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Jurnal Didanosine
Jurnal Didanosine
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
a r t i c l e
i n f o
Article history:
Received 1 September 2011
Revised 2 November 2011
Accepted 16 November 2011
Available online 25 November 2011
Keywords:
HIV-1
Vpr
Drug resistance
Didanosine
a b s t r a c t
Background: HIV-1 accessory Vpr protein is involved in the reverse transcription process and has been
shown to modulate the virus mutation rate. This process may play a role in the kinetics of appearance
of drug resistance mutations under antiretroviral treatment.
Methods: Vpr sequences were analyzed from plasma viruses derived from 97 HIV-1-infected individuals
failing antiretroviral treatment and 63 antiretroviral-nave patients. Vpr genetic variability was analyzed
for association with specic drug treatment and drug resistance mutations. Biological and virological
experiments were employed to characterize a mutation in Vpr found to be associated with virological
failure.
Results: E17A mutation located in the rst a-helix of Vpr was more prevalent in HAART-treated individuals compared to untreated individuals. E17A was associated with thymidine analog mutations (TAMs) in
reverse transcriptase M41L, L210W and T215Y and with the use of didanosine in the patients treatment
histories. E17A had no impact on the biochemical and functional properties of Vpr, and did not affect
kinetics of replication of wild-type or TAMs-containing viruses. However, its association with TAMs
and the use of didanosine was consistent with phenotypic susceptibility assays showing a signicant
3-fold decrease in didanosine susceptibility of viruses harboring Vpr E17A combined with TAMs compared to viruses harboring TAMs alone.
Conclusion: These ndings highlight a novel role of Vpr in HIV-1 drug resistance. Vpr E17A confers resistance to didanosine when associated with TAMs. Whether Vpr E17A facilitates excision of didanosine is
still to be determined.
2011 Elsevier B.V. All rights reserved.
1. Introduction
The high adaptive capacity of HIV-1 is demonstrated by its ability to escape from potent antiviral drugs in treated HIV-infected
individuals. Virological failure occurs when viruses with drug
resistance-associated mutations (RAMs) in the targeted viral
regions (reverse transcriptase, protease, integrase or envelope)
Corresponding author. Address: Department of Virology, CERVI Pitie-Salpetriere
Hospital, 83 boulevard de lHopital, 75013 Paris, France. Tel.: +33 142177418;
fax: +33 142177411.
E-mail address: slim.fourati@psl.aphp.fr (S. Fourati).
0166-3542/$ - see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.antiviral.2011.11.008
are selected and outgrow the more susceptible strains. The generation of resistant variants is a consequence of the error proneness
of the HIV-1 reverse transcriptase (RT) enzyme (Mansky and
Temin, 1995) and recombination (Jetzt et al., 2000; Levy et al.,
2004; Moutouh et al., 1996). In addition to gag, pol and env genes,
HIV-1 genome encodes a number of accessory proteins that may be
involved in viral diversication and generation of resistant variants. For example, Vif and Vpr have been shown to be indirectly
involved in the reverse transcription process (Bishop et al., 2004;
Mansky and Temin, 1995).
We recently demonstrated that some Vif variants are likely involved in the appearance of drug resistance-associated mutations
168
169
3. Results
3.1. Patient characteristics
Our population of patients failing HAART (n = 97) has the following characteristics: the median age was 46 years, 89% of the patients were men; the median CD4 cell count was 331 cells/mm3,
and the median plasma HIV-1 RNA was 4680 cp/ml at the time
of genotypic testing. Overall, median time of HIV-1 infection was
17 years (range: 425) and the median of prior ART exposure
was 13 years (range: 1.518). Patients received a median of eight
antiretroviral drugs in their history (range: 219). All patients
received at least one nucleoside/nucleotide reverse transcriptase
inhibitor (NRTI), 74% at least one non-nucleoside reverse transcriptase inhibitor (NNRTI) and 97% at least one protease inhibitor (PI)
in their history. At the time of analysis, 98% of viruses collected
from plasma from HAART-treated patients harbored at least one
drug resistance associated mutation (RAM) in reverse transcriptase
(RT) and/or protease (PR) (major mutations, as dened by the
IAS-USA guidelines).
