Accepted Manuscript Mycophenolic mofetil, an alternative antiviral and immunomodulator for the highly pathogenic avian influenza H5N1 virus infection Junhyung Cho, Hwajung Yi, Eun Young Jang, Mi-Seon Lee, Joo-Yeon Lee, Chun Kang, Chan Hee Lee, Kisoon Kim PII:

S0006-291X(17)32007-7

DOI:

10.1016/j.bbrc.2017.10.037

Reference:

YBBRC 38654

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 21 September 2017 Accepted Date: 6 October 2017

Please cite this article as: J. Cho, H. Yi, E.Y. Jang, M.-S. Lee, J.-Y. Lee, C. Kang, C.H. Lee, K. Kim, Mycophenolic mofetil, an alternative antiviral and immunomodulator for the highly pathogenic avian influenza H5N1 virus infection, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Mycophenolic mofetil, an alternative antiviral and immunomodulator for the highly pathogenic avian influenza H5N1 virus infection Junhyung Choa,d*, Hwajung Yia*, Eun Young Janga, Mi-Seon Leea,e, Joo-Yeon Leeb, Chun Kangc, Chan

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Hee Leed, Kisoon Kima†

a

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Author affiliation

Division of Viral Disease Research, bDivision of Emerging Infectious Disease Vector Research,

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Center for Infectious Diseases Research, Korea National Institute of Health, Cheongju, Republic of Korea. c

Division of Viral Diseases, Center for Laboratory Control of Infectious Diseases, Korea Centers for

Disease Control and Prevention, Cheongju, Republic of Korea.

e

Department of Microbiology, Chungbuk National University, Cheongju, Republic of Korea.

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d

Department of Life Science and Technology, Pai Chai University, Daejeon, Republic of Korea.

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†Correspondence

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*Both authors equally contributed to this work.

Kisoon Kim, Division of Viral Disease Research, Center for Infectious Diseases Research, Korea National Institute of Health, Korea Centers for Disease Control and Prevention, 187 Osongsaengmyeong2-ro, Heungduk-gu, Cheongju-si, Chungcheongbuk-do, 28159, Korea E-mail: [email protected]; Telephone:+82-43-719-8410, Fax: +82-43-719-8459

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ACCEPTED MANUSCRIPT Abstract Infection with the highly pathogenic avian influenza H5N1 virus results in a high incidence of mortality in humans. Severe complications from infection are often associated with

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hypercytokinemia. However, current neuraminidase inhibitors (NAIs) have several limitations including the appearance of oseltamivir-resistant H5N1 virus and the inability to completely ameliorate hyper-immune responses. To overcome these limitations, we evaluated the anti-viral activity of mycophenolic mofetil (MMF) against A/Vietnam/1194/2004 (H5N1) virus infection

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using MDCK cells and mice. The IC50 of MMF (0.94 µM) was comparable to that of zanamivir (0.87 µM) in H5N1 virus-infected MDCK cells based on ELISA. Time-course assays demonstrated

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that MMF completely inhibited H5N1 viral mRNA replication and protein expression for approximately 8 h after the initiation of treatment. In addition, MMF treatment protected 100% of mice, and lung viral titers were substantially reduced. The anti-viral mechanism of MMF against H5N1 virus infection was further confirmed to depend on the inhibition of cellular inosine

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monophosphate dehydrogenase (IMPDH) by exogenous guanosine, which inhibits viral mRNA and protein expression. Moreover, IL-1β, IFN-β, IL-6, and IP-10 mRNA expression levels were significantly downregulated in MDCK cells with MMF treatment. These results indicated that

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MMF could represent a novel inhibitor of viral replication and a potent immunomodulator for the

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treatment of H5N1 virus infection.

Keywords: Highly pathogenic avian influenza, H5N1 influenza virus, mycophenolic mofetil, immunomodulator, anti-influenza treatment

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1. Introduction Human infection with highly pathogenic avian influenza (HPAI) H5N1 virus has continuously

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threatened global public health. Human infection by this virus was reported in Hong Kong in 1997 [1], and from then until April of 2017, 858 human cases have been laboratory-confirmed and 453 people have died from H5N1 virus infection in Asia, Africa, Europe, and America [2]. Characteristics of the

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H5N1 infection include high mortality and severe complications [3, 4]. One reason for the higher mortality observed with this virus was suggested to be the through the excessive production of pro-

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inflammatory cytokines and chemokines [5-8].

Currently, neuraminidase inhibitors (NAIs) such as zanamivir and oseltamivir are common for the treatment of influenza infection. However, these have shown limited clinical efficacy against influenza virus [9, 10]. Not only has oseltamivir-resistant H5N1 virus isolated from human cases [11, 12], but this drug also has insufficient ability to ameliorate severe complications caused by

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hypercytokinemia. Moreover, administration within 48 hours is required for effective treatment [1317]. Thus, it is necessary to develop novel anti-influenza treatments that compensates for the

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limitations of currently available NAIs.

Mycophenolic mofetil (MMF), a mycophenolic acid (MPA) pro-drug, is a non-competitive

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inhibitor of inosine monophosphate dehydrogenase (IMPDH), and is FDA-approved for immune suppression. Its mechanism is through the repression of de novo synthesis of guanosine monophosphate by inhibiting IMPDH, leading to suppression of DNA synthesis [18, 19]. Accordingly, it has been reported that guanosine depletion by MPA inhibits viral RNA replication in HCV, chikungunya, and dengue viruses [20-23]. In addition, although the antiviral ability of MPA was recently reported for H1N1, H7N9, and influenza B virus [22], its efficacy as an immunomodulator during H5N1 infection, which results in excessive immune response compared to that with H7N9, has still not been evaluated [8].

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ACCEPTED MANUSCRIPT In this study, we first demonstrated that MMF inhibits H5N1 virus replication in MDCK cells and mice. Moreover, we demonstrated reduced proinflammatory cytokine and chemokine gene expression and confirmed the antiviral mechanism of MMF against H5N1 using MDCK cells.

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2. Materials and Methods 2.1. Virus, cells, and compound

A/Vietnam/1194/2004 (H5N1) virus was kindly provided by the CDC (Atlanta, U.S.A).

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Eleven-day-old specific pathogen free (SPF) embryonated chicken eggs were inoculated with H5N1 virus and incubated at 37 °C for 1 day. The eggs were chilled at 4 °C for 1 day and the virus titer was

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measured by performing plaque assays in Madin-Darby canine kidney (MDCK) cells cultured in MEM containing 10% v/v FBS and 1% v/v penicillin/streptomycin (Gibco, NY, USA) at 37 °C with 5% CO2. MMF (Sigma, MO, USA) and zanamivir (ZAN; Sigma) were dissolved in DMSO and PBS, respectively, and stored at −20 °C. All experiments were performed in biosafety level 3 facilities

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according to Korea Center for Diseases Control (KCDC) procedures.

2.2. Half maximal inhibitory concentration (IC50) measurements for H5N1 infection

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Confluent MDCK cells in 96-well plates were infected at a multiplicity of infection (MOI) of 0.1 of H5N1 virus for 1 h. Cells were washed and incubated with 0.125, 0.25, 0.5 and 1 µg/ml of

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MMF, ZAN, or DMSO in MEM with 1% BSA for 20 h. Virus infectivity was evaluated by ELISA with an influenza A NP protein antibody (Millipore, CA, USA), and the optical density was measured by SunriseTM (TECAN, Männedorf, Switzeland). The IC50 was determined using Graphpad Instat 4 Statistical Software.

2.3 Drug treatment and measurement of lung viral titers in mice Female 6- to 8-week-old BALB/c mice (Orient Bio, Gyeonggi-do, Korea) (eight mice/group)

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ACCEPTED MANUSCRIPT were intranasally infected with 3 times the mouse lethal dose (MLD50) of H5N1 virus (1 MLD50 of the H5N1 virus was previously reported as 0.66 pfu) [24]; animals were then intraperitoneally injected with 50 mg/kg/day of MMF or 25 mg/kg/day of oseltamivir, starting from 1 h post-infection (hpi). Mouse survival and body weight changes were monitored until 14 days post-infection (dpi). On day 3

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post-infection, three mice per group were sacrificed to measure lung viral titers. Mouse lungs were homogenized and supernatants were incubated with MDCK cells; viral titers were measured using the modified rapid culture assay [25]. Briefly, MDCK cells were seeded at 2.5 × 104 cells per well in 96-

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well cell culture plates and incubated overnight. Cells were infected with 2-fold serial dilutions of the supernatant (from 21 to 212) for 1 h. After removing the supernatant, cells were incubated with MEM

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with 1% BSA for 24 h and fixed with 80% acetone for 15 min. Cells were incubated for 1 h with antiinfluenza NP antibody (1:1,000, Millipore) in 5% skim milk and then with HRP-conjugated rabbit antibody (1:1,000, Millipore) for 1 h. Then, substrate solution (O-phenylenediamine (Sigma) dissolved in phosphate-citrate buffer with sodium perborate (Sigma)) was added to the cells and developed at room temperature. The infectious virus titer was determined as the reciprocal value of

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the maximum infectious dilution [26]. All mouse experiments were performed according to

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institutional guidelines of the Korea Center for Disease Control.

2.4. Quantitative real-time RT-PCR

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Total RNA was extracted from cells using TRIzolTM (Ambion, Leicestershine, UK) reagent. Subsequently, cDNA was synthesized from 1 µg of total RNA using a PrimeScriptTM RT reagent kit with gDNA Eraser (Takara, Shiga, Japan). Quantitative real-time PCR (qRT-PCR) was performed with Takara SYBR® Premix Ex TaqTM (Takara) and an ABI 7900HT (Applied Biosystems, CA, USA), following the manufacturer’s protocol. The sequence of primers used in this study are provided in Supplementary Table 1.

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ACCEPTED MANUSCRIPT 2.5. Western blotting MDCK cells were lysed in RIPA buffer (Biosesang, Gyeonggi-do, Korea) and 20 µg of proteins were separated using the BoltTM 4-12% Bis-Tris Plus (Invitrogen, MA, USA). Proteins were

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detected by probing the membranes with anti-NP (Sino Bio., Beijing, China), anti-NS1 (Santa Cruz), anti-M1 (Abcam, MA, USA) and anti-β-actin (Santa Cruz) antibodies. Membranes were incubated with rabbit anti-mouse (Santa Cruz) conjugated with horseradish peroxidase for 1 h, and membranes were washed five times with tris-buffered saline with 5% Tween 20. Blots were detected using Super

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2.6. Treatment with exogenous guanosine

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SignalTM (Thermo, MA, USA).

MDCK cells were infected at an MOI of 0.1 with H5N1 virus for 1 h. After incubation, cells were washed and treated with DMSO (CON), 100 µM guanosine, 1 µg/ml MMF, or MMF supplemented

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with 2-fold serial dilutions of guanosine (from 100 µM to 12.5 µM) for 6 h. After incubation, viral protein and gene expression was assessed by western blotting and qRT-PCR.

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2.7. Statistical analysis

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The data are expressed as the mean value ± SD (n = 3). The data were compared using the Student’s t tests and P < 0.05 was considered to indicate a significant difference.

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3. Results

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3.1. MMF has comparable efficiency to ZAN and protects mice from H5N1 virus infection

To evaluate the antiviral activity of MMF against H5N1 viral infection, we determined the IC50 of MMF for H5N1-infected MDCK cells. The expression of viral NP protein with 0.125, 0.25,

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0.5, and 1 µg/ml of MMF was 88.2 ± 5.5%, 76.9 ± 2.5%, 40.2 ± 1.5%, and 29.0 ± 3.3%, relative to that of the untreated control, respectively (Fig. 1A). The IC50 was determined to be 0.94 µM (410 ± 40

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ng/ml), which is comparable to that of ZAN (0.87 µM, 289 ± 40 ng/ml). The infectivity of MMF and ZAN were 29 ± 3.7% and 40.9 ± 1.8%, respectively at 1 µg/ml of treatment (Fig. 1). The cytotoxicity of MMF was determined to be > 25 µg/ml in MDCK cells (Supplementary Fig. 1). This result indicated that MMF is as effective as ZAN for the inhibition of H5N1 viral replication in MDCK cells.

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We next assessed the antiviral efficacy of MMF against H5N1 infection in mice. BALB/c mice were intranasally infected with three times the MLD50 of H5N1 and intraperitoneally administrated MMF at 50 mg/kg or oseltamivir phosphate (OSE) at 25 mg/kg/day once daily for 10

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days, initiated at 1 hpi. Survival rates for mice in the MMF and OSE treatment groups were 100% until 14 dpi, whereas all control group (PBS) mice died at 14 dpi (Fig. 1B). Body weights of animals

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in the MMF and OSE groups were maintained at over 90% of initial weight until 14 dpi (Fig. 1C). In addition, lung viral titers in the MMF and OSE treatment groups were significantly reduced compared to those in the PBS treatment group (Table 1). Based on these results, MMF has antiviral activity against H5N1 in mice.

3.2. MMF inhibits H5N1 viral mRNA replication and protein expression To clarify the antiviral activity of MMF, we investigated H5N1 virus mRNA and protein expression by performing time course experiments (Fig. 2). The expression of H5N1 NP-, M1-, and 7

ACCEPTED MANUSCRIPT HA-encoding mRNA decreased with MMF treatment to 0.3-, 0.2-, and 0.35-fold that of the CON group, respectively, at 4 hpi and were all approximately 0.1-fold relative to that of the CON group at 6 hpi. Accordingly, NP, M1, and NS1 protein expression in H5N1 infected cells was completely inhibited at 6 and 8 hpi, but was fully restored at 12 hpi. This indicated that MMF inhibits H5N1 virus

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mRNA replication and protein expression for approximately 8 h post-treatment.

protein expression in a dose-dependent manner

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3.3. Exogenous guanosine reverses the MMF-mediated inhibition of H5N1 mRNA replication and

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For HCV, dengue, influenza A (H1N1 and A/H7N9), and influenza B viruses, the antiviral mechanism of MMF is known to be depletion of intracellular guanosine by IMPDH, leading to inhibition of viral RNA replication [20, 22, 23]. Thus, we investigated whether this occurs with H5N1. Virus-infected MDCK cells treated with 1 μg/ml of MMF showed significantly reduced NP, M1, and

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HA mRNA expression, whereas exogenous guanosine at 12.5, 25, 50, and 100 µM resulted in a dosedependent recovery of NP, M1, and HA mRNA expression (Fig. 3A, B, and C). Accordingly, H5N1 NP, M1, and NS1 protein expression was completely inhibited by MMF, whereas exogenous

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guanosine resulted in a dose-dependent recovery of viral protein expression (Fig. 3D). These data indicated that inhibition of IMPDH is the major antiviral mechanism of MMF in response to H5N1

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infection.

3.4. MMF suppresses IL-6, IL-1β, IFN-β, and IP-10 mRNA expression in H5N1-infected cells To determine whether MMF can inhibit proinflammatory cytokine and chemokine mRNA expression in H5N1 infected MDCK cells, IL-6, IL-1β, IFN-β, and IP-10 mRNA expression levels were examined by qRT-PCR. Without MMF (CON), expression of IP-10, IL-6, IL-1β, and IFN-β continuously increased until 8 hpi, whereas with MMF, expression was markedly reduced by 0.5-, 0.6-, 0.4-, and 0.3-fold, respectively, compared to that in the CON group, at 8 hpi (Fig. 4A, B, C, and 8

ACCEPTED MANUSCRIPT D). These results suggested that MMF might have immunomodulatory effects during H5N1 infection of MDCK cells.

4. Discussion

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In this study, we first demonstrated the anti-viral activity of MMF against H5N1 virus infection in vitro and in vivo. Moreover, MMF was shown to inhibit proinflammatory cytokine and chemokine expression in response to H5N1 infection in MDCK cells. It was also confirmed that inhibition of

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IMPDH is the major anti-viral mechanism of MMF in response to H5N1 infection, which is consistent with the previously known mechanism of MPA for several viruses [20-23].

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Time course experiments showed drastic suppression of viral mRNA and protein expression until 8 hpi, which is consistent with a previous report showing that influenza A type (H1N1 and H7N9) replication is inhibited in the early stage by MPA [22]. However, viral protein expression was completely recovered by 12 hpi, which might result from the metabolism of MMF, as this compound is converted to the pharmacologically inactivate 7-O-glucuronide metabolite by UGT1A9 in MDCK

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cells [27, 28]; alternatively, guanosine synthesis might be restored by the salvage pathway [18]. Thus, inhibition of H5N1 mRNA replication and protein expression by MMF can be maintained for approximately 8 h in MDCK cells. There have been concerns that the antiviral activity of MMF might

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be abrogated after 12 hpi. However, the influenza life cycle, from infection to propagation in infected cells, occurs within 12 h [29, 30], suggesting that this is sufficient time to inhibit mRNA replication

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and consequently suppress viral propagation in H5N1 infected MDCK cells. There are several benefits associated with using MMF as a novel treatment against H5N1. First, repurposing an FDA approved drug can utilize previous research data including mechanisms of action, safety, characterization of cytokine regulation, and pharmacokinetic profiles, leading to a reduction in development timelines, costs, and substantial risks [31, 32]. Second, the IC50 of MMF (0.94 µM) might be suitable for human treatment. In humans, the general oral dose of MMF (1000 mg, for which the Cmax of MPA is 78 µM) is 70-fold higher than the concentration used in this study. In addition, this is comparable to the IC50 of ZAN (0.87 µM) used this study and the IC50 of MPA against H1N1 (0.62 9

ACCEPTED MANUSCRIPT µM), H3N2 (0.56 µM), and H7N9 (0.92 µM) in MDCK cells [22]. Third, a substantial reduction in IL-1β, IL-6, IFN-β, and IP-10 might ameliorate severe disease after H5N1 infection. It has been reported that upregulation of IL-6, IFN-β, and IP-10 in H5N1 patients is strongly associated with severe complications [5, 33], and that induction of IL-1β plays a critical role in lung inflammation and

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trypsin upregulation, resulted in multi-organ failure [34, 35].

In conclusion, this study first demonstrated that IMPDH suppression by MMF is the principal anti-viral mechanism of H5N1 virus replication inhibition, and showed efficient antiviral activity of

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this compound in mice. In addition, MMF showed potential as an immunomodulator by reducing inflammatory cytokine and chemokine gene expression. Collectively, this study provides new insight

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regarding the understanding of MMF as a novel therapeutic against H5N1 infection; further studies will be necessary to determine the immunomodulatory activity in vivo.

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Acknowledgements

A/Vietnam/1194/2004 (H5N1) virus was kindly provided by the CDC in the U.S.A. This research was supported by intramural grants from the Korea National Institute of Health (Grant number: 2013-

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NG43001-00 and 2016-NI43001-00).

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Figure legends

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Fig. 1. Antiviral efficacy of mycophenolic mofetil (MMF) with H5N1 virus infection in MDCK cells and mice. (A) Indicated concentrations of MMF (gray, circle), zanamivir (ZAN, dotted line, triangle), or vehicle (CON, black, square) were administrated for 24 h, after initiating infection for 1 h

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with H5N1 virus at an MOI of 0.1 in MDCK cells. Virus infectivity was evaluated by ELISA using an anti-influenza A NP antibody. Error bars indicate mean ± SD. Asterisks represent statistically

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significant differences between MMF and controls (without MMF) (*p < 0.05, ***p < 0.001). Eight mice per group were intranasally infected with 3 mLD50 of H5N1 virus. PBS (black, square), 25 mg/kg/day of oseltamivir (OSE, dotted line, triangle), or 50 mg/kg/day of MMF (gray, circle) in 100 µl of PBS was administrated by intraperitoneal (i.p.) injection daily for 10 days. (B) Survival rate of the mice (n = 3) based on Kaplan-Meier survival curves. (C) Body weight changes are shown as mean

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values (n = 3) until 14 dpi. As mice in the PBS group died at 11 and 12 dpi, body weight changes in

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the PBS group comprised data from a single mouse, from 12 to 14 dpi.

Fig. 2. Time course expression of H5N1 viral mRNA and proteins after mycophenolic mofetil

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(MMF) treatment. MDCK cells were infected with H5N1 virus at an MOI of 0.1, and 1 µg/ml of MMF was added. H5N1 viral mRNA, (A) NP, (B) M1, (C) HA, was measured at 4 and 6 h postinfection (hpi) by qRT-PCR and (D) viral proteins were assessed at 6, 8, and 12 hpi by western blotting. (A, B, C). Quantification of viral mRNA was normalized using GAPDH, and CON (virus infection without MMF) expression was set to 1. Asterisks represent significantly different values between CON and MMF groups, by Student t-test (***p < 0.001). (B) Anti-β-actin blots were used as loading controls.

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ACCEPTED MANUSCRIPT Fig. 3. Reversal of H5N1 viral RNA and protein expression with exogenous guanosine. MDCK cells were infected with H5N1 virus at an MOI of 0.1. Next, 1 µg/ml of mycophenolic mofetil (MMF) or MMF with the indicated guanosine concentration was added to the cells. The expression of H5N1 viral mRNA (A) NP, (B) M1, and (C) NS1, at 6 h post-infection (hpi) was measured by qRT-PCR.

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Viral mRNAs were normalized to GAPDH expression. The expression of CON was set to 1. Values are presented as mean ± SD. Statistically significant differences between CON and MMF are represented as ***p < 0.001, and between MMF only and MMF with 12.5, 25, 50, and 100 µM of

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guanosine as #p < 0.05 and ###p < 0.001. (D) The expression of viral NP, M1, and NS1 proteins at 8

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hpi was assessed by western blotting. Anti-β-actin blots were used as loading controls.

Fig. 4. Mycophenolic mofetil (MMF) inhibits proinflammatory cytokine and chemokine mRNA expression. MDCK cells were infected with H5N1 virus at an MOI of 0.1 and incubated with 1 µg/ml of MMF for the indicated time points. (A) IFN-β, (B) IL-6, (C) IL-1β, and (D) IP-10 mRNA was

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measured at 4, 6, and 8 h post-infection (hpi) by qRT-PCR. mRNA expression was normalized to GAPDH levels. CON (without MMF treatment) at 4 hpi was set to 1. The values are shown as mean ± SD. The asterisks represent statistically significant differences between CON and MMF (*p < 0.05,

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**p < 0.01, and ***p < 0.001).

Supplementary Fig. 1. Cell viability after mycophenolic mofetil (MMF) treatment. MDCK cells were treated with the indicated doses (serial diluted from 25 µg/ml to 0.1 µg/ml) of MMF and incubated for 24 h. Cell viability was measured using a Ez-CyTox kit (Daeil biotech, Seoul, Korea), according to manufacturer’s protocol. The O.D. value of no treatment was set to 100% and all other values were plotted as relative values.

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ACCEPTED MANUSCRIPT

Table 1. Lung virus titers in mice at 3 days post-infection (dpi) Virus titer in lungs at 3 dpi (GMT/g)

PBS (10% DMSO, v/v)

3657143

3011765

2133333

Oseltamivir (25mg/kg/day)

0

0

0

MMF (50mg/kg/day)

133

5333

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Compounds (mg/kg/day)

1454

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GMT, geometric mean of reciprocal dilution in end point titer; MMF, mycophenolic mofetil

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Supplementary Table 1. Primer sequences used in this study 5´- 3´

Sequence

GAPDH

forward

CCTTCATTGACCTCCACTACATGGT

reverse

CCACAACATACGTAGCACCACGAT

forward

TGAACCAAAGTGCTGTTCTTATT

reverse

ACGATGGACTTGCAGGAATC

forward

TCCTGGTGATGGCTACTGCTT

reverse

GACTATTTGAAGTGGCATCATCCTT

IL-1β

forward reverse

IFN-β

forward

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H5N1 hemagglutinin (HA)

GCAGGGCTTCTTCAGCTTCTC CCAGTTCCAGAAGGAGGACA

reverse

TGTCCCAGGTGAAGTTTTCC

forward

CCTGCTTGTGTGTACGGACT

reverse

TTGAAGCAGGCGGAAAGGAT

forward

TGCAGATTCACAGCATCGGT

reverse

GCCATCTGCTCCATAGCCTT

forward

TCGACAGAGCAGGTTGACAC

reverse

ATCGCAGAGCTTCCCATTGT

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H5N1 matrix1 gene (M1)

TCTCCCACCAGCTCTGTAACAA

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H5N1 nucleoprotein (NP)

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IL-6

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IP-10

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Primer

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ACCEPTED MANUSCRIPT

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120

* MMF

***

40 20 0 0

0.125

0.25

0.5

1

Concentration (μg/ml)

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120

B

ZAN

***

100

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80 60

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40 20 0 0

1

2

3

4

5

6

7

8

9 10 11 12 13 14

Day post-infection (dpi)

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CON

60

C

110 100

PBS OSE MMF

Body weight (%)

80

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Infectivity (% of control)

100

Survival rate (%)

Figure 1.

A

90

PBS OSE

80

MMF

70 60 0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15

Day post-infection (dpi)

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0.8 CON

0.6

MMF

***

0.4 0.2

***

0 4

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1.2 1 0.8

0.4 0.2

MMF

***

***

0

1 0.8 0.6 0.4

6

EP

1.2

4 Hours post-infection (hpi)

***

AC C

C

6h MMF; NP M1

CON

0.6

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Relative M1 expression

B

D

6

Hours post-infection (hpi)

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1

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Relative NP expression

1.2

Relative HA expression

Figure 2.

A

0.2

***

0 4 Hours post-infection (hpi)

6

CON MMF

NS1 β-actin

-

8h +

-

+

12 h +

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Figure 3. B ***

1.2

Relative M1 expression

#

1 0.8 0.4 0.2 0 CON

100μM

MMF

1 0.8 0.6 0.4 0.2 0

100μM

50μM

25μM

12.5μM

guanosine MMF + guanosine

CON

MMF

100μM

100μM

50μM

25μM

12.5μM

guanosine MMF + guanosine

D

1.4

#

***

1.2

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C

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1 0.8 0.6 0.4 0.2 0 CON

100μM guanosine

MMF

100μM

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Relative HA expression

1.2

###

***

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0.6

1.4

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1.4

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Relative NP expression

A

50μM

25μM

MMF + guanosine

MMF (µg/mL);

0

0

1

1

1

1

Guanosine (µΜ);

0

100

0

100

50

25

NP M1 NS1

12.5μM

β-actin

1 12.5

B

10

CON

***

MMF

8 6 4 2

*

4

6

8

C

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Hours post-infection (hpi)

IL-1β

12

CON

MMF

*

8 6 4 2

CON MMF

10 8 6 4 2 0 4

AC C

**

6

Hours post-infection (hpi)

4

6

8

Hours post-infection (hpi)

IP-10

D

***

EP

14

10

**

0

0

16

12

IL-6

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12

14

Relative IP-10 expression

14

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Relative IFN-β expression

IFN-β

Relative IL-6 expression

A

Relative IL-1β expression

Figure 4.

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14 12 10

** CON MMF

8 6 4 2

**

*

0 8

4

6 Hours post-infection (hpi)

8

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Supplementary Figure 1.

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140

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100 80 60 40

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Cell Viability (% of untreated)

120

20 0

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CON

0.1

AC C

Concentration of MMF (μg/ml);

25

ACCEPTED MANUSCRIPT Highlights 1. MMF inhibits H5N1 replication in vitro and in vivo. 2. Inhibition of H5N1 replication by MMF mediated through guanosine depletion.

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MMF has immunomodulatory activity during H5N1 infection

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3.

ACCEPTED MANUSCRIPT [Conflict of Interests]

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None of the authors or relevant organizations had any conflicts of interest regarding the generation of data or the subjective materials reported in this manuscript or in their publication.