chou, personal communication,
1999). A recent study from
another group reported a patient who
developed an 18HL insertion. That
patient also had a history of
IDV and NFV treatment, and the 18HL
insertion appeared following
the first year of IDV treatment
(
17). Protease gene inserts
have not been found so
far in HIV-1 and HIV-2 genotypes from protease
inhibitor-naïve
patients at the Stanford HIV database (
25)
and other
available databases. These patients' histories suggest
that
insertions, like point mutations, may be selected in vivo
during
protease inhibitor therapy but much more
infrequently.
There are several theories about how these insertions could have been
generated. Relatively to the strand transfer mechanism,
hairpin
structures and local sequence context can cause RT to
pause during
replication, leading to higher rates of mutation
in specific areas
(
8,
12,
34). During reverse transcription,
the finger
domain in HIV-1 RT (p66) is in intimate contact with
its template up to
6 nucleotide positions ahead of the catalytic
site, and the effect on
pausing of the RNA secondary structure
ahead of the enzyme might be
offset 5' on the template by approximately
6 nucleotides
(
8). Investigation suggests that hairpin loops
are common
features of the protease RNA secondary structure, especially
in the
region encompassing bases 87 through 99, which correspond
to codons 29 to 33 (data not shown). Given that 18 of the 22 isolates
in this study
possessed one, two, or five amino acid insertions
a few bases upstream
from this region, it is likely that the area
of insertion, codons 35 through 38, is affected by this process.
Further studies of the
secondary structure of protease RNA may
offer more insight regarding
the possible mechanisms of the insertion
patterns in HIV-1 protease
(
8).
HIV-1 replicating in vivo may find multiple molecular pathways to
increase its fitness. Despite the low prevalence of insertions
in the
protease gene of HIV-1, the results presented in this report
demonstrate that insertions are acquired in vivo and likely confer
an
advantage in terms of fitness. Further studies are needed to
characterize the factors that cause the selection and the biochemical
properties of these insert-containing proteases. A recent report
indicating that an insert-containing virus can be transmitted
between
patients (
6) suggested that such strains will be
encountered
in the future and may be important if they acquire drug
resistance
or in vivo replicative
advantages.
| |
ACKNOWLEDGMENTS |
We thank S. Chou, University of Oregon, Portland, for kindly giving
us one of the insertion isolates and that patient's treatment history.
L. C. Kovari, Wayne State University, Detroit, Mich., helped us
with his thoughts about structural analysis and other related topics.
We thank R. Lobato and R. Shafer, Stanford University, Stanford,
Calif., for helpful comments and criticism of the manuscript.
This research was supported by a National Foundation for Cancer
Research grant to T. C. Merigan for a project titled
"Drug resistance in infection with HIV" and by the Korea Science
and Engineering Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for AIDS
Research, Stanford University School of Medicine, 300 Pasteur Dr., Grant Building, S-156, Stanford, CA 94305-5107. Phone: (650) 724-4614. Fax: (650) 725-2395. E-mail: eunyoung{at}stanford.edu.
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270:325-332[Abstract/Free Full Text].
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Journal of Virology, November 2001, p. 11227-11233, Vol. 75, No. 22
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.22.11227-11233.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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