Saturday, June 26, 2010

'Raltegravir Is a Potent Inhibitor of XMRV

'Raltegravir Is a Potent Inhibitor of XMRV, a Virus Implicated in
Prostate Cancer and Chronic Fatigue Syndrome'
Ila R. Singh1*, John E. Gorzynski1, Daria Drobysheva1, Leda Bassit2,
Raymond F. Schinazi2

1 Department of Pathology, University of Utah, Salt Lake City, Utah,
United States of America, 2 Center for AIDS Research, Laboratory of
Biochemical Pharmacology, Department of Pediatrics, Emory University
School of Medicine and Veterans Affairs Medical Center, Decatur,
Georgia, United States of America

Citation: Singh IR, Gorzynski JE, Drobysheva D, Bassit L, Schinazi RF
(2010) Raltegravir Is a Potent Inhibitor of XMRV, a Virus Implicated
in Prostate Cancer and Chronic Fatigue Syndrome. PLoS ONE 5(4): e9948.
doi:10.1371/journal.pone.0009948

Full text- http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0009948


Abstract

Background
Xenotropic murine leukemia-related retrovirus (XMRV) is a recently
discovered retrovirus that has been linked to human prostate cancer
and chronic fatigue syndrome (CFS). Both diseases affect a large
fraction of the world population, with prostate cancer affecting one
in six men, and CFS affecting an estimated 0.4 to 1% of the
population.

Principal Findings
Forty-five compounds, including twenty-eight drugs approved for use in
humans, were evaluated against XMRV replication in vitro. We found
that the retroviral integrase inhibitor, raltegravir, was potent and
selective against XMRV at submicromolar concentrations, in MCF-7 and
LNCaP cells, a breast cancer and prostate cancer cell line,
respectively. Another integrase inhibitor, L-000870812, and two
nucleoside reverse transcriptase inhibitors, zidovudine (ZDV), and
tenofovir disoproxil fumarate (TDF) also inhibited XMRV replication.
When combined, these drugs displayed mostly synergistic effects
against this virus, suggesting that combination therapy may delay or
prevent the selection of resistant viruses.

Conclusions
If XMRV proves to be a causal factor in prostate cancer or CFS, these
discoveries may allow for rational design of clinical trials.




Editor: Peter Sommer, Institut Pasteur Korea, Republic of Korea


Received: February 18, 2010; Accepted: March 11, 2010; Published: April 1, 2010

Copyright: © 2010 Singh et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source
are credited.

Funding: This work is supported in part by NIH grant 2P30-AI-50409
(CFAR to RFS), and by the Department of Veterans Affairs (RFS). The
funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.

Competing interests: RFS is a founder and major shareholder of RFS
Pharma, LLC that is developing Amdoxovir clinically and he receives
royalties from the sales of 3TC. This does not alter the authors'
adherence to all the PLoS ONE policies on sharing data and materials.

* E-mail: ila.singh@path.utah.edu



---------------------------------------


Introduction Top
Xenotropic murine leukemia-related retrovirus (XMRV) is a recently
discovered infectious agent [1] that has been linked to human prostate
cancer [2] and chronic fatigue syndrome (CFS) [3]. Both diseases
affect a large fraction of the world population, with prostate cancer
affecting one in six men, and CFS affecting an estimated 0.4 to 1% of
the population [4], [5]. XMRV nucleic acid or proteins are found in
27% of prostate cancers and in 68% of chronic fatigue syndrome
patients, and in less than 4–6% of normal controls, suggesting an
association between the virus and human disease [2], [3].

CFS, a disease characterized by severe debilitating fatigue, has had
an uncertain etiology since its recognition. While a series of viral
agents and environmental toxins have been proposed to be associated
with CFS, no clear evidence for these has ever been presented
(reviewed in [6]). The recent association of XMRV with CFS from the
Whittemore Peterson Institute in Reno, Nevada, while far from being
proven causal, is the strongest viral association to be made yet.
Three recent reports, using plasmid DNA as positive controls, did not
find XMRV in CFS patients in Europe [7] [8] [9]. The prevalence of
XMRV in prostate cancer in Europe is uncertain, with one German group
reporting the presence of XMRV in human prostates [10], and the other
not detecting any [11]. However, the notion that a retrovirus might be
involved in both cancer and a neuroimmune illness in humans is not
without precedence. Human T-cell lymphotrophic virus, type 1 (HTLV-1),
another retrovirus, causes both T-cell lymphoma/leukemia as well as
tropical spastic paraparesis, a myelopathy due to immune defects
resulting from the viral infection.

Infectious XMRV has been isolated from sera of CFS patients [3]. The
presence of circulating infectious retrovirus particles in the blood
invokes a scenario not unlike infection with another retrovirus, human
immunodeficiency virus type 1 (HIV-1). Since there is no effective
treatment for this profoundly debilitating illness, the use of
antiretroviral agents that have proven to be reasonably safe for human
use, might be of benefit. The discovery of effective antiretroviral
agents against XMRV would allow for rational design of clinical trials
to prevent progression of prostate cancer or to treat CFS.

