Role of the human immunodeficiency virus type 1 envelope gene in viral fitness.
ABSTRACT A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.
- SourceAvailable from: Margaret Hosie[Show abstract] [Hide abstract]
ABSTRACT: Following long-term infection with virus derived from the pathogenic GL8 molecular clone of feline immunodeficiency virus (FIV), a range of viral variants emerged with distinct modes of interaction with the viral receptors CD134 and CXCR4, and sensitivities to neutralizing antibodies. In order to assess whether this viral diversity would be maintained following subsequent transmission, a synthetic quasispecies was reconstituted comprising molecular clones bearing envs from six viral variants and its replicative capacity compared in vivo with a clonal preparation of the parent virus. Infection with either clonal (Group 1) or diverse (Group 2) challenge viruses, resulted in a reduction in CD4+ lymphocytes and an increase in CD8+ lymphocytes. Proviral loads were similar in both study groups, peaking by 10 weeks post-infection, a higher plateau (set-point) being achieved and maintained in study Group 1. Marked differences in the ability of individual viral variants to replicate were noted in Group 2; those most similar to GL8 achieved higher viral loads while variants such as the chimaeras bearing the B14 and B28 Envs grew less well. The defective replication of these variants was not due to suppression by the humoral immune response as virus neutralising antibodies were not elicited within the study period. Similarly, although potent cellular immune responses were detected against determinants in Env, no qualitative differences were revealed between animals infected with either the clonal or the diverse inocula. However, in vitro studies indicated that the reduced replicative capacity of variants B14 and B28 in vivo was associated with altered interactions between the viruses and the viral receptor and co-receptor. The data suggest that viral variants with GL8-like characteristics have an early, replicative advantage and should provide the focus for future vaccine development.PLoS ONE 01/2013; 8(1):e54871. · 3.53 Impact Factor
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ABSTRACT: The feline and human immunodeficiency viruses (FIV and HIV) target helper T cells selectively, and in doing so they induce a profound immune dysfunction. The primary determinant of HIV cell tropism is the expression pattern of the primary viral receptor CD4 and co-receptor(s), such as CXCR4 and CCR5. FIV employs a distinct strategy to target helper T cells; a high affinity interaction with CD134 (OX40) is followed by binding of the virus to its sole co-receptor, CXCR4. Recent studies have demonstrated that the way in which FIV interacts with its primary receptor, CD134, alters as infection progresses, changing the cell tropism of the virus. This review examines the contribution of the virus-receptor interaction to replication in vivo as well as the significance of these findings to the development of vaccines and therapeutics.Current opinion in virology. 08/2013;
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ABSTRACT: Human immunodeficiency virus type-1 (HIV-1) fitness has been associated with virus entry, a process mediated by the envelope glycoprotein (Env). We previously described Env genetic diversification in a Zambian, subtype C infected, slow-progressor child (1157i) in parallel with an evolving neutralizing antibody response. Because of the role the Variable-3 loop (V3) plays in transmission, cell tropism, neutralization sensitivity, and fitness, longitudinally isolated 1157i C2-V4 alleles were cloned into HIV-1NL4-3-eGFP and -DsRed2 infectious molecular clones. The fluorescent reporters allowed for dual-infection competitions between all patient-derived C2-V4 chimeras to quantify the effect of V3 diversification and selection on fitness. 'Winners' and 'losers' were readily discriminated among the C2-V4 alleles. Exceptional sensitivity for detection of subtle fitness differences was revealed through analysis of two alleles differing in a single synonymous amino acid. However, when the outcomes of N = 33 competitions were averaged for each chimera, the aggregate analysis showed that despite increasing diversification and divergence with time, natural selection of C2-V4 sequences in this individual did not appear to be producing a 'survival of the fittest' evolutionary pattern. Rather, we detected a relatively flat fitness landscape consistent with mutational robustness. Fitness outcomes were then correlated with individual components of the entry process. Env incorporation into particles correlated best with fitness, suggesting a role for Env avidity, as opposed to receptor/coreceptor affinity, in defining fitness. Nevertheless, biochemical analyses did not identify any step in HIV-1 entry as a dominant determinant of fitness. Our results lead us to conclude that multiple aspects of entry contribute to maintaining adequate HIV-1 fitness, and there is no surrogate analysis for determining fitness. The capacity for subtle polymorphisms in Env to nevertheless significantly impact viral fitness suggests fitness is best defined by head-to-head competition.PLoS ONE 01/2013; 8(4):e63094. · 3.53 Impact Factor
