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1. Retrovirology
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quot;Shock and killquot; effects of class I-selective histone deacetylase inhibitors in
combination with the glutathione synthesis inhibitor buthionine sulfoximine in
cell line models for HIV-1 quiescence
Retrovirology 2009, 6:52 doi:10.1186/1742-4690-6-52
Andrea Savarino (andrea.savarino@iss.it)
Antonello Mai (antonello.mai@uniroma1.it)
Sandro Norelli (sandro.norelli@iss.it)
Sary El Daker (saryeldaker@yahoo.it)
Sergio Valente (sergio.valente1977@libero.it)
Dante Rotili (danterotili@libero.it)
Lucia Altucci (antonello.mai@uniroma1.it)
Anna Teresa Palamara (microbiologia.farmaceutica@uniroma1.it)
Enrico Garaci (presidenza@iss.it)
ISSN 1742-4690
Article type Short report
Submission date 7 April 2009
Acceptance date 2 June 2009
Publication date 2 June 2009
Article URL http://www.retrovirology.com/content/6/1/52
This peer-reviewed article was published immediately upon acceptance. It can be downloaded,
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2. “Shock and kill” effects of class I-selective histone deacetylase
inhibitors in combination with the glutathione synthesis inhibitor
buthionine sulfoximine in cell line models for HIV-1 quiescence
Andrea Savarino*1§ Antonello Mai,*2 Sandro Norelli1, Sary El Daker1, Sergio
Valente,2 Dante Rotili,2 Lucia Altucci,3 Anna Teresa Palamara2,5 and Enrico Garaci6.
Address: 1Dept. of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità,
Viale Regina Elena, 299, 00161, Rome, Italy, 2Pasteur Institute, Cenci-Bolognetti Foundation,
Dept. of Drug Chemistry and Technologies, Sapienza University of Rome, P.le A. Moro, 5, 00185,
Rome, Italy, 3Dept. of General Pathology, 2nd University of Naples, Vico L. De Crecchio 7, 80138
Naples, Italy, 5Pasteur Institute, Cenci-Bolognetti Foundation, Dept. of Public Health Sciences,
Sapienza University of Rome, P.le A. Moro, 5, 00185, Rome, Italy, 6Dept. of Experimental
Medicine, University of Rome Tor Vergata, Rome, Italy.
E-mail: Andrea Savarino: andrea.savarino@iss.it; Antonello Mai: antonello.mai@uniroma1.it; Sary
El Daker: saryeldaker@yahoo.it; Sandro Norelli: sandro.norelli@iss.it; Sergio Valente:
sergio.valente1977@libero.it; Dante Rotili: danterotili@libero.it; Lucia Altucci:
lucia.altucci@unina2.it; Anna Teresa Palamara: microbiologia.farmaceutica@uniroma1.it; Enrico
Garaci: presidenza@iss.it
*Equal contribution
§
Corresponding author

3. 2
Abstract
Latently infected, resting memory CD4+ T cells and macrophages represent a major obstacle to the
eradication of HIV-1. For this purpose, “shock and kill” strategies have been proposed (activation
of HIV-1 followed by stimuli leading to cell death). Histone deacetylase inhibitors (HDACIs)
induce HIV-1 activation from quiescence, yet class/isoform-selective HDACIs are needed to
specifically target HIV-1 latency. We tested 32 small molecule HDACIs for their ability to induce
HIV-1 activation in the ACH-2 and U1 cell line models. In general, potent activators of HIV-1
replication were found among non-class selective and class I-selective HDACIs. However, class I
selectivity did not reduce the toxicity of most of the molecules for uninfected cells, which is a major
concern for possible HDACI-based therapies. To overcome this problem, complementary strategies
using lower HDACI concentrations have been explored. We added to class I HDACIs the
glutathione-synthesis inhibitor buthionine sulfoximine (BSO), in an attempt to create an
intracellular environment that would facilitate HIV-1 activation. The basis for this strategy was that
HIV-1 replication decreases the intracellular levels of reduced glutathione, creating a pro-oxidant
environment which in turn stimulates HIV-1 transcription. We found that BSO increased the ability
of class I HDACIs to activate HIV-1. This interaction allowed the use of both types of drugs at
concentrations that were non-toxic for uninfected cells, whereas the infected cell cultures
succumbed more readily to the drug combination. These effects were associated with BSO-induced
recruitment of HDACI-insensitive cells into the responding cell population, as shown in Jurkat cell
models for HIV-1 quiescence. The results of the present study may contribute to the future design
of class I HDACIs for treating HIV-1. Moreover, the combined effects of class I-selective HDACIs
and the glutathione synthesis inhibitor BSO suggest the existence of an Achilles’ heel that could be
manipulated in order to facilitate the “kill” phase of experimental HIV-1 eradication strategies.

