Inhibitory Effects of Statins on Cytomegalovirus Production in Human Cells: Comprehensive Analysis of Gene Expression Profiles

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ORIGINAL ARTICLE

Inhibitory Effects of Statins on Cytomegalovirus Production in Human Cells:

Comprehensive Analysis of Gene Expression Pro

ﬁles

Tsugiya Murayama

1

, Changxiao Bi

1

, Ying Li

1

, Yasuhito Ishigaki

2

, Fumihide Takano

3

, Tsutomu Takegami

4

,

Tomihisa Ohta

3

, Hiroyuki Sumino

5

, Kimiko Ubukata

6

, Takashi Takahashi

7 *

1_{Department of Microbiology and Immunology, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Ishikawa, Japan} 2_{Division of Core Facility, Medical Research Institute, Kanazawa Medical University, Kahoku-gun, Ishikawa, Japan}

3_{Department of Pharmacognosy and Chemistry of Natural Products, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Ishikawa, Japan} 4_{Division of Molecular Oncology and Virology, Medical Research Institute, Kanazawa Medical University, Kahoku-gun, Ishikawa, Japan}

5_{Department of Clinical Laboratory, Gunma University Hospital, Maebashi, Gunma, Japan}

6_{Laboratory of Molecular Epidemiology for Infectious Agents, Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo, Japan} 7_{Laboratory of Infectious Diseases, Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo, Japan}

a r t i c l e i n f o

Article history: Received: Aug 22, 2010 Revised: Oct 11, 2010 Accepted: Oct 17, 2010 KEY WORDS: DNA microarray; human cytomegalovirus; human embryonic lung; statins;

viral replication

Background/Purpose: Inhibitory effects of statins on human immunodeﬁciency virus-1 or poliovirus replication were reported. Our aim was to clarify whether statins could inhibit the replication of cyto-megalovirus (CMV) in human cells and to determine the changes in gene expression proﬁles in host cells treated with statins using a DNA microarray.

Methods: Human embryonic lung (HEL) fibroblast cells were infected with CMV (Towne strain) at a multiplicity of infection of 1 and were simultaneously treated with mevastatin, simvastatin, lovastatin, or pravastatin (0.001e10mM). HEL cells were incubated for 6 days, and progeny viral titers were quan-tified by plaque assay. Time-dependent effects of mevastatin or simvastatin (1mM) on CMV replication were also examined. We determined the effects of mevastatin or simvastatin at concentrations ranging from 0.1mM to 10mM on the expressions of CMV immediate-early-1 (IE-1) and late proteins using Western blotting. Comprehensive analysis of gene expression profiles in HEL cells treated with mevas-tatin (1mM) was performed with a DNA microarray 1 day after infection.

Results: The 50% effective concentration values for the inhibition of CMV titers by mevastatin, simvas-tatin, lovassimvas-tatin, and pravastatin were 0.0006mM, 0.0055mM, 0.04mM, and 2.55mM, respectively. Inhibi-tion of viral replicaInhibi-tion by mevastatin was observed when added 24 hours after infecInhibi-tion, whereas that by simvastatin was observed when added 48 hours after infection. Mevastatin decreased the expression of the IE-1 protein, and simvastatin inhibited the expression of the late protein. We observed signiﬁcant changes of cellular growth/differentiation-associated gene expressions (e.g., downregulated cdk2 mRNA) in HEL cells treated with mevastatin.

Conclusion: Our data suggest that treatment with mevastatin could inhibit CMV replication at IE phase through altered expressions of cellular growth/differentiation-associated genes.