JOURNAL OF VIROLOGY, Aug. 2003, p. 9069–9073
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 16
Role of the Human Immunodeficiency Virus Type 1
Envelope Gene in Viral Fitness
Hector R. Rangel,1Jan Weber,1Bikram Chakraborty,1Arantxa Gutierrez,2
Michael L. Marotta,1Muneer Mirza,1Patti Kiser,1Miguel A. Martinez,2
Jose A. Este,2and Miguel E. Quin ˜ones-Mateu1,3*
Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation,1and
Center for AIDS Research, Case Western Reserve University,3Cleveland, Ohio, and
Laboratori de Retrovirologia, Fundacio irsiCaixa, Hospital Universitari
Germans Trias i Pujol, 08916 Badalona, Spain2
Received 21 April 2003/Accepted 30 May 2003
A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1
(HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene.
Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study
we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of
subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution
of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness
values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r ? 0.93;
P ? 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry
into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1.
In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness
in viruses resistant to novel antiretroviral entry inhibitors.
Human immunodeficiency virus type 1 (HIV-1) fitness, the
replicative adaptation of the virus to its environment (10), is
the result of the interaction of a multitude of viral and host
factors (reviewed in references 22 and 23). Among the viral
factors, many biological processes in the HIV-1 life cycle (e.g.,
cell entry, genome replication, protein synthesis and process-
ing, and particle assembly and release from cells) may affect
viral fitness. It is evident, however, that the envelope (env)
gene plays a major role in the competitive ability of the virus.
For example, the env gene is associated with viral transmission
(13, 15, 28) and host cell tropism (4, 14) and is the main target
of the host immune response (19, 27, 31). Consequently, many
studies have evaluated its direct contribution to viral replica-
tion and HIV-1 pathogenesis (2, 4, 5, 12, 19, 24, 28). In addi-
tion, a whole new generation of antiretroviral drugs is being
developed with the env gene as a primary target (e.g., HIV
entry inhibitors that involve viral env glycoproteins and their
cellular receptors) (8, 21). A recent study showed preliminary
evidence that the efficiency of host cell entry may be the factor
with the greatest impact on HIV-1 fitness in the absence of
drug selective pressure (3). In this study, we have used growth
competition experiments and TaqMan real-time PCR to mea-
sure fitness of both HIV-1 isolates and autologous env-recom-
binant viruses. Our results reveal the impact of the env gene on
the replication capacity of wild-type (wt) subtype B HIV-1
strains and the way in which host cell entry seems to define ex
vivo HIV-1 fitness in the absence of any unusual alterations
affecting other steps of the HIV-1 life cycle (e.g., deletions on
the HIV-1 nef gene  and the presence of drug resistance
mutations in the pol gene ).
HIV-1 isolates and env-pseudotyped viruses from eight sub-
type B HIV-1 strains with different biophenotypes (i.e., syncy-
tium-inducing [SI] or non-syncytium-inducing [NSI] and
CCR5-tropic [R5], CXCR4-tropic [X4], or dual-tropic [R5/X4]
viruses) were analyzed (Table 1). Two HIV-1 primary isolates
harboring similar env genes but with distinct patterns of drug
resistance mutations in the pol genes (F96 and F98) were
obtained from an HIV-1-infected individual treated at the
Hospital Universitari Germans Trias i Pujol in Badalona,
Spain (7). Two HIV-1 primary isolates that became resistant to
the CXCR4 antagonist AMD3100 and their parental strains
(i.e., CI-1, CI-1?, CI-2, and CI-2?) were obtained from a
previous study (11). Finally, two SI X4 HIV-1 isolates (labo-
ratory-adapted strain HIV-1B-HXB2and primary isolate HIV-
1B-92USO76) were obtained from the AIDS Research and Ref-
erence Reagent Program. This collection of viruses covers a
broad genotypic and phenotypic selection (i.e., wt strains, mul-
tidrug-resistant variants, and phylogenetically related viruses
with different coreceptor usage patterns), which allowed us to
analyze the contribution of the HIV-1 env gene to viral fitness.
Recombinant viruses carrying env genes corresponding to
those of these eight HIV-1 strains were constructed as previ-
ously described (6) (Fig. 1A). Briefly, A3.01/CCR5-F7 cells
* Corresponding author. Mailing address: Cleveland Clinic Founda-
tion, Lerner Research Institute, Department of Virology/NN10, 9500
Euclid Ave., Cleveland, OH 44195. Phone: (216) 444-2515. Fax: (216)
444-2998. E-mail: firstname.lastname@example.org.
(obtained from Q. Sattentau through the Centralised Facility
for AIDS Reagents, Medical Research Council) were trans-
fected by electroporation with a mixture of the env-defective
HIVHXB2plasmid pJJ5 (9) and the corresponding PCR-am-
plified HIV-1 env fragment. PCR amplification of the complete
gp160-encoding sequence (the region from 5580 to 8586 of the
HIV-1HXB2genome) was performed by nested PCR by using
the following external primers: Rec2F, 5?-GATAAAGCCAC
CTTTGCCTAGT-3? (nucleotide [nt] position 5514), and env2,
5?-TTCTAGGTCTCGAGATACTGCT-3? (nt position 8889).
The following primers were used for the second PCR: Rec1F,
5?-AAGGGCCACAGAGGGAGCCATA-3? (nt position 5580),
and E270R, 5?-GCGTCCCAGAAGTTCCACAA-3? (nt posi-
tion 8566). Before transfection, the pJJ5 plasmid was digested
with NcoI and BamHI at positions 5675 and 8475 of the HIV-
1HXB2genome. Open plasmid and PCR products were copre-
cipitated and resuspended in water. After transfection, infec-
tious viruses were recovered from the supernatants of cell
cultures and stored at ?80°C until use. All viral stocks (i.e.,
HIV-1 isolates and env-recombinant viruses) were propagated
in phytohemagglutinin-stimulated, interleukin-2-treated pe-
ripheral blood mononuclear cells (PBMC) as previously de-
scribed (24). The tissue culture dose for 50% infectivity was
determined for each isolate in triplicate with serially diluted
supernatants from each viral propagation. Reverse transcrip-
tase activity (29) in culture supernatants, on day 8 of culture,
was used to calculate the tissue culture dose for 50% infectivity
by using the Reed and Muench method (26). The MT-2 assay
(28) was used to analyze the viral phenotype (i.e., SI or NSI).
Coreceptor usage was determined by using viral stocks to in-
fect U87 cells expressing CD4 and either CCR5 or CXCR4
chemokine receptors as previously described (3) (Table 1).
Finally, nucleotide sequence analysis of the complete gp160-
encoding region of the env gene was used to verify the identity
of all viral stocks (i.e., HIV-1 isolates and env-recombinant
viruses) as previously described (24).
To estimate ex vivo HIV-1 fitness, growth competition ex-
periments were carried out as previously described (30).
Briefly, each subtype B HIV-1 primary isolate or env-recom-
binant virus competed against two different non-subtype B
HIV-1 control strains (HIV-1A-92UG029and HIV-1AE-CMU06)
FIG. 1. Schematic representation of the construction of HIV-1 re-
combinant viruses, growth competition experiments, and TaqMan re-
al-time PCR detection. (A) Patient PBMC samples were used to (i)
isolate HIV-1 and (ii) PCR amplify and clone the whole gp160-encod-
ing region of the env gene to construct HIV-1 env-recombinant viruses
(see Materials and Methods for details). (B) Individual dual infec-
tions with a subtype B query HIV-1 isolate and one control strain
(HIV-1A-92UG029or HIV-1AE-CMU06) were performed at a multiplicity
of infection (MOI) of 0.01 infectious U/cell. Wells I and III correspond
to positive controls for the query and control viruses, respectively.
(C) Three sets of subtype-specific primers and probes were designed to
quantify the proportion of both HIV-1 variants in the dual infection.
Subtype B-specific primers Bgag-S and Bgag-AS3 and probe pBgag-
ROX were used to PCR amplify and recognize a conserved region
within the subtype B HIV-1 gag gene. Similarly, subtype A-specific
primers A2env-S and A2env-AS2 plus probe pA2env-FAM and
CRF01_AE-specific primers E1env-S and E1env-AS, together with
probe pE1env-FAM, were used to PCR amplify and recognize con-
served regions in the HIV-1 env genes of clade A and the circulating
recombinant form CRF01_AE, respectively (30).
TABLE 1. HIV-1 isolates used to evaluate the role of
env in viral fitness
PR and RT resistant
aHIV-1 isolates F96 and F98 were obtained from a single patient in February
1996 and April 1998, respectively (7). CD4 counts and viral loads at these time
points were as follows: F96, 10 cells/?l and 891,205 HIV RNA copies/ml; F98,
388 cells/?l and 66,070 HIV RNA copies/ml. HIV-1 clinical isolates CI-1?and
CI-2?were obtained after in vitro selection with AMD3100 (11). CI-1 and CI-2
correspond to parental HIV-1 strains from the same individuals. HXB2 and
92US076 correspond to laboratory strain HIV-1B-HXB2and primary isolate HIV-
bwt indicates an absence of mutations in the pol gene associated with drug
resistance. After a history of antiretroviral treatment that included zidovudine,
didanosine, lamivudine, stavudine, nevirapine, indinavir, ritonavir, and saquina-
vir, the F98 HIV-1 isolate showed multiple protease (PR) (10I, 48V, 54V, 63P,
71V, 77I, 82A, and 90M) and reverse transcriptase (RT) (41L, 67N, 181C, 184V,
190A, 215Y, and 219E) drug resistance mutations (30) (http://www.iasusa.org).
cViral fitness values are from the averages of two relative fitness values
corresponding to the competitions of each HIV-1 isolate with two HIV-1 control
strains (see text for details) and are calculated relative to the fitness of the
wild-type HIV-192US076virus control (100%).
in a 1:1 initial proportion with a multiplicity of infection of 0.01
infectious U/cell (Fig. 1B). One milliliter of these viral mix-
tures was incubated with 106PBMC for 2 h at 37°C and 5%
CO2. Subsequently, the cells were washed three times with 1?
phosphate-buffered saline and then resuspended in culture
medium (106/ml). Cells were washed and fed with medium
after 4 days. Supernatants and cells were harvested at day 8,
resuspended in dimethyl sulfoxide-fetal bovine serum, and
stored at ?80°C for subsequent analysis. To determine viral
fitness, the final ratio of the two viruses produced from each
growth competition experiment was determined by TaqMan
real-time PCR and compared to viral production from the
monoinfections as previously described (30). Three sets of
subtype-specific primers and probes were designed (Fig. 1C).
These sets of primers and probes allowed subtype-specific
PCR amplification and hybridization so that cross-hybridiza-
tion between subtype B, A, and AE probes did not occur (30).
A relative fitness value for each virus in the competition was
estimated by using the production of each individual HIV-1
strain in the dual infection (24, 25). A total relative fitness
value was calculated as the average of the two relative fitness
values corresponding to the competition between each subtype
B HIV-1 isolate or recombinant virus and each of the non-
subtype B HIV-1 control strains. The total relative fitness
values were then compared and expressed as percentages
of the fitness of a wt subtype B HIV-1 primary isolate
(HIV-1B-92US076, with a fitness value set at 100%) (25, 30).