4. 3
Findings
Given the inability of antiretroviral therapy (ART) to eradicate HIV-1 from the body (even after
decade-long periods of therapy), and the absence of effective vaccines on the horizon, novel
approaches to HIV-1 eradication are needed. To this end, the so-called “shock and kill” strategies
have been proposed [1]. These strategies consist of inducing, through drugs, HIV-1 activation from
quiescence (i.e. the “shock” phase), in the presence of ART (to block viral spread), followed by the
elimination of infected cells (i.e. the “kill” phase), through either natural means (e.g. immune
response, viral cytopathogenicity) or artificial means (e.g. drugs, monoclonal antibodies, etc.) [1].
For the “shock” phase, histone deacetylase inhibitors (HDACIs) have been proposed [2]. Histone
deacetylases (HDACs) contribute to nucleosomal integrity by maintaining histones in a form that
has high affinity for DNA [3]. Physiologically, this activity is counteracted by histone acetyl
transferases (HATs) which are recruited to gene promoters by specific transcription factor-
activating stimuli [3].
Several of the currently available HDACIs activate HIV-1 from quiescence in vitro [4, 5]. However,
this activity is associated with a certain degree of toxicity [6], given that these inhibitors are not
class-specific and compromise a large number of cellular pathways [7, 8]. Class I HDACs comprise
HDAC1-3 and 8; they are predominantly nuclear enzymes and are ubiquitously expressed [9]. Class
II HDACs include HDAC4-7, 9 and 10 and shuttle between the nucleus and the cytoplasm [10, 11].
HDACs are recruited to the HIV-1 promoter by several transcription factors, including NF-κB
(p50/p50 homodimers), AP-4, Sp1, YY1 and c-Myc [12-14]. Identification of class/isoform-
selective HDACIs with increased potency and lower toxicity [3] and drugs able to potentiate their
effects is believed to be important for HIV-1 eradication.
To identify novel HDACIs capable of activating HIV-1, we first tested the HIV-1 activating ability
of our institutional library of HDACIs [see Additional file 1] in cell lines in which HIV-1 is
inducible (i.e. T-lymphoid ACH-2 cells and monocytic U1 cells). The potency of these molecules to
activate HIV-1 was assessed in terms of p24 production, as measured by ELISA (Perkin-Elmers,

5. 4
Boston, MA), following incubation with a drug concentration of 1 µM (generally used as a
threshold for selection of lead compounds). As a positive control, we used TNF-α (5 ng/ml), a
cytokine that activates HIV-1 transcription through NF-κB (p65/p50) induction [1]. As a reference
standard for the comparison of results, we used suberoylamide hydroxamic acid (SAHA; also
referred to as “vorinostat”), a non-specific inhibitor of both classes of HDACs when used in the
upper-nanomolar/micromolar range of concentrations [15].
The results revealed a number of compounds capable of activating HIV-1; and, for the most potent
compounds, there was good agreement between the results in the ACH-2 and U1 cells (Figure 1).
Only non-class selective and class I-selective HDACIs were significantly active (Figure 1), and
potent class I-selective HDACIs enhanced HIV-1 replication in the nanomolar range in a dose-
dependent manner (Figure 2). In general, class I selectivity was insufficient for eliminating toxicity,
although some of the compounds (e.g. MC2211) induced adequate HIV-1 activation and low-level
toxicity (Figure 1, 2). Of note, the class I-selective HDACIs that activated HIV-1 included MS-275,
an HDAC1-3-selective inhibitor currently being tested in phase II clinical trials as an anticancer
drug [15].
A previous study showed a trend towards higher toxicity of the HDACI trichostatin in ACH-2 cells
than in their uninfected counterparts and linked this phenomenon to the cytotoxicity of activated
HIV-1 replication in lymphoid cells [16]. In our experiments, three different class I HDACIs (i.e.
MS-275, MC2113 and MC2211) displayed lower CC50 in ACH-2 cells (Figure 2D) than in
uninfected CD4+ T cells (data from Jurkat cells are shown as an example in Figure 2E), yet the
extent of the difference did not support the possibility of a “therapeutic window”. The same
compounds displayed non-significant toxicity in U1 cells at concentrations up to 1 µM (Figure 2F).
In these experiments, an incubation period of 72 hours was preferred to shorter periods, because of
the intrinsically slow mode of action of epigenetic modulators, which only indirectly induce HIV-1
activation. This was confirmed by our experiments using Jurkat cell clones with an integrated green
fluorescence protein (GFP)-encoding gene under control of the HIV-1 LTR [17]. In these Jurkat cell

6. 5
clones, GFP induction by HDACIs was evident only in a fraction of cells at 24 hours of incubation
and increased over time [see Additional file 2].
To focus on the structural requirements for the most potent class I-selective HDACIs, we then
performed a structure/activity relationship (SAR) study. SAR studies relate the effect or the potency
of bioactive chemical compounds with their chemical structure and help to understand the structural
requirements for obtaining a desired effect. HDACIs are structured according to a general
pharmacophore model (i.e. quot;a molecular framework that carries the essential features responsible
for a drug’s biological activityquot; [18]) (Figure 3A). This pharmacophore model comprises a cap
group (CAP), a polar connection unit (CU), and a hydrophobic spacer (HS), which carries at its end
a Zn2+ binding group (ZBG), able to complex the Zn2+ at the bottom of the cavity [19]. The ZBG
consists of a hydroxamate, a sulfhydryl, or a benzamide moiety (Figure 3A shows a benzamide
inhibitor complexed with HDAC2). A general scaffold describing the characteristics of the most
potent HDACIs from our library is presented in Figure 3B, C. The differences in the general
structural requirements for the two main chemical types of HDACIs in our library (hydroxamates
and benzamides) can probably be attributed to the hydrophobicity/hydrophilicity balance (the more
hydrophobic benzamides require less hydrophobic CAP groups than hydroxamates do). The
molecular docking simulations, conducted as previously described [20,21], highlighted particular
requirements for the CU (Figure 3D). These requirements consisted of a uracil group or an amide
group in a cis-conformation, which presented the nitrogen-bond hydrogen and the carbonylic
oxygen on the same side of the molecule (usually amide groups are in a trans-conformation, with
the nitrogen-bond hydrogen and the carbonylic oxygen oriented in opposite directions) (Figure 3D).
SAHA, consistent with its non-specific inhibitory activity on HDACs [15], did not match the
characteristics of our pharmacophore model [see Additional file 3].
Given that class I selectivity, in general, did not markedly decrease the toxicity of HDACIs, we
have begun studies on complementary strategies that might increase the efficacy of class I HDACIs
at non-toxic concentrations. It is well known that HIV-1 induces a pro-oxidant status which in turn

7. 6
enhances the levels of HIV-1 transcription [22-25]. There are probably many mechanisms behind
HIV-1-induced oxidative stress, and the signals that it sparks are still far from being fully
understood [26]. In general, oxidative stress tilts the balance of HAT/HDAC activity towards
increased HAT activity and DNA unwinding, thus facilitating the binding of several transcription
factors [27]. The HIV-1–induced pro-oxidant status is in part mediated by decreased intracellular
levels of reduced glutathione [26, 28]. The depletion of reduced glutathione has been linked to
activation of viral replication [29], whereas the administration of this cofactor results in
antiretroviral effects [26]. We hypothesized that glutathione depletion might create an intracellular
environment that facilitates HIV-1 activation by HDACIs. To test this hypothesis, we evaluated the
HIV-1 activating effects of buthionine sulfoximine (BSO), which depletes glutathione by inhibiting
γ-glutamyl cysteine synthetase (a limiting step in glutathione synthesis) [27, 30].
BSO, at concentrations of up to 500 µM, did not significantly raise the p24 concentrations; yet it
increased the HIV-1 promoting effects of class I HDACIs, such as MS-275 (Figure 4A) and
MC2113 (data not shown) in ACH-2 cells (Figure 4A) and U1 cells (data not shown). According to
the literature, the concentrations of MS-275 and BSO adopted here are clinically achievable [31,
32]. The results shown in Figure 4A are based on a 24 hour incubation time, given the marked
cytotoxicity shown by the drug combination in the ACH-2 cells at 72 hours of incubation (Figure
4B). Since HIV-1 replicating cell cultures display decreased levels of reduced glutathione [29], their
poor tolerance to an inhibitor of glutathione synthesis is not surprising. This concept is supported by
experiments in uninfected Jurkat cells and Jurkat cell clones (6.3 and 8.4), which contain a
quiescent HIV-1 genome (with the GFP gene) under control of the LTR [17]. We found that the 6.3
cell clone succumbed more readily to the MS-275/BSO combination than its uninfected counterpart
(Figure 4 C, D). Similar results were obtained with the 8.4 clone (data not shown).
The Jurkat model for HIV-1 quiescence showed that BSO recruited HDACI-insensitive cells into
the responding cell population (Figure 5). These results are derived from the A1 Jurkat cell clone,
which has an integrated GFP/Tat construct under control of the HIV-1 LTR, which is quiescent in

8. 7
the majority of cells [17]. This clone was chosen because this type of analysis could not be
conducted in the 6.3 or 8.4 clones, since, at 24 hours of incubation with the drugs, these clones
displayed only a small proportion of cells expressing GFP, and a correct estimate of the expression
of this protein at subsequent time points was biased by the autofluorescence of dying cells. The A1
clone, which does not have a complete HIV-1 genome, was less sensitive to the toxic effects of the
MS-275/BSO combination than the 6.3 and 8.4 clones (data not shown).
To sum up, the combination of a class I-selective HDACI and BSO activates HIV-1 at
concentrations that show low toxicity in uninfected cells, and it induces cell death in infected cell
cultures. These results are consistent with a model in which BSO would favor the HIV-1 activating
effects of HDACIs by lowering the intracellular levels of reduced glutathione [30] and would
induce the death of infected cells by preventing replenishment of the reduced glutathione pools that
are further “consumed” by the virus activated from quiescence [28, 29]. If these results are
confirmed, the decreased pool of reduced glutathione may become an Achilles’ heel of the infected
cells, and its manipulation may open new avenues to their elimination.
This strategy will of course require optimization, and several issues still have to be addressed. First,
not all of the cells with a quiescent provirus respond to the treatment. A variegated phenotype after
activation, with only a fraction of the cell population becoming activated in response to a global
signal, was also shown by Jordan et al. [17], who attributed this phenomenon to the different local
chromatin environments. A thorough investigation of the molecular signals sparked by the
BSO/class I-selective HDACI combination (currently in progress in our laboratories) is expected to
provide insight into these phenomena. Moreover, the “therapeutic window” (i.e. the differential
toxicity in uninfected vs. infected cells) still needs to be widened. In this regard, the general
structural requirements for the HIV-1 activating HDACIs presented in our study, as well as the
recent identification of HDAC2 as a potential target for HIV-1 reactivation strategies [33], may
represent a good starting point for developing next-generation class I HDACIs with increased
selectivity and decreased toxicity. Finally, we are currently searching for novel γ-glutamyl-cysteine

9. 8
synthetase inhibitors acting in the nanomolar range and displaying lower toxicity than BSO in
uninfected cells.
The concept to activate provirus transcription to target latency is not new, and several clinical trials
have been conducted in the past years along this line, ranging from the administration of IL-2 to the
utilization of valproic acid [34-36]. The results of these trials have been largely disappointing so far.
Valproic acid, a relatively weak HDACI, was tested in a small clinical trial in combination with
antiretroviral therapy intensified with the fusion inhibitor enfuvirtide [35, 36], but some more recent
studies have failed to show a decay of resting CD4+ T cell infection in individuals under valproic
acid treatment for clinical reasons while also receiving standard ART [37]. Our study provides a
potentially more powerful approach for the “shock” phase of experimental HIV-1 eradicating
strategies and a potential tool for the “kill” phase. Notwithstanding the aforementioned need for
amelioration, it is interesting to point out that both MS-275 and BSO have passed class I clinical
trials for safety in humans and are therefore ready for testing in animal models. Such testing would
be important at a time when no proof-of-concept exists for the “shock and kill” theory. In this
regard, even a partial response (e.g. a reduction in latently infected cells) would be a valuable
indicator of the validity of this approach. The possible efficacy of the “shock and kill” approach is
still a matter of debate. For example, a recent study of Jeeninga et al. suggests that there are
different cellular reservoirs for HIV-1 latency and that each reservoir may require a specific
activation strategy [38]. Viral factors, along with cellular factors, may contribute to HIV-1
quiescence, and these factors may not be controlled by strategies using HDACIs.
Competing interests
AS, AM, ATP, and EG have requested patent rights on several compounds described in the present
study and on the MS-275/BSO combination.

10. 9
Authors’ contributions
AS conceived and coordinated the study, supervised the generation of biological data, conducted
the molecular docking, analyzed the data and drafted the manuscript. AM conceived the majority of
the structures described in the present study, supervised their synthesis and participated in
manuscript drafting. SN and SED conducted the biological testing and contributed to molecular
modeling and data analysis. SV, DR, and LA conducted synthesis and development of the HDACi.
LA conducted the HDAC inhibitory assays. ATP and EG contributed the idea of using BSO for
HIV-1 escape from latency and participated in the experimental planning.
Acknowledgements
The authors are thankful to Mr. Federico Mele, Rome, Italy, and Ms. Dora Pinto, ibidem, for
technical help, Ms. Maria Grazia Bedetti, ibidem, for administrative support, and Dr. Mark Kanieff,
ibidem, for the linguistic revision. This work was partially supported by grants from Special Project
AIDS-Italian Ministry of Health (AS), FIRB 2006 (ATP), PRIN 2006 (AM), European Union
(Epitron LSHC-CT2005-518417; Apo-sys HEALTH-F4-2007-200767) (LA), and PRIN 2006 and
AIRC (LA). Special thanks to Dr. Marco Sgarbanti, Rome, Italy, and Dr. Marina Lusic, Trieste,
Italy, for providing reagents and illuminating discussion. We finally would like to acknowledge the
AIDS Reagent Program (Bethesda, MD) as the source of the Jukat clones used in this study.
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15. 14
Figure legends
Figure 1. Potencies of different HDACIs in terms of activation of HIV-1 replication in U1 and
ACH-2 cells, and toxicity in uninfected Jurkat T-cells.
Panel A: Cells were incubated with the test compounds (1 µM), and p24 production was measured
by ELISA in cell culture supernatants at 72 hours post-infection (means ± SEM; 3 experiments).
Asterisks show the significant differences in comparison to untreated control cultures according to
repeated-measures ANOVA using Dunnet’s multiple comparison post-test (a Log transformation of
p24 values was applied to restore normality). Panel B: Uninfected Jurkat T cells were incubated for
72 h under similar conditions, and toxicity was measured by the methyl tetrazolium (MTT) method.
Results are presented as a percentage of the O.D. (λ = 550) of untreated controls subtracted of
background (means ± SEM; 3 experiments). Asterisks show the significant differences in
comparison to untreated control cultures according to repeated-measures ANOVA using Dunnet’s
multiple comparison post-test.
Figure 2. Dose-dependent activation of HIV-1 replication by class I-selective HDACIs and
corresponding toxicity in U1 and ACH-2 cells
Panels A,B: Concentration-dependent stimulation of HIV-1 p24 production in the latently infected
cell lines U1 (A) and ACH-2 (B) at 72 hours of incubation with MS-275, MC2211, MC2113 (class
I-selective HDACIs) and SAHA (a non-class-selective HDACI used as a positive control). Mean
values are from three independent experiments (error bars are not shown for better clarity). Dotted
lines represent the average p24 levels found in untreated controls in the same experiments. Panel C.
Effective concentrations for increasing viral replication to 500% of the basal levels of untreated
controls (EC500). Panel D: Cell viability of ACH-2 cells, as measured by the methyl tetrazolium
(MTT) method. Results are presented as a percentage of the O.D. (λ = 550) of untreated controls
subtracted for background (means ± SEM; 3 experiments). Panel E: Cell viability of uninfected

16. 15
Jurkat T cells incubated for 72 hours with the same drugs is shown as comparison. Panel F. 50%
cytotoxic concentrations (CC50). For the symbols in panels D,E, the reader should refer to those of
panels A,B.
Figure 3. Structural characteristics of HIV-1 activating HDACIs
Panel A: Docking of the HDACI MC2211 at the catalytic cavity of HDAC2, a class I enzyme. The
different portions of the inhibitor [i.e. the CAP portion (CAP), the connection unit (CU), the
hydrophobic spacer (HS), and the zinc-binding group (ZBG)] are mapped to the molecule
represented in the picture. The enzyme is shown as semi-transparent Connolly surface. The Zn++ ion
embedded in the catalytic cavity is shown as a dotted sphere. The inhibitor is shown according to
CPK colouring. General formulas for HDACIs capable of inducing HIV-1 activation from
quiescence. Panel D: Structural superimposition of the best docking poses for the HDACIs MC2113
and MC2211 within the catalytic cavity of HDAC2. Inhibitors are shown in CPK (MC2113: carbon
backbone in white; MC2211: carbon backbone in cyan). The enzyme backbone is shown as
cartoons. The Zn++ ion is shown as a gray sphere. Amino acids D100, H141 and G150 (important
for hydrogen bonding with the inhibitors) are shown as orange sticks.
Figure 4. Effects on HIV-1 replication and cell viability of class I-selective HDACIs, MS-275
and buthionine sulfoximine (BSO), alone or in combination. Panel A: HIV-1 p24 concentrations
in ACH-2 cell culture supernatants at 24 hours of incubation with the drugs. Panels B-D: Cell
viability values at 72 hours of incubation, as determined by the methyl tetrazolium (MTT) method:
ACH-2 cells (B), Jurkat 6.3 cells (C), uninfected Jurkat cells (D). Results are presented as
percentages of the absorbance (λ = 550) in untreated controls subtracted for background (means ±
SEM; 3 experiments). Asterisks show the significant differences found between BSO treatments
and matched treatments in the absence of BSO (* P < 0.05; ** P < 0.01; *** P < 0.001). Statistical
significance was calculated using repeated-measures, two-way ANOVA and Bonferroni’s post-test,

17. 16
following an appropriate transformation to restore normality, where necessary. The higher drug
concentrations adopted in Panels C,D serve as comparisons with the experiment in Figure 5.
Figure 5. Stimulation of HIV-1 LTR-controlled expression of green fluorescent protein (GFP)
by MS-275 and buthionine sulfoximine (BSO), alone or in combination in a Jurkat cell clone
(A1). The A1 cell clone, derived from T-lymphoid Jurkat cells, is a model for latent HIV-1
infection. This clone has an integrated GFP/Tat construct under the control of the HIV-1 LTR and
displays a basal proportion of cells expressing GFP, which increase following stimuli activating the
HIV-1 promoter. A1 cells were incubated for 72 hours with the different treatments, and GFP
expression was monitored by standard flow-cytometric techniques and assessed as the percentage of
fluorescent cells (indicated for each histogram) beyond the threshold value established using control
non-transfected Jurkat cells. One experiment out of three with similar results is shown. The
histograms derived from double-drug treatments were found to be significantly different (P < 0.01)
from those derived from treatments with a single drug at matched concentrations (Kolmogorov-
Smirnoff statistics). Differences between the drug concentrations adopted in this experiment and
that in Figure 4A are derived from adjustments due to the different nature of the cell lines adopted.
Additional files
Additional file 1
File format: DOC
Title: Additional file 1
Description: Structures and HDAC inhibiting activity of the cited HDACIs. Where data on human
HDACs are unavailable, data on maize HD1-B (homologous with human class I HDACs) and HD1-
A (homologous with human class II HDACs), or relevant references, are provided.
Additional file 2

18. 17
File format: PPT
Title: Additional file 2
Description: To study the HDACI response in a cell population, we used quiescently infected T-
lymphoid Jurkat cell clones. Two types of cell clones were used: 1) A1, and A2, which have an
integrated GFP/Tat construct under control of the HIV-1 LTR; 2) 6.3, and 8.4, which contain the
entire HIV-1 genome under control of the LTR and have the GFP gene replacing nef. The 6.3 cells
display insignificant basal levels of GFP expression. Cells were incubated with the different
treatments, and GFP expression was monitored in gated live cells at 12, 24 and 72 hours by
standard flow cytometric techniques. Results are presented as fluorescence histograms. Each
histogram reports the percentage of fluorescent cells beyond a threshold value established using
non-infected Jurkat cells.
Additional file 3
File format: PNG
Title: Additional file 3
Description: Structural superimposition of MC2211 (carbon backbone in cyan) and SAHA
(vorinostat; carbon backbone in yellow) docking at the HDAC2 catalytic site. SAHA, a non-
selective HDACI, displays an amide group in a conformation that does not match that of the class I-
selective HDACIs (Figure 2). The other molecular players are displayed in the same fashion as in
Figure 3.

21. Figure 3

24. Additional files provided with this submission:
Additional file 1: additional file 1.doc, 175K
http://www.retrovirology.com/imedia/1713791664267162/supp1.doc
Additional file 2: additional file 2.ppt, 251K
http://www.retrovirology.com/imedia/3051428352671626/supp2.ppt
Additional file 3: additional file 3.png, 239K
http://www.retrovirology.com/imedia/1164111384267162/supp3.png