In this study, we report the effect of 45 compounds on XMRV
replication in MCF-7 and LNCaP cells, cell lines generated from human
breast and prostate cancers, respectively. We studied drugs used in
the treatment of HIV-1 infections, as well as compounds used to treat
other viral infections in humans. XMRV is a gammaretrovirus, closely
related to the murine leukemia viruses (MLV) [1]. At the amino acid
level, it shares considerable identity with sequences of Moloney
murine leukemia virus (MoMLV), a prototype MLV. The maximum similarity
between XMRV and MoMLV proteins is found in the sequences for viral
protease (96% identity), and the least similarity is between the two
envelope proteins (66% identity) [1]. Unfortunately, not many
actively-used antiretroviral agents have been tested for activity
against MLV, with the exception of ZDV, which effectively suppresses
MLV [12], and was recently demonstrated to be effective against XMRV
as well [13]. In contrast, while there is a lot of information on
antiviral activity against essential HIV-1 proteins, there is very
little similarity between HIV-1 and XMRV proteins, with the proteases
(PR) of the two viruses sharing 28% identity at the amino acid level,
the reverse transcriptase proteins (RT) sharing 17% and the integrase
(IN) proteins sharing just 14% identity. This low sequence similarity
makes it difficult to predict which, if any, of the antiretroviral
agents that are effective against HIV-1 would be effective against
XMRV. We chose several drugs from each major class of antiretroviral
agents: nucleoside and non-nucleoside RT inhibitors (NRTIs and
NNRTIs), IN inhibitors, and PR inhibitors (PI). The envelope proteins
of the XMRV and HIV-1 are widely divergent in size (70 kD and 160 kD
respectively), utilize different receptors for viral entry and do not
share any significant similarity. Therefore, peptidomimetics that act
on the HIV-1 envelope protein to prevent viral entry were not included
in our study. A few inhibitors that are known to inhibit replication
of viruses other than retroviruses were also evaluated. A significant
number of compounds tested in our study, viz. 28 out of a total of 45,
are already FDA-approved for the treatment of infection with HIV-1 or
other viruses. We report here for the first time that the integrase
inhibitor, raltegravir (RAL), is extremely potent and selective
against XMRV, when used at low submicromolar concentrations in both
cell culture systems. Another IN inhibitor, L-000870812, and two
NRTIs, ZDV and tenofovir disoproxil fumarate (TDF), also inhibit XMRV
replication, but at higher concentrations. When combined, these
compounds display synergistic effects, suggesting combined modalities
to treat XMRV infection, thus delaying or preventing the selection of
resistant viruses.

Results Top
We tested a total of 45 compounds belonging to different classes of
HIV-1 inhibitors, and a few inhibitors of viruses other than
retroviruses, for their ability to inhibit XMRV replication in
cultured cells. LNCaP and MCF-7 cells were chosen for their ability to
support robust in vitro replication of XMRV. MCF-7 cells, because of
their better growth properties in culture were initially used to test
all 45 compounds (Figure 1). Compounds with anti-XMRV activity were
subsequently tested in both LNCaP and MCF-7 cells (Table S1). To
determine if a reduction in viral release might be due to toxicity of
the compound and not due to specific antiretroviral activity, cellular
morphology was monitored every 24 h by microscopic examination, and an
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphen​yltetrazolium bromide]
colorimetric assay was used to measure potential cytotoxicity produced
by the compounds. Supernatants were collected every 24 h and assayed
for viral release by measuring RT activity. Inhibition of RT activity
(see Figure 2) was averaged over 3–6 experiments, each performed in
duplicate, and used to calculate the median (EC50) and 90% effective
concentrations (EC90) for each compound (Figure 1). For comparison
purposes, all compounds evaluated in these studies were also tested
against HIV-1LAI in primary human lymphocytes.

Figure 1. EC50, EC90 and CC50 values of compounds tested in
XMRV-infected MCF-7 cells, and in HIV-1 infected peripheral blood
mononuclear cells.