1. Introduction

Endothelial and smooth muscle cells infected with human

cyto-megalovirus (CMV) may cause chronic inﬂammation in vessels,

suggesting an association between arteriosclerosis and CMV infection. However, latently infected CMV is frequently activated in immunosuppressed patients, such as those with AIDS or organ

transplants, thereby causing severe morbidity and eventual

mortality.1Symptomatic CMV infection has been treated

success-fully with ganciclovir (GCV), but the emergence of GCV-resistant strains is a current issue in the treatment of immunocompromised subjects with CMV infection. Although either foscarnet or cidofovir has been used as an alternative therapy against GCV-resistant

strains, these treatments are not always successful.2There is a need

for the development of new or alternative anti-CMV agents.3

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, are used as lipid-lowering agents in clinical settings. It is demonstrated that statins play a multifactorial role in the

mainte-nance of transplanted organs.4Nie et al5report that statins exert

immunosuppressive effects in rats undergoing heterotopic limb * Corresponding author. Laboratory of Infectious Diseases, Graduate School of

Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

E-mail:[email protected](T. Takahashi).

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allografts by inhibiting activation and proliferation of T cells. In addition to these immunomodulatory actions, statins are shown to have antiviral effects. Fluvastatin is described to inhibit CMV

replication through a decrease of nuclear factor-

k

B binding activity

in human endothelial cells.6However, there is very limited

infor-mation regarding the inhibitory actions of various statins against CMV replication in other human cell lines. We aimed to clarify whether the activities of various agents can suppress CMV production in human embryonic lung (HEL) cells and to determine

signiﬁcant alterations of genetic proﬁles in cells treated with statins

using a DNA microarray. 2. Methods

2.1. Virus, cells, and reagents

CMV Towne strain was applied throughout our investigation.7CMV

was propagated in HEL cells, and the clariﬁed supernatant was

stored in liquid nitrogen until use. Viral infectivity was titrated

using a plaque assay method as previously reported.8Four statins,

mevastatin, simvastatin, lovastatin, and pravastatin, were

commercially available (Calbiochem Inc., San Diego, CA, USA). These reagents were dissolved in a cell culture medium.

2.2. Cell culture

HEL cells9were cultured in Dulbecco’s modiﬁed Eagle’s minimal

essential medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan), supplemented with 10% heat-inactivated fetal calf serum (Z.L.

Bocknek Laboratory, Ontario, Canada), L-glutamine (0.3 mg/mL),

gentamicin (50 mg/mL), and amphotericin B (2.5 mg/mL). All cell

cultures were maintained in a humidiﬁed incubator at 37_{C in 5%}

CO2/95% air.

2.3. Viral production

When HEL cells in 24-well plates (IWAKI Microplate; IWAKI Glass

Co., Funahashi, Japan) reached con_{ﬂuency, the cells were}

inocu-lated with CMV at a multiplicity of infection of 1. After adsorption

for 1 hour, the cells were supplemented with 1 mL of Dulbecco’s

modiﬁed Eagle’s minimal essential medium containing 2% fetal calf

serum in the presence or absence of various concentrations of statins for indicated time intervals. The infectious virus produced in

culture supernatants was titrated using a plaque assay.8

2.4. Inhibitory effects of statins on cytomegalovirus production The CMV-infected cells were treated with four statins (mevastatin, simvastatin, lovastatin, or pravastatin) at different concentrations

ranging from 0.001

m

M to 10

m

M. All cells were incubated for 6 days

after viral inoculation, and then, the CMV titers were measured. Based on the curves showing relationships between relative virus titers (a percentage of the control titers) and statin concentrations

used, 50% effective concentration (EC50) was calculated for each

statin.

2.5. Time-dependent effects of statins on cytomegalovirus production

The cells were inoculated with CMV, and treatment with

mevas-tatin (at a concentration of 1

m

M) was started at indicated times

(1, 8, 24, and 48 hours) after infection. All cells were incubated for 5

or 7 days after viral inoculation, and the viral titers were quantiﬁed.

Similarly, simvastatin (1

m

M) was added at various time points

(1, 24, 48, and 72 hours) after infection, and the CMV titers were determined at Day 7 after infection.

2.6. Cytotoxic activity of statins

Subconﬂuent monolayers of the uninfected cells were cultured on

24-well plates in the absence or presence of different

concentra-tions ranging from 0.01

m

M to 10

m

M of mevastatin or simvastatin.

They were incubated under conditions of 37C and 5% CO2for 3 or 6

days. The number of viable cells was determined using trypan-blue exclusion dye staining, and the ratio of the viable cells under exposure to the statins to viable negative control cells was calcu-lated for each concentration.

