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J Virol, May 1998, p. 3534-3538, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Human Immunodeficiency Virus Type 1 Subtype F
Reverse Transcriptase Sequence and Drug Susceptibility
Cristian
Apetrei,1,2,*
Diane
Descamps,1
Gilles
Collin,1
Ibtisam
Loussert-Ajaka,1
Florence
Damond,1
Mihai
Duca,2
François
Simon,1 and
Françoise
Brun-Vézinet1
Laboratoire de Virologie, Hôpital
Bichat-Claude Bernard, 75018 Paris, France,1 and
Virus Laboratory, Microbiology Department, School of
Medicine, Gr. T. Popa University, 6600 Iasi, Romania2
Received 21 August 1997/Accepted 15 January 1998
 |
ABSTRACT |
We sequenced and phylogenetically analyzed the reverse
transcriptase (RT) regions of the pol genes of 14 human
immunodeficiency virus type 1 (HIV-1) isolates from Romanian patients,
which were classified as subtype F on the basis of
env gene structure. The RT sequences showed that the
strains clustered phylogenetically and were equidistant from other
HIV-1 subtypes as shown by the neighbor-joining and maximum-likelihood
methods, allowing us to define HIV-1 subtype F according to the
pol classification. The subtype F RT sequences differed
from reported group M RT sequences by 10.94% (for nucleotides) and
7.6% (for amino acids). Phenotypic analysis of subtype F
susceptibility to three classes of antiretroviral compounds showed an
increase in the 50% inhibitory concentration of the
tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2-(1H)-one and -thione (TIBO) derivate R82913 for one strain which was naturally resistant to this compound. This first report of subtype F
pol sequences confirms the perfect correlation between the
phylogenetic positions determined by env and
pol analyses and suggests that virus variability might
influence the efficacy of antiretroviral treatments. This
finding warrants a global evaluation of the phenotypic and genotypic
susceptibility of HIV-1 subtypes to antiretroviral drugs.
| |
INTRODUCTION |
Most human immunodeficiency virus
type 1 (HIV-1) drug susceptibility studies have involved subtype B. Little information on the impact of viral diversity on natural
susceptibility to antiretroviral drugs has been reported to date.
However, HIV-1 group O viruses are naturally resistant to
nonnucleoside reverse transcriptase (RT) inhibitors (8), as
is HIV-2 (28). Subtypes are defined on the basis of the
env (24, 25) or gag (19)
gene. Most RT sequences reported to date belong to subtype B strains,
which prevail in North America and western Europe (25),
i.e., regions where antiretroviral drugs are developed and
clinical trials are conducted. RT sequences of subtypes A to D
are also available (25, 32, 34). The full sequences of
Thai strains defined as subtype E according to env
classification and defined as subtype A on the basis of the
gag sequence (19) also corresponded to subtype A
on the basis of the pol gene (5, 14). By
contrast, full sequence analysis of a subtype G strain ruled out
recombination events in the pol gene (6), in
keeping with a previous report on the RT sequences of subtype G
(17). To date, no information has been published on the
pol genes of the other HIV-1 group M subtypes.
We present the first RT gene sequences and data on the phenotypic
susceptibility to antiretroviral drugs of subtype F strains from
Romanian patients. This is the dominant subtype in Romanian children
and adults (1, 11) and is also a minor viral form in other
countries, such as Brazil (23), Argentina (4),
Cameroon (27), Russia (18), Taiwan
(7), Martinique (10), Cyprus (16),
France (33), Belgium (13), and The Netherlands
(20). Recombinant F/B strains have also been reported
(21, 30).
| |
MATERIALS AND METHODS |
Study population.
We studied 14 HIV-1 subtype F strains
isolated from Romanian children (n = 9) and adults
(n = 5). All but one of the infected children were
nosocomially infected (by injections with nonsterile, reused needles
and syringes); the remaining one was vertically infected. Clinical and
epidemiological data are described elsewhere, together with virus
isolation, env subtype determination, and strain
codification (1). None of the patients had received antiretroviral therapy.
HIV-1 RT sequencing.
DNA was extracted with
phenol-chloroform from cocultured peripheral blood mononuclear cells
from (PBMC) infected patients, precipitated with ethanol, and
quantified spectrophotometrically. The pol gene was then
amplified in a nested PCR with outer primers RT-18 and RT out and inner
primers RT-19 and RT-20 as previously described (26).
Each nested-PCR product (1,008 bp) was subjected to direct population
sequencing with sense primer A20
(5'-ATTTTCCCATTAGTCCTATT-3') and antisense primer
NE120 (5'-ATGTCATTGACAGTCCAGCT-3'). Sequencing reactions were run with the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase (FS; Perkin-Elmer) on
an automated sequencer (Applied Biosystems 373A).