Despite differences in viral phenotypes and coreceptor usage
patterns, our growth competition and real-time PCR method
was able to accurately determine fitness of both HIV-1 isolates
and env-recombinant viruses (Table 1). For example, when the
B-92US076 env-recombinant virus competed in PBMC against
both HIV-1 controls, followed by real-time PCR detection, a
relative fitness value similar to that observed with the HIV-1
isolate was obtained (1.45 and 1.62 for the env recombinant
and the HIV-1 isolate, respectively, corresponding to 97 and
100% of the fitness of the HIV-1 control) (Fig. 2). When the
rest of the viruses were analyzed, a strong, statistically signifi-
cant correlation was observed between the fitness values cal-
culated for wt HIV-1 isolates and those for the env-recombi-
nant viruses (r ? 0.86; P ? 0.01; Pearson product moment)
(Fig. 3), suggesting that the env gene may be driving viral
fitness in wt HIV-1 strains. It is important to note that the
multidrug-resistant F98 isolate and the corresponding env-re-
combinant virus were not included in this correlation. This
highly mutated HIV-1 isolate showed impairment in fitness,
which was not evident when its env gene was introduced into a
wt HIV-1 backbone (Fig. 3A). A recent study analyzed the
fitness levels of both wt F96 and multidrug-resistant F98 vi-
ruses by using HIV-1 isolates and recombinant viruses carrying
FIG. 2. Fitness of HIV-1B-92US076and the autologous env-recombinant virus. (A) Growth competition experiments with the HIV-1B-92US076
isolate and each one of the control viruses (HIV-1A-92UG029and HIV-1AE-CMU06) in PBMC, with TaqMan real-time PCR to measure viral
production. Similar experiments were performed by using the B-92US076 env-recombinant virus and both non-subtype B control viruses. TaqMan
real-time PCR amplification plots and charts showing proportions of virus production are indicated. (B) Relative fitness relationships between
query viruses (i.e., HIV-1 isolates or env recombinants) and non-subtype B HIV-1 controls. Numbers in parentheses under B-92US076 correspond
to the relative fitness value in each condition (24). Arrow directions indicate fitness differences (positive or negative) between both viral strains
(24). For example, the HIV-1B-92US076isolate is 9.2-fold more fit than HIV-1AE-CMU06. Numbers in black boxes correspond to the fitness values
relative to the that of the wt HIV-192US076control (100%) as described in Table 1.
VOL. 77, 2003NOTES9071
the protease gene, the reverse transcriptase gene, and the 3?
end of the gag gene (30). Fitness values of the wt F96 viruses
were similar to that of the HIV-1 control (i.e., viral isolate,
96%; pol recombinant, 102%). Analyses of the drug-resistant
F98 HIV-1 isolate and the autologous pol-recombinant virus
showed a comparable reduction in fitness (i.e., viral isolate,
25%; pol recombinant, 19%) (30). Thus, a considerable de-
crease in replication capacity due to selection and accumula-
tion of drug-resistant mutations in the pol gene seems to have
overcome the effect of other viral genomic regions (e.g., the
env gene) on the overall ex vivo fitness of the F98 virus.
A previous study showed that the addition of the CXCR4
antagonist AMD3100 to PBMC infected with R5/X4 HIV-1
isolates resulted in the selection of R5 HIV-1 variants or
AMD3100-resistant viruses without a change in coreceptor
usage (11). Here we determined the fitness of four HIV-1
primary isolates (i.e., CI-1, CI-1?, CI-2, and CI-2?) and the
corresponding env-recombinant viruses (Table 1 and Fig. 3). In
agreement with results in a previous report (1), both
AMD3100-resistant viruses, regardless of coreceptor usage,
showed a decrease in fitness compared with their parental
strains. Interestingly, fitness values of all four env-recombinant
viruses (i.e., wt parental and AMD3100 resistant) mimic the
values of the autologous HIV-1 isolates (Fig. 3). Finally, a
slight reduction in fitness values was observed for most of the
env-recombinant viruses compared to those of their parental
HIV-1 isolates (Table 1; Fig. 3). Although this may stress the
importance of relative harmony between different viral
genomic regions in order to maintain optimal viral fitness, it
may also be a consequence of the env recombination proce-
dure. Nevertheless, this phenomenon did not affect the corre-
lation between fitness values determined with HIV-1 isolates
and env-recombinant viruses.