2.7. Detection of viral proteins

The CMV-infected cells were treated with the indicated

concen-trations ranging from 0.1

m

M to 10

m

M of mevastatin or simvastatin

until 1 day after viral inoculation and were harvested for Western blot analysis to detect the expression of the immediate-early-1 (IE-1) protein. In another trial, the cells were treated with the same concentrations of the same statins until 3 or 6 days after infection and were harvested for Western blotting analysis with the antibody against the late protein. Western blot analysis for the detection of the structural IE-1 and late proteins was performed as previously

described.3The

b

-actin protein was applied as an internal control.

2.8. Detection of viral mRNA

The infected cells were incubated with different concentrations

ranging from 0.1

m

M to 10

m

M of mevastatin until 24 hours after

infection. Total RNA was extracted using a standard method and was reversely transcribed after eliminating residual genomic DNA.

The cDNA products were ampliﬁed for IE-1/-2 and

b

-actin gene

expressions through real-time reverse-transcription polymerase

chain reaction (PCR) with each speciﬁc primer set and iQ SYBR

Green Supermix (Bio-Rad Laboratories Inc., Tokyo, Japan) for 34

cycles (10 seconds at 95C, 20 seconds at 55C, and 20 seconds at

72C) by a Mini Opticon real-time PCR using Gene Expression

Macro software (Bio-Rad Laboratories Inc.). The PCR primers for the

ampliﬁcation of IE-1/-2 genes were used as previously reported.10

The

b

-actin gene was used as an internal control. The

compara-tive expression levels of the IE-1/-2 mRNAs were calculated

according to the expression levels of the

b

-actin mRNA.

2.9. Determination of gene expression proﬁles in cells by DNA

microarray analysis

Total RNA was isolated from the cells using a commercially available kit (RNeasy Mini Kit; QIAGEN GmbH, Hilden, Germany). The

extracted RNA was quantiﬁed by photometry at 260/280 nm, and

the quality of RNA was determined by the ratio of 18S/28S ribo-somal band intensities in an ethidium bromide containing 1% agarose gel through electrophoresis. Comprehensive analysis of

gene expression proﬁles in the infected cells was performed using

a Human U133 Plus 2.0 GeneChip microarray system (Affymetrix

Inc., Santa Clara, CA, USA) as previously reported.11The amounts of

probe-speciﬁc transcripts were determined based on the average of

the differences between the perfect-match and mismatch intensi-ties. Because replicate assays were not performed, the signal intensity of selected genes that were up- or downregulated by at least twofold changes compared with that of a control group were extracted by the GeneSpring GX software package version 7.3.1 (Agilent Technologies Inc., Santa Clara, CA, USA). Ingenuity Pathway Analysis (Ingenuity Systems Inc., Redwood City, CA, USA) was used

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as an additional method for evaluating the functional signiﬁcance

of the induced gene expression proﬁles.

2.10. Validation of data by DNA microarray analysis

Real-time reverse-transcription PCR analysis was performed as described regarding the detection of viral mRNA, using prepared RNA samples. Four target genes (cdk2, arf1, xrcc2, and psmb4) in the infected cells treated with mevastatin or simvastatin at different

concentrations ranging from 0.1

m

M to 10

m

M until 24 hours after

infection were selected among those having signiﬁcantly up- or

downregulated changes based on the gene expression proﬁles.

The oligonucleotide sequences of the forward/reverse primer set for

each gene ampli_{ﬁcation were described previously.}12e15

Compara-tive expression levels of the selected genes were calculated according

to the expression levels of the

b

-actin mRNA. The expression levels of

the four mRNAs are expressed as fold changes relative to the baseline expression levels in the untreated control group. Together with the

validation of data by DNA microarray analysis, we also conﬁrmed the

changes of viral loads (IE-1 mRNA expression). 2.11. Data analyses

Data were analyzed using statistical analysis methods and graphing software (KaleidaGraphTM; Synergy Software, Tokyo, Japan). All

data were expressed as mean standard deviation. The

signiﬁ-cance of the differences among values was determined by

Dun-nett’s multiple tests after one-way analysis of variance in

comparison with control cultures, and p values less than 0.05 were

considered to indicate signiﬁcance. Furthermore, Fisher’s exact test

was used to determine the probability that biological function assigned to each network could be explained by chance alone. 3. Results