All compounds were evaluated in duplicate at least three times. Values
shown are average of replicate assays.
*(4S)-8-Chloro-4-methyl-5-(3-methylbut-2-eny​l)-3,4,5,6-tetrahydro-1H-
[1], [3]diazepino[4,5,6-cd]indole-2(2aH)-thione.

doi:10.1371/journal.pone.0009948.g001
Inhibitors of HIV-1 reverse transcriptase
The following NRTI inhibitors of HIV-1 RT were tested in our XMRV
replication assays: ZDV, 3′-azido-3′-deoxyadenosine (AZA),
3′-azido-3′-deoxyguanosine (AZG), 3′-azido-3′-deoxy-5-methyl-cytidine
(CS-92), lamivudine (3TC), emtricitabine [(-)-FTC], tenofovir (TNV)
and its prodrug form TDF, 9-(β-D-1,3-dioxolan-4-yl)guanine (DXG) and
its prodrug form, amdoxovir (DAPD, AMDX), (-)-carbovir (CBV),
stavudine (D4T) and its corresponding cytosine analog (D4C), videx
(ddI), zalcitabine (ddC), and 3′-fluoro-3′-deoxythymidine (FLT). We
also tested the NNRTIs efavirenz, and a TIBO derivative that was shown
to be effective in a murine system [14]. Among these, the most potent
XMRV inhibitors were ZDV and TDF (Figure 2, A, B). The EC50 and EC90
in MCF-7 cells were 0.11 µM and 7.3 µM for ZDV, and 0.24 µM and 15.3
µM for TDF respectively (see Figure 1). The EC50 and EC90 values were
also determined in LNCaP cells, a prostate cancer cell line, and there
was a consistent difference of up to 5 fold between the two cell
lines, which may be related to their differing rates of nucleoside
uptake and bioconversion to the active nucleoside triphosphate analog.
The EC50 and EC90 in LNCaP cells were 0.14 µM and 1.1 µM for ZDV, and
0.9 µM and 4.2 µM for TDF, respectively. CBV, AZA, FLT and D4T all
showed greater than 70% inhibition of XMRV replication (see Table S1),
but at the much higher concentration of 100 µM. AZG, CS-92, (-)-FTC,
3TC, ddI, DAPD and DXG were essentially inactive at 100 µM (Figure 1).
TFV was also ineffective against XMRV, probably due to its polar
nature, which may not allow sufficient drug to penetrate into the
cells. The NNRTIs efavirenz (EFV) and the TIBO derivative, did not
demonstrate any major activity against XMRV.

Figure 2. Inhibition of XMRV replication in LNCaP cells in the
presence of increasing concentrations of antiviral agents.

Viral release from XMRV-infected LNCaP cells in the presence of
increasing concentrations of (A) ZDV (B) TDF (C) RAL and (D)
L-000870812, was determined by measuring RT activity in the
supernatants. Percent inhibition was calculated based on infected
cells exposed to DMSO alone being set to 0% inhibition, and naïve
cells in the absence of any compounds set at 100% inhibition. Cell
viability was checked by microscopy, quantified by the MTT assay, and
represented by shaded bars. Data for each compound were derived from
an average of at least three independent experiments, each performed
in duplicate.

doi:10.1371/journal.pone.0009948.g002
Inhibitors of HIV-1 Integrase
IN inhibitors raltegravir (RAL or MK-0518) and L-000870812 [15] were
also evaluated for their ability to inhibit XMRV in the two cell
systems (Figure 1, Figure 2 C and D). Of all the compounds tested, RAL
was the most potent, with an EC50 of 0.005 µM and an EC90 of 3.5 µM in
MCF-7 cells, and an EC50 of 0.03 µM and an EC90 of 0.46 µM in LNCaP
cells (Table S1). L-000870812, showed activity against XMRV
replication at considerably higher concentrations, with an EC50 and
EC90 of 0.16 µM and 26.9 µM in MCF-7 cells, and 0.7 µM and 4.5 µM in
LNCaP cells, respectively.

Inhibitors of HIV-1 Protease
Nine known HIV-1 PIs were evaluated for activity against XMRV (Figure
1). The most effective was nelfinavir, albeit with an EC50 of 34.3 µM.
The following PIs had very modest anti-XMRV activities: atazanavir
(EC50 of 64.8 µM), amprenavir (EC50 of 68.0 µM), lopinavir (EC50 of
72.2 µM), and ritonavir (EC50 of 76.4 µM). Darunavir, indinavir,
saquinavir and tipranavir were essentially ineffective against XMRV in
vitro, when tested up to 100 µM.

Inhibitors of viruses other than HIV-1
A select number of antiviral agents known to inhibit viruses other
than retroviruses were also evaluated. These included the
anti-herpetic drugs acyclovir (ACV), ganciclovir (GCV), vidarabine
(ara-A), 5-Iodo-2′-deoxyuridine (IdUrd), penciclovir (PCV), foscarnet
(PFA), vistide (HPMPC); the anti-hepatitis drugs entecavir (ETV),
telbivudine (LdT), and ribavirin (RBV). ETV was also selected because
it was recently reported to inhibit HIV-1 replication, both in vitro
and in humans [16]. Other compounds claimed to be effective against
XMRV, MLV, HIV-1 and other viruses, such as chloroquine [17],
dehydroepiandrosterone (DHEA) [18], methylene blue and aspirin were
also evaluated for anti-XMRV activity in vitro. Methylene blue is
known to have antiherpetic activity and also can inactivate HIV-1
[19]. Unfortunately, most of the compounds listed above, except IdUrd
were ineffective against XMRV, or were effective at toxic
concentrations (Figure 1). IdUrd demonstrated a low therapeutic index
(TI, the ratio of CC50/EC50) and cannot be considered as a specific
antiviral agent against XMRV.