Phylogenetic analysis.
DNA sequences were analyzed with the
multiple-sequence editor Clustal W (35) and improved by
visual inspection. The sequences were gapstripped and a pairwise matrix
based on 591 sites was generated with the DNADIST program of the PHYLIP
package, version 3.56 (12). Tree topology was inferred by
the neighbor-joining method with the Kimura two-parameter distance
matrix (PHYLIP) and a transition/transversion ratio of 2. Bootstrap
analysis was performed with the SEQBOOT (100 resamplings), DNADIST,
NEIGHBOR, and CONSENSE programs (PHYLIP package). Phylogenetic analysis was also performed by the maximum-likelihood method, using the DNAML
program (12). The tree outliers were the HIV-1 group O sequences (MVP5180 and ANT70). In the tree construction we also included the RT sequences of six subtype B HIV-1 isolates (LAI, SF2,
MN, OYI, JRFL, and JRCSF), three subtype D sequences (ELI, NDK, and
Z2Z6), two subtype A sequences (UG037 and U455), the sequence of an A/G
recombinant strain (IBNG) (14), and the sequence of a
presumed A/D recombinant strain (MAL) from the Los Alamos National
Laboratory database (25). The newly reported sequences for
strains CM240 (5), 90CR402, and 93TH253 (14),
recombinant A/E strains, were also used in the phylogenetic analyses.
Phenotypic susceptibility assay.
The phenotypic
susceptibilities of the cellular HIV-1 subtype F isolates were analyzed
in a PBMC assay, taking into account the replication kinetics of each
strain (3). After conventional isolation of HIV from frozen
PBMC, the cell-free HIV-1 subtype F supernatants corresponding to peak
RT activity were serially diluted (100 to
10
4) and incubated with fresh normal
phytohemagglutinin-stimulated PBMC. After being washed, the infected
cells were placed in 96-well plates containing six serial dilutions of
the antiretroviral drugs. Each dilution was tested in triplicate. On
days 5, 7, and 10, the supernatants were collected and half the medium
was replaced with fresh drug-containing medium. The 50% tissue culture
infective dose was assessed by measuring RT activity in control
drug-free supernatants collected on the same days. At the peak of RT
activity we calculated the drug concentrations inhibiting 50 and 90%
(IC50 and IC90, respectively) of the RT
activity of 100 50% tissue culture infective doses. For zidovudine
(ZDV), an IC50 cutoff of 0.05 µM has been defined to
classify the virus isolates as ZDV sensitive or ZDV resistant, based on
phenotypic analyses of several isolates from treated and untreated
patients and on comparisons of these results to genotypic data. For the
other antiretroviral drugs, it has not been possible to determine any
cutoff value. In this study, phenotypic resistance to these compounds
was defined as at least a fivefold increase in IC50s for
the HIV-1 subtype F isolates compared to those for HIV-1 subtype B
strains.
Antiretroviral agents.
We tested the nucleoside RT
inhibitors ZDV (Wellcome, Dartford, United Kingdom) and lamivudine
(3TC; Glaxo-Wellcome, Dartford, United Kingdom); the nonnucleoside RT
inhibitors
tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) derivate R82913 (Janssen, Beerse, Belgium), delavirdine (DLV; Upjohn, Kalamazoo, Mich.), and nevirapine (NVP; Boehringer Ingelheim Pharmaceuticals, Ridgefield, Conn.); and the
protease inhibitors saquinavir (SQV; Roche, Welwyn Garden City, United
Kingdom) and ritonavir (RTV; Abbott, Abbott Park, Ill.). The purified
drugs were kindly provided by the manufacturers.
Nucleotide sequence accession numbers.
The nucleotide and
amino acid sequences of codons 33 to 235 of the RT genes and proteins
from the 14 HIV-1 subtype F isolates have been submitted to GenBank
(accession no. Y16138 to Y16151).
| |
RESULTS |
Genetic analysis.
Figure 1 shows
the amino acid alignment of codons 33 to 235 of the RT genes of the
different subtype F isolates. The subtype F consensus sequence differed
from that of subtype B in 11 residues (V35T, T39A, E40D, D131E, I135L,
S162Y, K173T, Q174K, T200A, Q207A, and R211K). Two of these mutations
(A200 and K211) have been reported to occur in subtype B isolates.
Although not yet reported for subtype B isolates, one residue (D40) of
the subtype F consensus sequence has been described as occurring in
consensus sequences of subtype A. The sequence T173-K174 has been
described for the IBNG subtype A/G isolate (25). Residues
A39, E131, L135, and Y162, which are frequently present in subtype F
strains, have never been encountered in other HIV-1 group M subtypes.