3.1. Inhibitory effects of statins on cytomegalovirus production

The EC50values for inhibition of CMV production by mevastatin,

simvastatin, lovastatin, and pravastatin were 0.0006

m

M, 0.0055

m

M,

0.04

m

M, and 2.55

m

M, respectively. Mevastatin showed the most

pronounced suppressive effect.

3.2. Time-dependent effects of statins on cytomegalovirus production

Based on the EC50data, mevastatin and simvastatin were selected

for the evaluation of time-dependent actions against CMV

production. Mevastatin (1

m

M) exhibited signiﬁcantly inhibitory

effects on viral replication over a period of 5 or 7 days when started at 1, 8, and 24 hours after CMV infection but not when started at 48

hours after viral inoculation (Figure 1A). On the other hand,

sim-vastatin (1

m

M) achieved a signiﬁcant suppression of CMV

produc-tion over a period of 7 days when started 48 hours as well as 1 and

24 hours after the infection (Figure 1B).

3.3. Cytotoxic activity of statins

We determined the cytotoxic activity of mevastatin or simvastatin

without CMV infection according to the EC50data. Ratio of viable

cells under exposure to the statins (0.01e10

m

M) to viable negative

control cells for 3 days was more than 90%, and those under exposure

to mevastatin and simvastatin (10

m

M) for 6 days were

approxi-mately 73% and 89%, respectively. These observations suggest that

decreased viral titers by these statins simply reﬂect inhibitory

actions of CMV replication without direct statin cytotoxicity.

3.4. Viral protein

Because mevastatin and simvastatin showed different time-dependent effects, we selected these reagents for examination of variations in the production of early and late CMV proteins during

statin exposure (0.1e10

m

M). Mevastatin inhibited the expression of

the IE-1 protein at 1 day after viral inoculation, whereas simvastatin inhibited the expressions of the late protein at 3 and 6 days after

infection (Figure 2).

3.5. Viral mRNA

We determined the ability of mevastatin to inhibit expressions of the IE-1/-2 mRNAs when incubated until 24 hours after infection,

based on the protein expression data. At a concentration of 0.1

m

M,

mevastatin signiﬁcantly inhibited the expression of only IE-1

mRNA, whereas at 1

m

M, it signiﬁcantly inhibited the expressions of

both IE-1 and IE-2 mRNAs (Figure 3).

3.6. Gene expression proﬁles in cells by DNA microarray analysis

We performed a comprehensive analysis of gene expression

proﬁles in the infected cells treated with mevastatin (1

m

M) until 24

hours after infection. Table 1 shows mRNAs that were up- or

downregulated by at least twofold compared with the control. According to Ingenuity Pathway Analysis categories, we observed

signi_{ﬁcant changes of cellular growth/differentiation-associated}

gene expressions in the infected cells treated with mevastatin. 3.7. Validation of data by DNA microarray analysis

Based on the results of gene expression proﬁles in the cells, we

selected four up- or downregulated genes (i.e., cdk2, arf1, xrcc2, and

A

B

Time of addition (hr): Time of addition (hr): Simvastatin Mevastatin 7 d.p.i. 7 d.p.i. 5 d.p.i. 5 d.p.i.

Figure 1 Time-dependent effects of mevastatin (A) or simvastatin (B) at 1mM on human cytomegalovirus replication. *Signiﬁcantly different levels compared with those in the controls. Meva¼ mevastatin; Simva ¼ simvastatin; d.p.i ¼ days postinfection.