Numerous studies have addressed HIV-1 fitness and the
potential effects on viral load, resistance to protease inhibitors
(PI) and/or reverse transcriptase inhibitors (RTI), and disease
progression (22, 23). However, great effort is being devoted to
the development of new drugs that may inhibit the entry of
HIV-1 into susceptible cells (8, 21). In addition, current clinical
trials involve treatment of HIV-infected individuals with com-
binations of PI, RTI, and viral entry inhibitors (e.g., enfu-
virtide, formerly T-20) (18). Therefore, studies to estimate
fitness of HIV-1 with genes for potential multidrug resistance
(i.e., pol and env) must be designed. The growth competition
and real-time PCR assay has been demonstrated to be useful in
analyzing fitness of wt (3, 24) and drug-resistant HIV-1 isolates
and pol-recombinant viruses (1, 25, 30). In this case, our ex vivo
assay may be useful for analyzing fitness of both wt HIV-1
isolates and isolates resistant to PI, RTI, and/or entry inhibi-
tors. These studies are currently under way in our laboratory.
In this study, we have analyzed the fitness of different sub-
type B HIV-1 isolates with distinct phenotypes and coreceptor
usage patterns, showing a statistically significant correlation
with the fitness of the corresponding autologous env-recombi-
nant viruses. Although HIV-1 fitness is the result of many
biological processes in the virus life cycle (i.e., cell entry, re-
verse transcription, integration, gene expression, and virion
assembly and release from cells), our results suggest that early
events in the life cycle of wt HIV-1 isolates (e.g., viral entry)
may make a major contribution to overall viral fitness. Recent
studies have proposed that differences in fitness levels among
HIV-1 subtypes may map to the env gene, perhaps having an
impact on disease progression as well as transmission, evolu-
tion, and diversification of HIV-1 in different regions of the
world (1, 3, 16, 20, 23). Further studies on the role of the wt
HIV-1 env gene in viral fitness will help us to understand its
effects on viral tropism, replication, and/or persistence in a
variety of microenvironments within the human host, in addi-
tion to contributing to the development of novel antiretroviral
FIG. 3. Contribution of the env gene to HIV-1 fitness. (A) Relative fitness of wt subtype B HIV-1 isolates and autologous env-recombinant
viruses (constructed in an HIV-1HXB2backbone). Values are percentages of the fitness of the wt HIV-1B-92US076control (100%). (B) Correlation
between relative fitness values measured for HIV-1 isolates and those for the corresponding env-recombinant viruses in an HIV-1HXB2background.
The multidrug-resistant F98 HIV-1 isolate and the corresponding env-recombinant virus were not included in this analysis (see text for details).
The Pearson product moment correlation coefficient was used to determine the strength of association or correlation between ex vivo HIV-1 fitness
values of HIV-1 isolates and those of env-recombinant viruses.
H.R.R. and J.W. contributed equally to the experiments described in
Research performed at the Cleveland Clinic Foundation (M.E.Q.-
M.) was supported by research grants from the National Heart, Lung,
and Blood Institute, NIH (5-KO1-HL67610-03), and the Center for
AIDS Research (AI36219) at Case Western Reserve University. Re-
search at the Fundacio irsiCaixa was supported by research grants
from la marato ´ de TV3 (FIS 01/0067-02 and Red Tematica Coopera-
tiva de Investigacion en Sida) and from Fundacio ´n para la Investiga-
cio ´n y la prevencio ´n del SIDA en Espan ˜a (FIPSE 36293/02 and
36207/01 [M.A.M.] and BFM-2000-1382 and FIS01/1116 [J.A.E.]).
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