Combination effects of active compounds on XMRV replication
Binary combinations of the most potent compounds, viz. RAL,
L-000870812, TDF and ZDV were tested for activity against XMRV in
LNCaP cells. Compounds were tested at increasing concentrations, in
three different sets, with the ratio of the two compounds kept
constant. The data were analyzed using the CalcuSyn method originally
described by Chou and Talalay [20]. A summary of results for all
combinations evaluated in LNCaP cells is presented in Figure 3.

Figure 3. Evaluation of drug-drug interactions against XMRV at 50%,
75%, 90%, and 95% inhibition.

Combination Index (CI) values were determined for a mutually exclusive
interaction using CalcuSyn program, where CI <1 indicates synergism,
CI = 1 indicates additive effect, and CI >1 indicates antagonism.
Weighted average CI value (CIwt) was assigned as [CI50 + 2CI75 + 3CI90
+ 4CI95]/10. RAL, raltegravir; TDF, tenofovir disoproxil fumarate;
ZDV, zidovudine.

doi:10.1371/journal.pone.0009948.g003
For the combinations tested (RAL with TDF or ZDV or L-000870812; TDF
with ZDV or L-000870812; L-000870812 with ZDV), an additive or mostly
synergistic interaction was noted at all effect levels without
apparent cytotoxicity at the highest concentrations used. In the
computational analysis for either one of the four drugs (RAL, TDF,
L-000870812, or ZDV), the linear correlation coefficient (r values) of
the median-effect plot or for their constant ratio combinations ranged
from 0.92 to 0.99 (data not shown), matching the law of mass-action.
The in vitro effect of the combination of RAL with either TDF or ZDV,
showed a favorable dose reduction at all ratios. In addition, all
Combination Index (CI) values (see Materials and Methods) were less
than 1, suggesting synergy when the combination ratios of either TDF
or ZDV and RAL were analyzed (Figure 3). In addition, dual
combinations of either ZDV and L-000870812 or TDF were also tested.
The weighted CI (CIwt) values of 0.1 to 0.5 for TDF + ZDV, indicated
synergistic effects at all ratios tested (Figure 3). Moreover, the
dual combination of L-000870812 and ZDV at ratio 1:2 indicated a
nearly additive effect (CIwt of 1.0); however at ratio of 1:0.4, the
CI value was 0.3, indicating synergism (Figure 3). Of significance was
that all dual combinations containing RAL, the most potent antiviral
agent against XMRV, demonstrated marked synergy at all effect levels
without any apparent cytotoxicity. Interestingly, the combination of
the two IN inhibitors was not antagonistic or additive, but was found
to be synergistic, suggesting that these two compounds may have
different antiviral mechanisms (see discussion).

Discussion Top
In the absence of a clear etiology, the treatment of CFS has been both
empirical and unconventional. Therapies have included immunostimulant
therapy through injections of staphylococcus toxoid [21], intravenous
immunoglobulin therapy [22] [23],[24], and hydrocortisone [25] each
with uneven results. Interferon-β and TNF-α inhibitors have been tried
in very small numbers of patients. Anti-depressants, NSAIDs,
anxiolytic drugs, stimulants, anti-allergy drugs and anti-hypotensive
drugs have all been used, but are not universally beneficial [26]. The
lack of effective therapy has led to use of plant extracts [27],
homeopathy [28], [29], hypnosis [30], acupuncture [31], and whole body
periodic acceleration stress [32], none with sustained benefits. 1The
only modalities of treatment that have any proven benefits are
cognitive behavioral therapy and graded exercise programs, both of
which appear to aid by improving coping skills rather than reduce
symptoms [33]. If, XMRV proves to have a causal association with human
disease, then the knowledge that certain antiretroviral agents inhibit
XMRV at submicromolar concentrations in vitro, and have synergistic
effects when combined, as shown in this study, might lead to clinical
trials. We found that RAL, L-000870812, ZDV, and TDF strongly inhibit
XMRV in cell culture, with RAL being the most potent, at an EC50 of
0.005 µM, and others such as L-000870812 (EC50 = 0.16 µM), ZDV (EC50 =
0.11 µM) and TDF (EC50 = 0.24 µM) being quite effective as well. In
addition, these compounds had high therapeutic indices, with values
for ZDV = 591; TDF = 218; RAL = 18,460 and L-000870812 = 378,
indicating that it should be possible to achieve therapeutic antiviral
levels with minimal toxicity.