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psmb4) in the infected cells treated with mevastatin or simvastatin

(0.1e10

m

M) until 24 hours after infection. The reasons why these

four genes were selected for validation of DNA microarray data

were signiﬁcant levels of fold changes and estimated functions of

genes. Mevastatin dose dependently induced elevations in expressions of PSMB4 and XRCC2 mRNAs and decreased

expres-sions of CDK2, ARF1, and IE-1 mRNAs (Figure 4A and 4B). Similarly,

simvastatin dose dependently increased or decreased expressions of three of the four genes except for ARF1 mRNA, together with the

decreased expression of IE-1 mRNA (Figure 4C and 4D).

4. Discussion

Our observations suggest that treatment with mevastatin,

simvas-tatin, or lovastatin at various EC50 concentrations of 0.0006

m

M,

0.0055

m

M, or 0.04

m

M could inhibit CMV production. Clinical trial

conﬁrmed achievable plasma concentration (0.10e3.92

m

M) of

lovastatin administered at a dose of 25 mg/kg/d, which corresponds to the dose range that could trigger apoptosis of sensitive tumor

types in vitro.16Based on theﬁndings of our experiment, the statin

interventions should be further developed as novel or alternative strategies for treatment of CMV infection.

Statins are reported to play a crucial role in the management of sepsis in a clinical setting. Administration of statins in patients with atherosclerosis is shown to be associated with a decreased risk of subsequent sepsis, and randomized clinical trials of statins for

prevention of sepsis are warranted.17In addition to the

manage-ment of sepsis, there are several descriptions concerning the inhibitory effects of reagents on viral replication, including

polio-virus,18HIV-1,19 and hepatitis C virus.20Furthermore, combined

treatment with a statin and caffeine effectively ameliorates lung damage, inhibits viral replication, and is at least as effective as the therapy with oseltamivir and ribavirin in H5N1-, H3N2-, and

H1N1-infected mice.21This combination seems to be more effective when

administered preventatively rather than as a treatment. Therefore, it will also be important to clarify the antiviral actions of statins

against inﬂuenza viruses in future investigations.

A potential mechanism of how the viral replication in CMV-infected cells could be inhibited by mevastatin seems to be cellular arrest, similar to statin effects on malignant cells in previous

report.16 _{Comprehensive analysis of gene expression pro}_{ﬁles in}

CMV-infected cells revealed that mevastatin induced signiﬁcant

changes of cellular growth/differentiation-associated gene expres-sions. In particular, this statin dose dependently induced both reduced expressions of CDK2/ARF1 mRNAs and increased expres-sions of PSMB4/XRCC2 mRNAs, leading to decreased expression of CMV IE-1 mRNA. CDK2 is a member of Ser/Thr protein kinase families, shows activity restricted to G1-S phase, and is essential for cell cycle G1/S phase transition. Transcriptional activation of the cdc2 is associated with Fas-induced apoptosis of human

hemato-poietic cells.12 Interestingly, mevastatin is said to inhibit cellular

growth of a prostate cancer cell line through inhibition of CDK2.22

Moreover, inhibition of CDK2 activity by roscovitine inhibits CMV DNA synthesis, production of infectious progeny, and viral antigen

expression in infected cells in a dose-dependent manner.23ARF1,

Figure 2 Expressions of viral proteins, including immediate-early-1 (IE-1) and late proteins, in human cytomegalovirus-infected cells treated with mevastatin (left) or simvastatin (right) at 0.1mM, 1mM, and 10mM. b-Actin protein was applied as an internal control. CMV¼ human cytomegalovirus; d.p.i ¼ days postinfection; meva ¼ mevastatin; simva¼ simvastatin.

Figure 3 Expressions of viral mRNAs, including immediate-early-1 and -2 (IE-1 and IE-2) mRNAs, in human cytomegalovirus-infected cells treated with mevastatin (0.1mM, 1mM, or 10mM) until 24 hr after infection. *Signiﬁcantly different levels compared with those in the controls. Relative expression levels of IE-1 and IE-2 mRNAs were calculated according to the expression levels ofb-actin mRNA.