170
Fig. 1. Frequency of mutations in the three a-helix functional domains of HIV-1 Vpr. The amino acids variation shown is based on 63 sequences derived from drug-nave
patients and 97 sequences derived from patients failing on HAART. The variation in the three a-helix functional domains is shown for each of the two patient populations.
Substitution at position 17 is indicated by . HxB2 was used as reference sequence.
Table 1
Genetic variations in E17, L22 and R32 positions of the rst a-helix of Vpr that
were found more polymorphic in pretreated patients (p < 0.05, entropy program
http://www.hiv.lanl.gov/cgi-bin/ENTROPY). E17A and R32K mutations were more
prevalent in pretreated patients compared to untreated patients.
Position
Amino acid
Untreated
17
E (wild type)
A
Q
D
G
V
71 (73%)
17 (18%)
4 (4%)
3 (3%)
1 (1%)
1 (1%)
59 (93%)
2 (3%)
2 (3%)
0
0
0
22
L (wild type)
F
I
R
88 (91%)
4 (4%)
4 (4%)
1 (1%)
62 (98%)
0
1 (2%)
0
32
R (wild type)
K
84 (86%)
13 (14%)
61 (97%)
2 (3%)
171
Fig. 2. Percentage of patients having received nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) in their history and harboring either E17 (WT) or A17. We observed
a unique association between E17A and didanosine. Similar analyses were performed for other drug classes: non-nucleoside reverse transcriptase inhibitors (NNRTI), protease
inhibitors (PI), anti-integrase but failed to show any association (Supplementary le 1). ddI: didanosine, ddC: zalcitabine, 3TC: lamivudine, FTC: emtricitabine, D4T: stavudine,
ABC: abacavir, TDF: tnofovir disoproxil fumarate.
Fig. 3. Biochemical characterization and intracellular distribution of the Vpr E17A variant. (A) Packaging assay of the HA-tagged Vpr proteins into virus particles. Virions were
produced from 293T cells co-transfected with an HIV-1-based packaging vector lacking the vpr gene in combination with the empty plasmid (mock) or with plasmids for
expression of the wild type or mutated HA-tagged Vpr protein. 48 h later, proteins from cell and virion lysates were separated by SDSPAGE and analyzed by Western blotting
with anti-HA and anti-CAp24 antibodies. (B) Subcellular distribution of the HA-tagged Vpr proteins. HeLa cells were transfected with the empty plasmid (mock) or plasmids
for expression of the wild type or mutated HA-Vpr protein and were then analyzed 48 h after transfection by immunouorescence. Cells were xed, permeabilized, and
subsequently stained with anti-HA and DAPI (40 ,60 -diamidino-2-phenylindole). Cells were analyzed by epiuorescence microscopy, and images were acquired by using a
charge-coupled device camera. (C) In vitro pulldown assay for Vpr binding to UNG2. Lysates from 293T cells transfected with the empty plasmid (mock) or plasmids for
expression of the wild type or mutated HA-Vpr protein (right panel) were incubated with equal amounts of either GST or GST-UNG2 (upper panels, Ponceau red) immobilized
on GSHSepharose beads. Bound proteins were then analyzed by immunoblotting with anti-HA (lower panels). (D) Co-precipitation analysis of Vpr and UNG2 proteins. 293T
cells transfected with the empty plasmid (mock) or plasmids for expression of the wild type or mutated HA-Vpr protein in combination with either GFP or GFP-UNG2
expression plasmids (lower panel, Cell lysate) were lyzed and subjected to immunoprecipitation with anti-GFP. Precipitates were then analyzed by immunoblotting with
anti-HA (upper panel) and anti-GFP (middle panel).