Several compounds that we evaluated had a limited effect on XMRV
replication in vitro. Some of these effects can be explained by
currently understood mechanisms. For example, both 3TC and (-)-FTC
need a functional YMDD motif in RT to be active. The M184V mutation in
HIV-1 RT makes the virus resistant to 3TC and (-)-FTC [34]. These
drugs are ineffective against MoMLV, because in MoMLV RT, V is the
natural residue in this motif in place of M. Similarly, V is also the
natural residue at this location in XMRV RT, making 3TC and (-)-FTC
ineffective. Why none of the HIV-1 PIs were effective against XMRV (a
finding that has been reported for selected PIs before [13]) remains
unclear at this time, but could be related to the size of the PI
pocket as well as other biochemical and structural factors.

There was a difference in activities of compounds when tested in
different cell types, which may be related to drug uptake by cells,
the different levels of natural dNTP in the cells, as well as
different intracellular phosphorylation capacity [35]. In general, the
EC50 for the active compounds listed above were lower in MCF-7 than
LNCaP cells suggesting greater potency. Relative to HIV-1, the
compounds were generally less potent against XMRV than HIV-1,
especially at the EC90 level.

The use of monotherapy for treating HIV infections has lead to the
appearance of drug resistant virus [36], [37]. The finding that RAL,
L-000870812, TDF and ZDV have strong synergistic effects when combined
in dual combination bodes well for combination therapy in case of XMRV
infection. If XMRV infection parallels other retroviral infections,
then the use of combination antiretroviral therapy might maintain XMRV
suppression, prevent the emergence of resistance to antiretroviral
agents and possibly also cause amelioration of disease. For HIV-1,
combination therapy works especially well when the combined drugs have
different viral protein targets, or in the case of nucleosides,
utilize different kinases for their activation to NTP analogs [31].
We, therefore, judiciously selected drug combinations that inhibit
XMRV, such as RAL with ZDV or TDF or L-000870812. When the data were
analyzed using the robust method of Chou and Talalay, additive or
synergistic interactions were found at all effect levels when these
agents were tested in LNCaP cells. Of significance was that no
antagonism was noted for any of the combinations evaluated in these
cells. To our surprise, even the two IN inhibitors displayed a
synergistic effect. Both IN inhibitors act by inhibiting the strand
transfer reaction, but if their mechanism of action were to be
identical, they would display an additive effect in combination. A
synergistic effect suggests that there might be subtle mechanistic
differences in the actions of these two IN inhibitors, a finding that
is corroborated by unpublished biochemical experiments (personal
communication, Dr. Daria Hazuda, Merck Research Laboratories, West
Point, PA). It is important to note here, that XMRV differs from HIV-1
in one aspect that is significant for these studies: XMRV isolates
show very limited sequence diversity compared to HIV-1 or MLV. Of all
the sequenced XMRV isolates that currently exist, both from cases with
prostate cancer as well as CFS, obtained from geographically distant
parts of the United States, the two least related genomes differ from
each other in only 27 out of a total of over 8,100 nucleotides. A
similar degree of limited genetic diversity has been found for HTLV-1
[38], another retrovirus implicated in both cancer and neuroimmune
illness. It has been suggested that this lack of diversity in XMRV
sequences implies that the number of replication cycles within one
infected individual is limited [39]. This would suggest that XMRV has
a considerably lower potential for developing drug-resistant
mutations, as compared to HIV-1. Furthermore, it is likely that a
combination of just two drugs might be sufficient for preventing the
emergence of drug-resistant mutant virus, though this would need to be
tested before any therapeutic recommendations can be made. We have
attempted to select for RAL resistant viruses in culture for several
months now, and have not yet been successful at isolating drug
resistant viruses.

When an assay to measure XMRV viral loads becomes available, virus
levels in the blood might become an objective surrogate marker for an
effective response to antiviral drugs, in addition to clinical
outcomes. While it is not yet clear if any illnesses are directly
caused by XMRV, our data indicates that XMRV infections might be
prevented or treated with specific antiviral agents. In the presence
of any evidence of causality of human disease, our findings should
provide the basis for designing clinical trials to treat them.