None of the sequences analyzed contained mutations previously linked to resistance to nucleoside or nonnucleoside RT inhibitors. The sequence of the RO-BCI23 isolate was more similar to that of the subtype B
consensus sequence in the first part of the RT gene (subtype B
structure in sequences encoding T39, E40, and I135) but was more
variable than the other subtype F isolates in the second half of the RT
gene (close to the binding pocket). Compared to the subtype B consensus
sequence, the RO-BCI23 protein bore four mutations close to the active
site of the RT protein (E169D, K173A, Q174K, and D177E).
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FIG. 1.
Alignment of the deduced amino acid sequences of HIV-1
subtype F RT and the consensus (Cons) sequences of HIV-1 group M and
group O strains. Strains RO-BCI17 to RO-BCI23 were isolated from
HIV-1-infected adults. *, established following the analysis of the
pol sequences originating from 11 Zimbabwean seroconverters
(32). **, naturally resistant to TIBO derivate.
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|
At position 60, all but one of the strains originating in
nosocomially infected Romanian children (RO-BCI7, RO-BCI8,
RO-BCI9,
RO-BCI11, RO-BCI12, RO-BCI15, and RO-BCI16) bore a
valine (as
in the subtype B consensus sequence), whereas the
strains isolated
from adults (RO-BCI17, RO-BCI18, RO-BCI19,
RO-BCI20, and RO-BCI23),
a vertically infected child (RO-BCI13), and a
nosocomially infected
child from whom the strain was isolated in 1994 (RO-BCI1) bore
an isoleucine. Conversely, at position 39, all but one
(RO-BCI17)
of the strains isolated from adults bore a threonine, like
subtype
B strains, whereas all those isolated from nosocomially
infected
children bore an alanine.
Phylogenetic analysis of HIV-1 RT sequences.
Phylogenetic
analysis of the RT nucleotide sequences by the neighbor-joining and
maximum-likelihood methods gave similar results. Phylogenetic trees
were constructed by using the 14 Romanian HIV-1 RT sequences,
representative RT sequences of subtype B strains, and sequences
belonging to different subtypes obtained from the database
(25). Figure 2 shows the
phylogenetic tree constructed by neighbor joining. High bootstrap
values were obtained at the relevant nodes, indicating that subtypes B,
D, and F each form a consistent clade. Subtype A was composed of five
different strains defined as A or E on the basis of the env
classification, whereas recombinant strains IBNG and MAL were subtype
outliers.
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FIG. 2.
Phylogenetic analysis comparing the RT regions of HIV-1
pol genes from different strains. Tree topology was inferred
by the neighbor-joining method. The tree was based on an
alignment of nucleotides from which columns containing gaps have been
deleted (597 nucleotides). The tree was rooted with HIV-1 group O
sequences. The numbers given at the branch points are the 50%
threshold majority consensus values for 100 bootstrap replicates.
Vertical distances are given for clarity. The cluster of sequences from
nosocomially infected children (*) had already been observed by
analyzing the env gene (1). Strain RO-BCI13
(**) was isolated from a vertically infected child. Strains
RO-BCI17, RO-BCI18, RO-BCI19, RO-BCI20, and RO-BCI23 were isolated from
HIV-1-infected adults.
|
|
The subtype F phylogenetic tree showed that the strains from
nosocomially infected Romanian children formed a cluster, as
was
previously observed by analyzing the
env sequence
(
1).
The RT sequence of a strain from a vertically infected
child (RO-BCI13)
clustered with the adult sequences. The RT sequence of
strain
RO-BCI23 formed a separate branch within the subtype F tree.
The average intrasubtype F sequence divergence (597 nucleotides) was
3.56% (range, 1.54 to 6.36%), and the distance between
the subtype F
sequences and the sequences of other subtypes belonging
to group M was 10.94% (range, 8.04 to 13.5%)
(
P < 0.001) (Table
1).
The divergence among the subtype F amino acid sequences (197
residues) was 3.7% (range, 1.57 to 6.7%), whereas the mean
distance
between the subtype F RT amino acid sequences and other group
M sequences was 7.6% (range, 6.00 to 11.59%).
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TABLE 1.
Nucleotide and amino acid divergences between subtype F
and other group M subtype and group O RT sequences
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Phenotypic susceptibility.
The IC50s of nucleoside
analogs ZDV and 3TC were similar to those for wild-type subtype B field
isolates. Although the IC50s were higher for the subtype F
strains than for the subtype B strains, all the subtype F isolates were
susceptible to protease inhibitors. All were also sensitive to the
nonnucleoside RT inhibitors NVP and DLV. Susceptibility to the third
nonnucleoside RT inhibitor, TIBO derivate R82913, was lower for two
isolates (Table 2); one isolate (RO-BCI1)
showed borderline susceptibility, with a moderate increase in
IC50 and IC90 (0.30 and 1.1 µM,
respectively), the second strain (RO-BCI23) showed a significant
increase in both IC50 and IC90 (0.53 and 2.02 µM, respectively). This phenotype was not associated with any of the
known mutations linked to TIBO resistance. However, the RO-BCI23
isolate showed the most variable RT sequence of the subtype F isolates
and presented different residues close to the active site of the RT. We
therefore assessed the phenotypic susceptibilities of all the available
subtype F isolates to TIBO. The TIBO IC50 (mean ± standard deviation) for 12 subtype F isolates was 0.07 ± 0.06 µM (range, 0.01 to 0.2 µM), and the IC90 was 0.4 ± 0.27 µM (range, 0.01 to 0.88 µM), meaning that all 12 strains
were susceptible.
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TABLE 2.
IC50s and IC90s of nucleoside and
nonnucleoside RT inhibitors and protease inhibitors of HIV-1 subtype F
isolates, reference HIV-1 isolates, and HIV-2
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| |
DISCUSSION |
Studies of HIV's genetic diversity have shown different rates of
variability for the different viral genes, the most conserved structural gene being pol (29, 31). Analysis of
the pol gene was not considered relevant for genotyping
(36), but several recent reports have shown that the
numerous selection pressures on the pol gene, generally
reflected by synonymous substitutions, make it suitable for
phylogenetic studies (32, 34). The different pol
subtypes described so far correspond to env or
gag subtypes (18, 25, 32, 34).
We analyzed the RT coding regions of 14 isolates originating in
different parts of Romania and characterized as subtype F on the basis
of the env sequence (1). Based on a
pol nucleotide and amino acid sequence comparison, these
strains clustered phylogenetically and were equidistant from other
HIV-1 subtypes. This first report of subtype F pol sequences
confirms the perfect correlation between the phylogenetic positions
determined by env and gag analysis. The similar
results obtained by the neighbor-joining and maximum-likelihood methods
support the reliability of the phylogenetic tree of HIV-1 RT sequences.
Furthermore, phylogenetic analysis of the pol gene of
Romanian HIV-1 isolates revealed a cluster similar to that obtained by
analyzing the env gene sequences. The sequences of isolates
from nosocomially infected children formed a separate branch within the
Romanian sequence cluster with a high bootstrap value (i.e., 73), which
supports our hypothesis of a unique introduction of HIV in horizontally
infected children from Romania, probably resulting from the use of an
infected blood product (1).
None of the patients in this study had ever received antiretroviral
therapy. To investigate the impact of viral variability on
susceptibility to antiretroviral drugs, we analyzed the phenotypic susceptibilities of four subtype F isolates to different classes of
antiretroviral drugs. All but two of the strains had drug
susceptibilities similar to those of wild-type subtype B field isolates
in the same assay (Table 2). One isolate (RO-BCI1) showed borderline susceptibility, i.e., there was a moderate increase in both the IC50 and IC90 of a nonnucleoside RT inhibitor,
TIBO, whereas another isolate (RO-BCI23) showed significantly
diminished susceptibility to TIBO. Although this compound is not used
to treat HIV infection, the phenotypic susceptibility analysis showed
that diversity within group M might affect drug susceptibility.
Nonnucleoside RT inhibitors are a promising class of antiretroviral
compounds for combined therapy because of their low toxicity, facility
of use, and reasonable cost. However, the use of these agents might be
hindered by the existence of naturally resistant variants. HIV-1 group
O isolates are naturally resistant to nonnucleoside RT inhibitors, as
is HIV-2 (9). Because point mutations can lead to marked
reductions in drug susceptibility (22), intra- or
intersubtype pol diversity might be reflected by particular
drug susceptibility profiles. HIV-1 subtype F is only a minor form of
HIV-1 but is widely distributed (4, 7, 10, 13, 16, 18, 20, 21, 23,
27, 33), with major circulation in Romania (1, 11,
15). Phylogenetic studies of isolated strains from different
countries have revealed that separate epidemiological events
contributed to the worldwide distribution of this subtype (1, 2,
16, 18). Our study revealed a natural reduction in susceptibility
to a nonnucleoside RT inhibitor in HIV-1 subtype F strains and
reinforces the need for global screening for HIV-1 group M isolates by
sequencing and phenotyping susceptibility analysis.
| |
ACKNOWLEDGMENTS |
This work was supported by Agence Nationale de Recherches sur le
SIDA (ANRS), France (grant 96009). C.A. is an ANRS postdoctoral fellow.
The samples were obtained with the support of the Romanian Association
against AIDS.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie, Hôp. Bichat-Claude Bernard, 46 Rue Henri Huchard,
75018 Paris, France. Phone: 33 1 40 25 88 94. Fax: 33 1 46 27 02 08. E-mail: francois.simon{at}bch.ap-hop-paris.fr.
| |
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0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