172
4. Discussion
Several studies have suggested a role for HIV-1 Vpr in modulating the viral mutation rate during the course of infection. Vpr has
been found to incorporate the nuclear form uracil-DNA glycosylase
2 (UNG2) into HIV-1 virions. UNG-2 is a cellular DNA-repair enzyme involved in nucleotide-excision repair, and its incorporation
in virions is correlated with the ability of Vpr to alter the mutation
Fig. 4. Virological characterization of the VPR E17A. (A) Replication kinetics of RT and Vpr mutants. Replication kinetics of full-length NL4-3 WT and mutants derived from
NL4-3: Vpr-E17A (E17A), RT-M41L, L210W, T215Y (TAMs) and Vpr-E17A + RT-M41L, L210W, T215Y (TAMs + E17A) viruses were determined using MT-2 T cells. Supernatants
were collected every 13 days and assayed for viral p24 antigen. (BC) Resistance of RT and Vpr mutants to didanosine. Hela-P4 Cells were infected, in triplicate, with NL4-3
WT (WT), and mutants derived from NL4-3: Vpr-E17A (E17A), RT-K65R (K65R), RT-[M41L, L210W, T215Y] (TAMs) and Vpr-E17A + RT-[M41L, L210W, T215Y] (E17A + TAMs)
viruses (equivalent of 5 ng of p24gag antigen). The single cycle titers of viruses were determined 48 h after infection by quantifying b-galactosidase activity in HeLa-P4 lysates
in a colorimetric assay (the CPRG assay). Cells were infected with viruses and grown in the presence of increasing concentrations of didanosine (B) or AZT (C). The 50%
inhibitory concentration (IC50) was determined as the drug concentration giving 50% inhibition of b-galactosidase levels with respect to untreated infected cells. Drug
susceptibility results were expressed as the fold change in susceptibility, dened as the ratio of the IC50 of the mutant variant to that of NL4-3 wild-type. Assays were
performed three times.
173
of viruses with TAMs in resistance to didanosine rather than a potential role in AZT resistance. Whether Vpr E17A increases the
excision process of didanosine induced by TAMs is still to be
determined.
In conclusion, our results clearly show an association between
Vpr E17A, TAMs (M41L, L210W, and T215Y) and the use of didanosine. Consistently with phenotypic drug susceptibility assays, the
observed association between TAMs and Vpr E17A in patients sequences is related to a potentializing effect of viruses with TAMs in
enhancing resistance to didanosine. One possible explanation is
that the use of didanosine in viruses harboring TAMs may select
for mutations in Vpr that, in turn, enhance resistance to didanosine. Alternatively, viruses harboring Vpr E17A before treatment
may be more likely to develop resistance to didanosine when this
drug is introduced into a regimen and when the patient is infected
with a virus containing TAMs. Longitudinal studies need to be conducted to elucidate the kinetic of appearence of these mutations.
As for mechanistic respects, functional and structural analysis of
interaction between Vpr and RT should better characterize this
association, specically to determine if Vpr plays a role in the excision process induced by TAMs. Similarly, because integrase is also
involved in the reverse transcription complex and has been shown
to interact with Vpr (Gleenberg et al., 2007) studies evaluating the
role of Vpr in resistance to integrase inhibitors should be
conducted.
Competing Interests
The authors have declared that no competing interests exist.
Acknowledgments
We thank G. Le Mallier and P. Grange for their technical
assistance.
This work was supported by Agence Nationale de recherche sur
les SIDA et les hpatites virales (ANRS), Sidaction, the Association
de Recherche en Virologie et Dermatologie (ARVD) and the European Communitys Seventh framework Program (FP7/2007-2013)
under the project Collaborative HIV and Anti-HIV Drug Resistance
Network (CHAIN).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.antiviral.2011.11.008.
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