Table 1 Gene expression proﬁles in virally infected cells treated with mevastatin by DNA microarray analysis

Upregulated mRNA Fold change Downregulated mRNA Fold change

PSMB4 4.76 RPS2 7.46

SLC25A37 4.472 CDK2 6.308

XRCC2 4.461 C7ORF11 5.224

ZNF652 4.322 HLA-B 5.066

NCRNA00094 3.972 DUT (includes EG:1854) 5.002

HELLS 3.971 ARF1 4.912

UBE2B 3.456 GADD45GIP1 4.835

RBBP6 (includes EG:5930) 3.427 IFI16 4.705

ATP6V0D2 3.408 ID1 4.697

FARP1 3.277 HSPB1 4.664

To validate the data generated by DNA microarray analysis, the italicized mRNAs were selected as four target genes for real-time reverse-transcription polymerase chain reaction assays.

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a member of RAS superfamily, is localized to Golgi apparatus, and has a central role in intra-Golgi transport. This molecule regulates

epidermal growth factoredependent growth and migration of

breast cancer cells through activation of phosphatidylinositol

3-kinase pathway.13PSMB4, a member of proteasome B-type family, is

distributed throughout the eukaryotic cells at a high concentration and cleaves peptides in an ATP/ubiquitin-dependent process in a nonlysosomal pathway. An expression survey of a panel of glioma cell lines demonstrated the expression of the PSMB4 and validity of the proteasome complex as a target for survival inhibition, which

was conﬁrmed in a series of glioma and nonglioma cell lines.24

XRCC2, a member of RecA/Rad51-related protein family, partici-pates in homologous recombination to maintain chromosome stability and to repair DNA damage. This molecule is important to preserve or restore replication forks during rapid clonal expansion of

developing lymphocytes.25There are no investigations of

associa-tions between mevastatin and ARF1, PSMB4, or XRCC2. Therefore, we need to determine the detailed up- and downstream networks of CDK2, ARF1, PSMB4, XRCC2, and other molecules involved in cellular growth/differentiation in CMV-infected cells under intervention of mevastatin.

Effects of the four statins on blood cholesterol and lipoprotein levels are quantitatively similar when administered at equivalent doses. We found that mevastatin, simvastatin, and lovastatin, but

not pravastatin, were able to efﬁciently inhibit CMV production in

HEL cells. It is unclear why pravastatin could exhibit less inhibitory effects on viral replication. Pravastatin only has hydrophilic

prop-erty and no

b

-hydroxy-

d

-lactone moiety that the other three statins

commonly include as chemical structures. In future basic

researches, it will be necessary to conﬁrm signiﬁcant differences in

the chemical structures and biochemical activities among the statin reagents in detail. Moreover, we should examine the antiviral effects of mevastatin and simvastatin in vivo, using an animal model of murine CMV infection to provide experimental evidence for our hypotheses.

Acknowledgments

The authors have no conﬂicts of interest to declare in relation to this

article. This study was funded, in part, by a Grant-in-Aid for Scientiﬁc

Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (number 21390306, to Dr T. Takahashi). References

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0 50 100 150 200 250

A

B

C

D

0 0.1 1 10 IE1 PSMB4 CDK-2

Relative expression to control (%)

Concentration of mevastatin (µM) 0 50 100 150 200 250 300 0 0.1 1 10 IE1 XRCC2 ARF-1 Concentration of mevastatin (µM)

Concentration of simvastatin (µM)

0 50 100 150 200 250 300 0 0.1 1 10 IE1 PSMB4 CDK-2 0 50 100 150 200 250 300 350 0 0.1 1 10 IE1 XRCC2 ARF-1

Concentration of simvastatin (µM)

Figure 4 Dose-dependently altered expressions of four selected genes, that is, cdk-2, arf1, xrcc2, and psmb4, in human cytomegalovirus-infected cells treated with mevastatin (A and B) or simvastatin (C and D) at 0.1mM, 1mM, and 10mM until 24 hr after infection. The expression levels of the four mRNAs are expressed as fold changes relative to the baseline expression levels in the untreated control group. Together with the validation of data by DNA microarray analysis, we also conﬁrmed the changes of viral loads. IE-1 ¼ immediate-early-1 mRNA.

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