Materials and Methods Top
XMRV, Cells and Infection with XMRV
293T cells (ATCC, CRL-11268) were transfected with pXMRV1, an
infectious clone of XMRV [2]. Virus released in the supernatant was
harvested and titrated by inoculating MCF-7 cells, a breast cancer
cell line (ATCC, HTB-22) at 70% confluence, with a series of ten-fold
dilutions of XMRV in serum-free medium, followed 36–48 hrs later by
fixation of cells in paraformaldehyde and processing for
immunofluorescence using a rabbit antiserum developed against
inactivated XMRV [2]. Typical virus preparations gave titers of
approximately 2–5×106 infectious units/ml. Virus was diluted in
serum-free medium and used to inoculate cells at a multiplicity of
infection (MOI) of approximately 3, in the presence of various
antiviral compounds as described below. LNCaP, a prostate cancer cell
line (ATCC, CRL-1740) and MCF-7 cells were grown to about 50%
confluence in DMEM containing 10% heat inactivated fetal bovine serum,
100 µg/ml penicillin, and 100 IU/ml streptomycin. Cells were washed
twice with Dulbecco's phosphate buffered saline (DPBS, Gibco), and
incubated with the viral inoculum for 90 min at 37°C in the presence
of 95% air and 5% CO2, cells were washed twice with DPBS, detached
with Trypsin-EDTA (0.25% Trypsin; Cellgro), and counted. One thousand
cells were added to each well of a 96-well plate, along with an equal
volume of medium containing the retroviral inhibitor at 2-fold the
desired concentration. Inhibitors were dissolved in DMSO or water,
depending on their solubility, and were all tested at 0.01 to 100 µM
in 10-fold increments. Where the drug was found to be active at 0.01
µM, further dilutions from 1 nM to 0.01 nM were tested. Each inhibitor
was tested in duplicate a minimum of three separate times in MCF-7
cells in a completely blind fashion using coded compounds, and the
results averaged. Active compounds were also evaluated for antiviral
activity in LNCaP cells to confirm activity in a secondary cell line
known to support XMRV replication. For controls, wells containing
water or DMSO at appropriate concentrations were used.

Assays for cytotoxicity and XMRV replication
Each well was carefully monitored for signs of cellular toxicity due
to the inhibitors by microscopic observation every 24 h. In addition,
cell viability was measured using the CellTiter 96 AQueous One
Solution cell proliferation assay according to the manufacturer
(Promega, Madison, Wis). Viral release from the cells was assayed by
measuring RT activity in the supernatant. For this, supernatant from
each well was collected every 24 h and frozen at −20°C until it was
analyzed by RT assay for viral release as described previously [40].
In brief, oligo(dT)·poly(rA) primer-template assays were performed in
the presence of radiolabeled [α-32P]dTTP and Mn2+. After incubating
the viral supernatants with the RT reaction mix for 1 h at 37°C,
samples were spotted onto DE81 DEAE cellulose paper (Whatman) and
unincorporated label washed away with 2× SSC (1× SSC = 0.15 M NaCl and
0.015 M sodium citrate). Virion-associated RT was analyzed using a
Typhoon 9410 PhosphorImager (GE Healthcare) and quantified with the
Image J software (http://rsbweb.nih.gov/ij/). Inhibition of viral
release as measured on day 6 after inoculation was averaged over 3–5
experiments and plotted. The antiviral EC50 and cytotoxic
concentrations (CC50,) was determined from the concentration–response
curve using the median effect method [20]. HIV-1 replication assays
were performed as described previously using peripheral blood
mononuclear cells (PBMC) obtained from the American Red Cross,
Atlanta, GA, that were stimulated with phytohemagglutinin (PHA) for 72
hr [41].

Combination studies
To evaluate whether the antiviral effects of dual drug combinations
of: RAL with TDF or ZDV or L-000870812; TDF with ZDV or L-000870812;
L-000870812 with ZDV were synergistic, additive or antagonistic, drug
combinations at several constant ratios were evaluated. RAL,
L-000870812, ZDV and TDF were first tested alone to determine the EC50
and EC90 values, at least three times, each in duplicate. For the
median-effect analysis, the compounds were combined at several ratios
based on multiples of their EC50 or EC90 values. For each drug (alone
or in combination), three to four independent experiments were
performed and all samples were processed in duplicate. Analysis was
performed using the software CalcuSyn (Biosoft, Ferguson, MO, USA)
(see Figure 3 for CI values), which allows automated simulation of
synergism and antagonism at all dose and effect levels and displays
the methods of Chou and Talalay [20], including median effect plot and
CI values. Because the high degree effects are more therapeutically
relevant than the low degree of effects, the additional weighted
average CI (CIwt) was calculated, which uses the formula: CIwt = [CI50
+ 2CI75 + 3CI90 + 4CI95]/10, where CI50, CI75, CI90, CI95 are the CI
values at 50%, 75%, 90% and 95% inhibition, respectively. [20], [42].

Supporting Information Top
Table S1.

EC50, EC90 and CC50 values of compounds active against XMRV in MCF-7
cells, as tested in XMRV-infected LNCaP cells. Compounds found to have
significant activity in MCF-7 cells were tested in LNCaP cells for
activity. All compounds were evaluated in duplicate at least three
times. Values shown are average of replicate assays. Corresponding
values in MCF-7 cells are shown for comparison.

(0.11 MB TIF)

Acknowledgments Top
We thank Mervi Detorio and Melissa Johns for their excellent technical
assistance, and Daniel Choe and Robert Schlaberg for their help with
preliminary experiments.

Author Contributions Top
Conceived and designed the experiments: IRS RFS. Performed the
experiments: JEG DD. Analyzed the data: IRS JEG LB RFS. Contributed
reagents/materials/analysis tools: IRS RFS. Wrote the paper: IRS RFS.

References Top
Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, et al. (2006)
Identification of a novel Gammaretrovirus in prostate tumors of
patients homozygous for R462Q RNASEL variant. PLoS Pathog 2: e25. Find
this article online
Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR (2009) XMRV is
present in malignant prostatic epithelium and is associated with
prostate cancer, especially high-grade tumors. Proc Natl Acad Sci U S
A 106: 16351–16356. Find this article online
Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, et al.
(2009) Detection of an infectious retrovirus, XMRV, in blood cells of
patients with chronic fatigue syndrome. Science 326: 585–589. Find
this article online
Hayat MJ, Howlader N, Reichman ME, Edwards BK (2007) Cancer
statistics, trends, and multiple primary cancer analyses from the
Surveillance, Epidemiology, and End Results (SEER) Program. Oncologist
12: 20–37. Find this article online
Jason LA, Richman JA, Rademaker AW, Jordan KM, Plioplys AV, et al.
(1999) A community-based study of chronic fatigue syndrome. Arch
Intern Med 159: 2129–2137. Find this article online
Devanur LD, Kerr JR (2006) Chronic fatigue syndrome. J Clin Virol 37:
139–150. Find this article online
Erlwein O, Kaye S, McClure MO, Weber J, Wills G, et al. (2010) Failure
to detect the novel retrovirus XMRV in chronic fatigue syndrome. PLoS
One 5: e8519. Find this article online
Groom HCT, Boucherit VC, Makinson K, Randal E, Baptista S, et al.
(2010) Absence of xenotropic murine leukaemia virus-related virus in
UK patients with chronic fatigue syndrome. Retrovirology 7:
10.1186/1742-4690-1187-1110. Find this article online
van Kuppeveld FJ, Jong AS, Lanke KH, Verhaegh GW, Melchers WJ, et al.
(2010) Prevalence of xenotropic murine leukaemia virus-related virus
in patients with chronic fatigue syndrome in the Netherlands:
retrospective analysis of samples from an established cohort. BMJ 340:
c1018. Find this article online
Fischer N, Hellwinkel O, Schulz C, Chun FK, Huland H, et al. (2008)
Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer.
J Clin Virol 43: 277–283. Find this article online
Hohn O, Krause H, Barbarotto P, Niederstadt L, Beimforde N, et al.
(2009) Lack of evidence for xenotropic murine leukemia virus-related
virus(XMRV) in German prostate cancer patients. Retrovirology 6: 92.
Find this article online
Ruprecht RM, O'Brien LG, Rossoni LD, Nusinoff-Lehrman S (1986)
Suppression of mouse viraemia and retroviral disease by
3′-azido-3′-deoxythymidine. Nature 323: 467–469. Find this article
online
Sakuma R, Sakuma T, Ohmine S, Silverman RH, Ikeda Y (2009) Xenotropic
murine leukemia virus-related virus is susceptible to AZT. Virology.
Ho W, Kukla MJ, Breslin HJ, Ludovici DW, Grous PP, et al. (1995)
Synthesis and anti-HIV-1 activity of
4,5,6,7-tetrahydro-5-methylimidazo-[4,5,​1-jk][1,4]benzodiazepin-
2(1H)-one (TlBO) derivatives. 4. J Med Chem 38: 794–802. Find this
article online
Markowitz M, Morales-Ramirez JO, Nguyen BY, Kovacs CM, Steigbigel RT,
et al. (2006) Antiretroviral activity, pharmacokinetics, and
tolerability of MK-0518, a novel inhibitor of HIV-1 integrase, dosed
as monotherapy for 10 days in treatment-naive HIV-1-infected
individuals. J Acquir Immune Defic Syndr 43: 509–515. Find this
article online
Yamada A, Sako A, Nishimura S, Nakashima R, Ogami T, et al. (2009) [A
case of HIV coinfected with hepatitis B virus treated by entecavir].
Nippon Shokakibyo Gakkai Zasshi 106: 1758–1763. Find this article
online
Naarding MA, Baan E, Pollakis G, Paxton WA (2007) Effect of
chloroquine on reducing HIV-1 replication in vitro and the DC-SIGN
mediated transfer of virus to CD4+ T-lymphocytes. Retrovirology 4: 6.
Find this article online
Schinazi RF (1990) Antiviral activity of dehydroepiandrosterone. In:
MKaW Regelson, editor. The Biological Role of Dehydroepiandrosterone
(DHEA). Berlin New York: Walter de Gruyter & Co. pp. 157–177.
Floyd RA, Schinazi RF (1998) Thiazine dyes used to inactivate HIV in
biological fluids. In: USPaT Office, editor. USPTO Patent Full-Text
and Image Database. USA: Oklahoma Medical Research Foundation.
Chou TC, Talalay P (1984) Quantitative analysis of dose-effect
relationships: the combined effects of multiple drugs or enzyme
inhibitors. Adv Enzyme Regul 22: 27–55. Find this article online
Zachrisson O, Regland B, Jahreskog M, Jonsson M, Kron M, et al. (2002)
Treatment with staphylococcus toxoid in fibromyalgia/chronic fatigue
syndrome–a randomised controlled trial. Eur J Pain 6: 455–466. Find
this article online
Lloyd A, Hickie I, Wakefield D, Boughton C, Dwyer J (1990) A
double-blind, placebo-controlled trial of intravenous immunoglobulin
therapy in patients with chronic fatigue syndrome. Am J Med 89:
561–568. Find this article online
Peterson PK, Shepard J, Macres M, Schenck C, Crosson J, et al. (1990)
A controlled trial of intravenous immunoglobulin G in chronic fatigue
syndrome. Am J Med 89: 554–560. Find this article online
Vollmer-Conna U, Hickie I, Hadzi-Pavlovic D, Tymms K, Wakefield D, et
al. (1997) Intravenous immunoglobulin is ineffective in the treatment
of patients with chronic fatigue syndrome. Am J Med 103: 38–43. Find
this article online
Cleare AJ (2003) The neuroendocrinology of chronic fatigue syndrome.
Endocr Rev 24: 236–252. Find this article online
Afari N, Buchwald D (2003) Chronic fatigue syndrome: a review. Am J
Psychiatry 160: 221–236. Find this article online
Tharakan B, Manyam BV (2006) Botanical therapies in chronic fatigue.
Phytother Res 20: 91–95. Find this article online
Weatherley-Jones E, Nicholl JP, Thomas KJ, Parry GJ, McKendrick MW, et
al. (2004) A randomised, controlled, triple-blind trial of the
efficacy of homeopathic treatment for chronic fatigue syndrome. J
Psychosom Res 56: 189–197. Find this article online
Ernst E (2004) A randomised, controlled, triple-blind trial of the
efficacy of homeopathic treatment for chronic fatigue syndrome. J
Psychosom Res :: 57–503; author reply 504. Find this article online
Gregg VH (1997) Hypnosis in chronic fatigue syndrome. J R Soc Med 90:
682–683. Find this article online
Mears T (2005) Acupuncture in the treatment of post viral fatigue
syndrome–a case report. Acupunct Med 23: 141–145. Find this article
online
Sackner MA, Gummels EM, Adams JA (2004) Say NO to fibromyalgia and
chronic fatigue syndrome: an alternative and complementary therapy to
aerobic exercise. Med Hypotheses 63: 118–123. Find this article online
Reid S, Chalder T, Cleare A, Hotopf M, Wessely S (2000) Chronic
fatigue syndrome. BMJ 320: 292–296. Find this article online
Schinazi RF, Lloyd RM Jr, Nguyen MH, Cannon DL, McMillan A, et al.
(1993) Characterization of human immunodeficiency viruses resistant to
oxathiolane-cytosine nucleosides. Antimicrob Agents Chemother 37:
875–881. Find this article online
Schinazi RF, Hernandez-Santiago BI, Hurwitz SJ (2006) Pharmacology of
current and promising nucleosides for the treatment of human
immunodeficiency viruses. Antiviral Res 71: 322–334. Find this article
online
Volberding PA, Lagakos SW, Grimes JM, Stein DS, Balfour HH Jr, et al.
(1994) The duration of zidovudine benefit in persons with asymptomatic
HIV infection. Prolonged evaluation of protocol 019 of the AIDS
Clinical Trials Group. JAMA 272: 437–442. Find this article online
Land S, McGavin C, Lucas R, Birch C (1992) Incidence of
zidovudine-resistant human immunodeficiency virus isolated from
patients before, during, and after therapy. J Infect Dis 166:
1139–1142. Find this article online
Van Dooren S, Pybus OG, Salemi M, Liu HF, Goubau P, et al. (2004) The
low evolutionary rate of human T-cell lymphotropic virus type-1
confirmed by analysis of vertical transmission chains. Mol Biol Evol
21: 603–611. Find this article online
Coffin JM, Stoye JP (2009) Virology. A new virus for old diseases?
Science 326: 530–531. Find this article online
Telesnitsky A, Blain S, Goff SP (1995) Assays for retroviral reverse
transcriptase. Methods Enzymol 262: 347–362. Find this article online
Schinazi RF, Sommadossi JP, Saalmann V, Cannon DL, Xie MY, et al.
(1990) Activities of 3′-azido-3′-deoxythymidine nucleotide dimers in
primary lymphocytes infected with human immunodeficiency virus type 1.
Antimicrob Agents Chemother 34: 1061–1067. Find this article online
Bassit L, Grier J, Bennett M, Schinazi RF (2008) Combinations of
2′-C-methylcytidine analogues with interferon-alpha2b and triple
combination with ribavirin in the hepatitis C virus replicon system.
Antivir Chem Chemother 19: 25–31.

No comments: