WO2013158820A1 - Compositions and methods for treating viral diseases - Google Patents

Abstract

The invention relates to compositions and methods for treating viral diseases. Specifically, the invention relates to activating AMP-Activated Kinase (AMPK) or inhibiting fatty acid synthesis to treat diseases caused by or associated with viruses that depend on fatty acid synthesis for their replication.

Description

COMPOSITIONS AND METHODS FOR TREATING VIRAL DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application 61/635,688, filed April 19, 2012, which is incorporated by reference herein in its entirety.

GOVERNMENT INTEREST

[0002] The work described in this application was, in part, supported by the United States Department of Health and Human Services, the National Institutes of Health, Grant Numbers HG000046, R01AI074951, and U54AI057168. United States Government has certain rights in this application.

FIELD OF THE INVENTION

[0003] The invention relates to compositions and methods for treating viral diseases. Specifically, the invention relates to activating AMP-Activated Kinase (AMPK) or inhibiting fatty acid synthesis to treat diseases caused by or associated with viruses that depend on fatty acid synthesis for their replication.

BACKGROUND OF THE INVENTION

[0004] Emerging and re-emerging arthropod-borne viral pathogens have lead to significant world-wide morbidity and mortality in humans and domestic animals, and are of medical and agricultural concern. Bunyaviruses are an important group of insect-borne RNA viruses that include disease causing members such as Sin Nombre, Hantavirus, Crimean-Congo hemorrhagic fever virus, and Rift Valley Fever Virus (RVFV). RVFV is a mosquito borne Category A agent initially endemic to sub-Saharan Africa. However, outbreaks of RVFV have recently occurred in Egypt and the Arabian Peninsula, indicating the potential of this virus to spread to new geographical areas. RVFV has particular importance as an agricultural pathogen, where infection of livestock can lead to significant morbidity and mortality among young animals, and cause catastrophic abortion rates. Most humans infected with RVFV develop self-limited febrile illness, although approximately 1-3% die from the disease due to hemorrhagic symptoms. No effective vaccines or antiviral therapies have yet been developed against RVFV. [0005] All viruses undergo sequential steps to complete their replication cycles. Bunyaviruses and other RNA viruses compartmentalize their RNA replication machinery on cellular membranes. An essential feature of these infections is the ability of viruses to rearrange and proliferate internal cellular membranes into distinct structures compartmentalizing the viral replication complex and supporting viral genome replication. Depending on the virus, these membrane modifications can be derived from distinct cellular sources, including ER, Golgi, endosomal, and mitochondrial membranes, and may have complex biogenesis pathways derived from multiple intracellular origins. Bunyamwera virus, a member of the Bunyavirus family related to RVFV, induces the formation of a new Golgi membrane-derived tubular structure with a globular head that harbors the viral replication complex. Disrupting the formation of this structure is associated with decreased levels of virus replication. While different families of viruses use membranes derived from different cellular sources, and create membranous structures with distinct morphologies, there are some similarities in these structures, suggesting that commonalities exist in the mechanisms by which disparate viruses depend upon lipid metabolism or trafficking. One clear point of overlap includes a requirement for cellular lipid biogenesis pathways and the generation of newly synthesized lipids. Furthermore, enveloped viruses, which include Bunyaviruses, require incorporation of cellular membranes into their lipid envelopes during virus assembly, in a process that may also involve lipid modifications.

[0006] Accordingly, there exists a need to develop compositions to treat diseases caused by viruses that depend on lipid metabolism or fatty acid synthesis for their replication.

SUMMARY OF THE INVENTION

[0007] In one embodiment, the invention provides a method for treating a viral disease in a subject, said disease caused by or associated with a virus that depends on fatty acid synthesis for its replication, the method comprising: administering to said subject a therapeutically effective amount of an AMP-Activated Kinase (AMPK) activator, a fatty acid synthesis inhibitor, or a combination thereof, thereby treating said viral disease in said subject.

[0008] In another embodiment, the invention provides a composition comprising: an AMPK activator, a fatty acid synthesis inhibitor, or a combination thereof, present in an amount effective to treat a viral disease caused by or associated with a virus that depends on fatty acid synthesis for its replication. [0009] In another embodiment, the invention provides a method for treating a Rift Valley Fever Virus (RVFV) infection in a subject, the method comprising: administering to said subject a therapeutically effective amount of an AMPK activator, a fatty acid synthesis inhibitor, or a combination thereof, thereby treating said RVFV infection in said subject.

[00010] In another embodiment, the invention provides a composition comprising: an AMPK activator, a fatty acid synthesis inhibitor, or a combination thereof, present in an amount effective to treat said RVFV infection.

[00011] In another embodiment, the invention provides a method for identifying a molecule to effectively treat a viral disease, said disease caused by or associated with a virus that depends on fatty acid synthesis for its replication, the method comprising: screening a plurality of AMPK activators or a plurality of fatty acid synthesis inhibitors to effectively treat said viral disease, thereby identifying a molecule to effectively treat said viral disease.

[00012] In another embodiment, the invention provides a method for identifying a molecule to effectively treat a viral disease, said disease caused by or associated with a virus that depends on fatty acid synthesis for its replication, the method comprising: testing an AMPK activator or a fatty acid synthesis inhibitor; determining whether said AMPK activator or fatty acid synthesis inhibitor effectively treats said viral disease, thereby identifying a molecule to effectively treat said viral disease.

[00013] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[00014] Figure 1: AMPK restricts RVFV infection. A. Plaque assays were performed on wild type (WT) and ΑΜΡΚα1/ΑΜΡΚα2^{_ ~} MEFs. Representative data from triplicate experiments is shown. B. Quantification of plaques from A. presented as the normalized mean+SD relative to the number of wild type plaques from three experiments. C. The diameter of 30 representative plaques in each duplicate well from A. was used to calculate the average plaque area, displayed as the normalized mean+SD in triplicate experiments. D. WT or ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs were infected with serial dilutions of RVFV, incubated for 16 hours, and processed for immunofluorescence. (RVFV-N, green; nuclei, blue). E. Quantification of D. presented as percent of infected cells. A representative of three experiments is shown. F. One-step growth curve of RVFV in WT or ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs. RVFV grown in WT or ΑΜΡΚα1/ΑΜΡΚα2 ^{_ ~} MEFs for 4, 8, or 12 hours was tittered on BHK cells and is presented as the normalized mean of triplicate experiments +SD. * indicates p<0.05.

[00015] Figure 2: AMPK activation restricts RVFV. A-B. U20S cells were pretreated with 10 mM 2DG, 10 μΜ oligomycin or PBS (untreated) for 1 hour and infected with serial dilutions of RVFV (A) for 10 hours or vaccinia virus (B) for 8 hours and processed for immunofluorescence. Data are displayed as the average percent infection relative to the highest concentration of virus in the untreated control + SD from triplicate experiments. C-D. U20S cells (C) or MEFs (D) were pretreated with 12 mM 2DG, 100 μΜ A769662, or PBS for 1 hour and infected with RVFV (MOI 1) for 10 hours. Infection was measured by immunofluorescence. Data are displayed as the normalized percent infection relative to the untreated control +SD in triplicate experiments; * indicates p<0.05.

[00016] Figure 3: LKB1 restricts RVFV infection: A. RVFV was plaqued on LKB1-/-;LKB1 and LKBl-/-;Vec MEFs. Representative data from triplicate experiments is shown. B. Quantification of plaques from A. presented as the normalized mean+SD of wild type plaques from three experiments. C. The diameter of 30 representative plaques each of three experiments was used to calculate the average plaque area, which is displayed as the normalized mean+SD in triplicate experiments. D. LKB1-/-;LKB1 or LKBl-/-;Vec MEFs were infected with serial dilutions of RVFV, incubated for 16 hours, and processed for immunofluorescence. (RVFV-N green; nuclei blue). A representative of triplicate experiments is shown. E. Quantification of D. presented as RVFV percent infection in LKB1-/-;LKB 1 and LKBl-/-;Vec MEFs. A representative of triplicate experiments is shown. F. Time course of RVFV infection in LKB1-/-;LKB1 and LKBl-/-;Vec MEFs. Cells were infected with RVFV (MOI 1), and fixed at indicated hours post infection. A representative of triplicate experiments is shown. G. LKB1-/-;LKB1 or LKBl-/-;Vec MEFs were pretreated with 100 μΜ A769662 or 10 μg/mL STO609 for 1 hour prior to infection with RVFV (MOI 1) for 10 hours and processed for immunofluorescence. Data are displayed as the average percent infection relative to the LKB1-/-;LKB1 untreated control + SD from triplicate experiments. * indicates p<0.05. [00017] Figure 4: AMPK restricts RVFV RNA replication. A. Northern blot of genomic S segment and N mRNA from RVFV (MOI 1) grown in WT or ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs for 4, 8, or 12 hours. A representative of triplicate experiments is shown. B-C. Quantification of RVFV mRNA (B) or genomic RNA (C) in WT or ΑΜΡΚα1/ΑΜΡΚα2 ^{_ ~} MEFs displayed as the normalized fold change from WT 4 hours. A representative of triplicate experiments is shown. D. RVFV binding assay. RVFV (MOI 10) was bound to WT or ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs at 4°C for 1 hour, then washed, and treated with PBS or trypsin to remove bound virus. qRTPCR was performed on isolated RNA to detect RVFV S genome. Data are displayed as the average AACT of triplicate experiments normalized to GAPDH control. * indicates p<0.05. E. 2DG (12 mM), A769662 (100 μΜ) or Ammonium Chloride (NH₄C1, 12 mM) was added either 1 hour prior to infection with RVFV (MOI 1), with infection, or 1, 2, or 4 hours post infection. After 10 hours of infection cells were fixed and processed for immunofluorescence. Data are displayed as the average percent infection relative to the post entry level of infection (NH₄C1 added at 4 hpi) + SD from triplicate experiments. * indicates p<0.05.

[00018] Figure 5: Acetyl-CoA Carboxylase Activity is Tightly Regulated by AMPK during RVFV Infection. A. Phosphorylation of AMPK and downstream effectors upon RVFV infection. WT MEFs were infected with RVFV (MOI 1) for 4 or 8 hours. Lysates were collected and assayed by immunoblot for phospho-AMPK, phospho-ACC, and phospho-eEF2. Total protein was assayed for each and Tubulin was measured as a loading control.

Representative blot of triplicate experiments is shown. B. Phosphorylation of AMPK and downstream effectors in WT and ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs. Cells were treated with AMPK activators 2DG (12 mM), oligomycin (OM, 10 μΜ), and A769662 (100 μΜ) for 4 hours. Lysates were collected and assayed by immunoblot as above. Representative blot of triplicate experiments shown. C. Phosphorylation of AMPK and ACC upon treatment with UV- inactivated RVFV. WT MEFs were infected with live or UV-inactivated RVFV (MOI 1) for 4 or 8 hours. Lysates were collected and assayed by immunoblot as above. Representative blot of triplicate experiments is shown. D. Blocking fatty acid synthesis inhibits RVFV infection. MEFs were treated with the fatty acid synthase inhibitors Cerulenin (45 pM) and C75 (12.5 μΜ) or the AMPK activator A769662 (100 μΜ), infected with RVFV (MOI 1), and processed for immunofluorescence. Data are displayed as the normalized average percent infection relative to the untreated control + SD in triplicate experiments. * indicates p<0.05. E. WT MEFs were treated with 100 μΜ A769662 for 10 hours and stained for cellular lipids with BODIPY lipophilic fluorescent dye. (BODIPY, red; nuclei, blue). Representative images from triplicate experiments are shown. F. Quantification of E. presented as integrated BODIPY intensity per cell relative to untreated control + SD in triplicate experiments. * indicates p<0.05. G. WT and ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs were grown overnight and stained for cellular lipids with BODIPY lipophilic fluorescent dye. (BODIPY, red; nuclei, blue). Representative images from triplicate experiments are shown. H. Quantification of G. presented as integrated BODIPY intensity per cell relative to WT + SD in triplicate experiments. * indicates p<0.05.

[00019] Figure 6: Addition of palmitate restores RVFV infection in the presence of A769662. A. U20S cells were pretreated with 100 μΜ palmitate overnight and 100 μΜ A769662 or PBS was added 1 hour prior to infection with RVFV (MOI 1). Cells were incubated for 10 hours, and processed for immunofluorescence. (RVFV-N, green; nuclei, blue) B. Quantification of A. Data are displayed as the normalized percent infection relative to the untreated control at MOI 1.25 +SD in triplicate experiments; * indicates p<0.05 compared to untreated vehicle control.

[00020] Figure 7: Additional arboviruses are restricted by AMPK. WT or ΑΜΡΚα1/ΑΜΡΚα2^{" _} MEFs were infected with serial dilutions of KUNV (A), SEW (E), or VSV (I) and processed for immunofluorescence. (Virus, green; nuclei, blue). Quantifications of the percent infection for KUNV (B), SINV (F) and VSV (J) are shown as representatives of triplicate experiments. LKB ^_;LKBl and LKBl ' jVec MEFs were infected with serial dilutions of

KUNV (C), SINV (G), and VSV (K) and processed for immunofluorescence. (Virus, green; nuclei, blue). Quantifications of the percent infection are shown for KUNV (D), SINV (H) and VSV (L) are shown as representatives of triplicate experiments.

[00021] Figure 8: AMPK restricts RVFV. A. Time course of RVFV infection in WT and ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs. Cells were infected with RVFV and fixed at indicated time post infection. (RVFV, green; nuclei, blue) B. Quantification of A. A representative of triplicate experiments is shown.

[00022] Figure 9:. AMPK inhibition leads to increased RVFV infection. A. U20S cells were pretreated with 10 μΜ Compound C or PBS (untreated) for 1 hour and infected with serial dilutions of RVFV for 10 hours and processed for immunofluorescence. Data are displayed as the average percent infection relative to untreated control + SD from triplicate experiments. * indicates p<0.05. B. Cellular Toxicity in response to drug treatment. U20S were pretreated with 10 mM 2DG, 10 μΜ oligomycin, 100 μΜ A769662, 10 μΜ Compound C, 10 μg/mL STO609 or PBS (untreated) for 1 hour, infected with RVFV, and processed for immunofluorescence 10 hpi. Cell nuclei were counted using automated microscopy as a measure of cytotoxicity. Data are displayed as the average number of nuclei relative the untreated control + SD from triplicate experiments.

[00023] Figure 10: Dose-dependent inhibition of RVFV infection.. U20S cells were pretreated with serial dilutions of A769662 (A), 2DG (B), or STO609 (C) prior to infection with RVFV (MOI 1), and processed for immunofluorescence 10 hpi. Data are displayed as the average percent infection relative to the 0 drug control + SD from triplicate experiments. * indicates p<0.05.

[00024] Figure 11: A769662 activates AMPK to restrict infection. A. WT and ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs were pretreated with 100 μΜ A769662 or PBS (untreated) for 1 hour, then infected with RVFV (MOI 1) for 10 hours and processed for immunofluorescence. Data are displayed as the average percent infection relative to the WT untreated control + SD from triplicate experiments. * indicates p<0.05. B. Cell numbers from (A) as a measure of cell toxicity. Data are displayed as the average number of nuclei relative to the untreated + SD from triplicate experiments.

[00025] Figure 12: Cellular ATP content is unchanged during RVFV infection. WT MEFs were treated with 2DG (12 mM), A769662 (μΜ), or infected with RVFV at MOI 2.5 or 12, spun at 1200 rpm for 1 hour, and incubated for 4 hours. ATP concentration was measured by luminescence. Data are displayed as average RLU relative to untreated control +SD from triplicate experiments. * indicates p<0.05.

[00026] Figure 13: AMPK's role in the type I interferon response. A-B. WT and ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs were infected with RVFV for 10 hours. Expression of IFN (A) and OAS 1 (B) were measured by qRT-PCR. Data are representatives of duplicate experiments. C. WT MEFs were treated with IFN for 15 minutes or 4 hours, lysed, and assayed by immunoblot for phospho-AMPK and phospho-ACC. Total AMPK and tubulin were assayed. A representative of triplicate experiments is shown. D. Quantification of C. using Image J software.

[00027] Figure 14: Quantification of Immunoblots using Image J software. A-D. Phosphorylation of AMPK and downstream effectors upon RVFV infection. WT MEFs were infected with RVFV (MOI 1) for 4 or 8 hours. Lysates were collected, assayed by immunoblot and quantified for phospho-AMPK (A), phospho-ACC2 (B), phospho-ACCl (C), and phospho-eEF2 (D) normalizing to the tubulin loading control. Data are displayed as the average density relative to untreated at 4 hours from triplicate experiments. E-H. Phosphorylation of AMPK and downstream effectors in WT and ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs. Cells were treated with AMPK activators 2DG (12 mM), oligomycin (OM, 10 μΜ), and A769662 (100 μΜ) for 4 hours. Lysates were collected, assayed by immunoblot, and quantified as above for phospho-AMPK (E), phospho-ACC2 (F), phospho-ACCl (G), and phospho-eEF2 (H) normalized to the tubulin loading control. Data are displayed as the average density relative to untreated at 4 hours from triplicate experiments.

[00028] Figure 15: UV-inactivated RVFV is replication incompetent. U20S cells were infected with live (MOI 1) and UV-inactivated virus (equivalent volume to MOI 1) for 10 hours, and processed for immunofluorescence. (RVFV-N, green; nuclei, blue).

[00029] Figure 16: AMPK is not activated by RVFV in LKB1 null MEFs.LKBl -/-;LKB1 and LKBl-/-;Vec MEFs were infected with RVFV (MOI 1) for 4 hours. Lysates were collected and assayed by immunoblot for phospho-AMPK. Total AMPK and tubulin were assayed. Representative blot of duplicate experiments is shown.

[00030] Figure 17: A: mTORCl is not required for AMPK-mediated restriction of RVFV. WT and ΑΜΡΚα1/ΑΜΡΚα2 ^{_ ~} MEFs were pretreated with 10 nM Rapamycin or PBS for 1 hour and infected with RVFV (MOI 1) for 10 hours and processed for immunofluorescence. A representative of duplicate experiments is shown. B. Autophagy does not restrict RVFV. RVFV was plaqued in MEFs expressing a control hairpin RNA or a hairpin against Atg5. C. Atg5 mRNA expression by qRT-PCR in MEFs expressing a control hairpin RNA or a hairpin against Atg5 normalized to GAPDH.

[00031] Figure 18: Palmitate treatment does not inhibit AMPK activation or signaling. U20S cells were treated with palmitate overnight, then treated with 2DG (12 mM) and A769662 (100 μΜ) for 10 hours. Lysates were collected and assayed by immunoblot for phospho- AMPK, and phospho-ACC. Total AMPK, ACC and tubulin were assayed. Representative blot of duplicate experiments is shown.

[00032] Figure 19: Addition of palmitate partially restores KUNV infection in the presence of A769662. A. U20S cells were pretreated with 100 μΜ palmitate and 100 μΜ A769662 or PBS 1 hour prior to infection with KUNV (MOI 1). Cells were incubated for 16 hours, and processed for immunofluorescence. (KUNV-Nsl, green; nuclei, blue) B. Quantification of A.

Data are displayed as the normalized percent infection relative to the non-A769662 treated control +SD in triplicate experiments; * indicates p<0.05. C. Quantification of non-drug treated samples in (A). Palmitate treatment inhibited KUNV infection. Data are displayed as the normalized percent infection relative to the untreated vehicle control +SD in triplicate experiments; * indicates p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

[00033] The invention relates to compositions and methods for treating viral diseases. Specifically, the invention relates to activating AMPK or inhibiting fatty acid synthesis to treat diseases caused by or associated with viruses that depend on fatty acid synthesis for their replication.

[00034] In one embodiment, provided herein is a method for treating a viral disease in a subject, said disease caused by or associated with a virus that depends on fatty acid synthesis for its replication, the method comprising: activating AMPK or inhibiting fatty acid synthesis or combination thereof.

[00035] AMPK can be activated by administering to a subject a therapeutically effective amount of an AMPK activator, known to one of skilled in the art. Fatty acid synthesis can also be inhibited by administering to a subject a therapeutically effective amount of a fatty acid synthesis inhibitor, known to one of skilled in the art.

[00036] Surprisingly and expectedly, the inventors of the instant application found that RNA viruses' dependence on fatty acid biosynthesis for their replication is a process that is tightly regulated by the energy sensor AMPK.

[00037] Specifically, the inventors of the instant application found that AMPK is antiviral against Rift Valley Fever Virus (RVFV), and this restriction is dependent on the upstream activator Liver Kinase Bl (LKB1). Furthermore, pharmacological activation of AMPK inhibited viral infection. AMPK was activated by RVFV infection, and in particular striking changes in Acetyl-CoA-Carboxylase (ACC) activity dependent on AMPK were observed, leading to discover that AMPK is antiviral through its role in fatty acid metabolism. Cells lacking AMPK had increased global lipid levels, while pharmacological activation of AMPK led to decreased cellular lipids, consistent with AMPK control of lipid availability as a restriction point for viral replication. Importantly, the inventors could bypass the antiviral effects of AMPK by feeding cells palmitate, the first fatty acid produced downstream of ACC. Since palmitate treatment restored RVFV infection, the inventors demonstrate that AMPK specifically restricts infection through its role in inhibiting fatty acid biosynthesis. Since many viruses are dependent upon fatty acid biosynthesis for their replication, the inventors tested whether AMPK restricted additional RNA viruses. Surprisingly and expectedly, the inventors found that AMPK has antiviral activity against multiple arboviruses from disparate families including: the Flavivirus Kunjin virus, the Togavirus Sindbis virus, and the Rhabdovirus Vesicular stomatitis virus. Taken together, data clearly show that AMPK activation is broadly anti- viral, and can provide for novel antiviral therapeutics.

[00038] As used herein, "AMPK activator" may refer to any molecule that is able to directly or indirectly activate AMP- Activated Kinase.

[00039] AMPK activators are well known in the art and fully described in US Patent Application Publications US 20110319497, US 20070244202, US 20110034505, US 20100221748, US 20100047177, US 20090203638, and US 20090042810, and PCT Patent Application Publications WO2008083124, WO2009132136, WO2009076631, WO2009135580, WO/2008/006432, WO2007005785, and WO2007002461, all of which are incorporated by reference herein in their entirety.

[00040] AMPK is a key target molecule to treat metabolic syndromes such as diabetes. In fact, an AMPK-activator, Metformin has been used as anti-type 2 diabetes drugs for more than 50 years. Metformin is one of the most popular anti-diabetic drugs in the United States and one of the most prescribed drugs overall, with nearly 35 million prescriptions filled in 2006 for generic Metformin alone.

[00041] Other known activators of AMPK that could be used with the methods and compositions of the present invention include, but are not limited to: 5-aminoimidazole-4- carboxamide-l-beta-D-ribofuranoside (AICAR), Resveratrol, and Thiazolidinedione. In the cell, AICAR is converted to ZMP, an AMP analog that has been shown to activate AMPK. Resveratrol increases the activity of SIRT1 and animal life span, and it also increases AMPK activity by SIRTl independent mechanism. Thiazolidinedione (TZD), a PPAR gamma activator, which activates AMPK in PPAR gamma-independent manner.

[00042] Additional examples of AMPK activator include, but are not limited to a biguanide (e.g., phenformin), N-substituted-heterocycloalkyloxybenzamide compounds, Carboxamide compounds, Sulfonamide and amine compounds, Thienopyridone derivatives, Imidazole derivatives, Thiazoles derivatives, and 3,4-substituted thiazoles.

[00043] In one embodiment, an AMPK activator is a direct AMPK activator (e.g., AICAR). In another embodiment, an AMPK activator is an indirect AMPK activator (e.g., phenformin). Activators of AMPK include small or large molecule activators.

[00044] The nucleotide sequence of the catalytic domain (alpha 1) of human AMPK has the nucleotide sequence set forth in GenBank Accession No. NM_206907 and encodes a protein having the amino acid sequence set forth in GenBank Accession No. NP_996790. The nucleotide sequence of the non-catalytic domain (beta 1) of human AMPK has the nucleotide sequence set forth in GenBank Accession No. NM_006253 and encodes a protein having the amino acid sequence set forth in GenBank Accession No. NP_006244. The nucleotide sequence of the non-catalytic domain (gamma 1) of human AMPK has the nucleotide sequence set forth in GenBank Accession No. NM_212461 and encodes a protein having the amino acid sequence sets forth in GenBank Accession No. NP_997626. The nucleic acid sequence or amino acid sequence described herein includes a homologue, a variant, an isomer, or a functional fragment thereof. Each possibility is a separate embodiment of the invention.

[00045] Fatty acid synthesis inhibitors are also well known in the art and fully described in US Patent Application Publication US 20100022630 which is incorporated by reference herein in their entirety. In one embodiment, fatty acid synthesis inhibitor is lipid lowering drug.

[00046] Methods for making AMPK activators and fatty acid inhibitors are also well known in the art. Any suitable method, known to a person of skilled in the art, can be used to make AMPK activators and fatty acid inhibitors.

[00047] Viral disease treated by the invention is a disease caused by or associated with a virus that depends on fatty acid synthesis for its replication. In one embodiment, a virus that depends on fatty acid synthesis for its replication is a Bunyavirus. Examples of a Bunyavirus, include, for example, but are not limited to, Sin Nombre, Hantavirus, Crimean-Congo hemorrhagic fever virus, and Rift Valley Fever Virus (RVFV).

[00048] A dependence on fatty acid synthesis or lipid biosynthesis and virally induced membrane modifications is not unique to Bunyaviruses; many RNA viruses require extensive membrane modifications and proliferations to support their replication complex. These RNA viruses are well known in the art.

[00049] Additional examples of a virus that depends on fatty acid synthesis for its replication include, for example, but are not limited to, Flavivirus Kunjin virus (KUNV), the Togavirus Sindbis virus (SINV), and the Rhabdovirus Vesicular stomatitis virus (VSV).

[00050] As used herein, the terms "treat" and "treatment" refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e. , where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented. In some embodiments, treatment may refer to preventing a viral infection.

[00051] In one embodiment, provided herein is a composition comprising an AMP-Activated Kinase (AMPK) activator, a fatty acid synthesis inhibitor, or a combination thereof, present in an amount effective to treat a viral disease caused by a virus that depends on fatty acid synthesis for its replication.

[00052] In another embodiment, provided herein is a pharmaceutical composition comprising an AMP-Activated Kinase (AMPK) activator, a fatty acid synthesis inhibitor, or a combination thereof.

[00053] The invention also provides a pharmaceutical composition comprising small molecule, antibody, nucleic acid, peptide, vector, host cell, or other agents of this invention and one or more pharmaceutically acceptable carriers. "Pharmaceutically acceptable carriers" include any excipient which is nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. The pharmaceutical composition may include one or additional therapeutic agents.

[00054] Pharmaceutically acceptable carriers include solvents, dispersion media, buffers, coatings, antibacterial and antifungal agents, wetting agents, preservatives, buggers, chelating agents, antioxidants, isotonic agents and absorption delaying agents.

[00055] Pharmaceutically acceptable carriers include water; saline; phosphate buffered saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphate, citrate and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; EDTA; salt forming counterions such as sodium; and/or nonionic surfactants such as

TWEEN, polyethylene glycol (PEG), and PLURONICS; isotonic agents such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride; as well as combinations thereof. Antibacterial and antifungal agents include parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.

[00056] The pharmaceutical compositions of the invention may be formulated in a variety of ways, including for example, solid, semi-solid (e.g., cream, ointment, and gel), and liquid dosage forms, such as liquid solutions (e.g. , topical lotion or spray), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. In some embodiments, the compositions are in the form of injectable or infusible solutions. The composition is in a form suitable for oral, intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, or topical administration. The composition may be formulated as an immediate, controlled, extended or delayed release composition.

[00057] More particularly, pharmaceutical compositions suitable for use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. , glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed. (1980).

[00058] In some embodiments, the composition includes isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00059] Sterile solutions can be prepared by incorporating the molecule, by itself or in combination with other active agents, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, one method of preparation is vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in US Appl. Publ. No. 2002/0102208 Al, which is incorporated herein by reference in its entirety. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from viral disease or disorders.

[00060] Effective doses of the compositions of the present invention, for treatment of conditions or diseases as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but non-human mammals (e.g., domestic animals) can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[00061] The pharmaceutical compositions of the invention may include a "therapeutically effective amount." A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.

[00062] The invention further provides a kit comprising a therapeutically effective amount of an AMPK activator or fatty acid synthesis inhibitor or both.

[00063] AMPK activator or fatty acid synthesis inhibitor may be administered alone, or in combination with one or more other therapeutically effective agents. In one embodiment, AMPK activator may be administered alone, or in combination with one or more therapeutically effective agents or treatments. In another embodiment, fatty acid synthesis inhibitor may be administered alone, or in combination with one or more therapeutically effective agents or treatments. The other therapeutically effective agent may be conjugated to

AMPK activator and/or fatty acid synthesis inhibitor, incorporated into the same composition as AMPK activator and/or fatty acid synthesis inhibitor, or may be administered as a separate composition. The other therapeutically agent or treatment may be administered prior to, during and/or after the administration of AMPK activator and/or fatty acid synthesis inhibitor.

[00064] In one embodiment, AMPK activator is co-administered with fatty acid synthesis inhibitor. In another embodiment, AMPK activator is administered independently from the administration of fatty acid synthesis inhibitor. In one embodiment, AMPK activator is administered first, followed by the administration of fatty acid synthesis inhibitor. In another embodiment, fatty acid synthesis inhibitor is administered first, followed by the administration of AMPK activator.

[00065] The administration of the AMPK activator and/or fatty acid synthesis inhibitor with other agents and/or treatments may occur simultaneously, or separately, via the same or different route, at the same or different times. Dosage regimens may be adjusted to provide the optimum desired response (e.g. , a therapeutic or prophylactic response).

[00066] In one example, a single bolus may be administered. In another example, several divided doses may be administered over time. In yet another example, a dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for treating mammalian subjects. Each unit may contain a predetermined quantity of active compound calculated to produce a desired therapeutic effect. In some embodiments, the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved.

[00067] The composition of the invention may be administered only once, or it may be administered multiple times. For multiple dosages, the composition may be, for example, administered three times a day, twice a day, once a day, once every two days, twice a week, weekly, once every two weeks, or monthly.

[00068] It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[00069] "Administration" to a subject is not limited to any particular delivery system and may include, without limitation, oral (for example, in capsules, suspensions or tablets), parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection), rectal, topical, and transdermal. Administration to a host may occur in a single dose or in repeat administrations, and in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition (described earlier). Once again, physiologically acceptable salt forms and standard pharmaceutical formulation techniques are well known to persons skilled in the art (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co.).

[00070] Another aspect of the invention is a method for identifying a molecule to effectively treat a viral disease, said disease caused by a virus that depends on fatty acid synthesis for its replication, the method comprising: screening a plurality of AMP-Activated Kinase (AMPK) activators or a plurality of fatty acid synthesis inhibitors to effectively treat said viral disease, thereby identifying a molecule to effectively treat said viral disease. Screening assays are well known in the art. Any suitable screening assay known to one of skilled in the art can be used. Yet another aspect of the invention is a method for identifying a molecule to effectively treat a viral disease, said disease caused by a virus that depends on fatty acid synthesis for its replication, the method comprising: testing a AMP-Activated Kinase (AMPK) activator or a fatty acid synthesis inhibitor; determining whether said AMPK activator or fatty acid synthesis inhibitor effectively treats said viral disease, thereby identifying a molecule to effectively treat said viral disease.

[00071] The term "subject," as used herein, may refer to any mammal, for example, a human or an animal (e.g., domestic animal).

[00072] Any patent, patent application publication, or scientific publication, cited herein, is incorporated by reference herein in its entirety.

[00073] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. EXAMPLES

MATERIALS AND METHODS

Cells, Antibodies, Reagents, and Viruses

[00074] MEFs, BHK and U20S cells were maintained at 37°C in DMEM supplemented with 10% FBS (Sigma), 100 μg/mL penicillin/streptomycin, 2 mM L-glutamine, and lOmM Hepes. LKB1^{_ "} MEFs were complemented with MIGR (Vector) or FLAG-LKB 1 -MIGR (LKB1 cDNA) retrovirus and sorted on GFP+ cells by FACS as previously described. Rift Valley fever virus MP- 12 was grown in Vero-E6 cells supplemented with 10% FBS. RVFV was UV- inactivated in a Stratalinker. KUNV was grown in BHK cells. VSV-GFP was grown in BHK cells as described. SINV-GFP virus was grown in C636 cells. All viruses were tittered by plaque assay in BHK cells. Antibodies were obtained from the following sources: anti-RVFV ID8 (gift from C. Schmaljohn USAMRIID), anti-KUNV 9NS1, anti-tubulin (Sigma), and anti- P-AMPK, t-AMPK, P-ACC, t-ACC, P-eEF2, t-eEF2 (Cell Signaling Technology). Fluorescently labeled secondary antibodies and BODIPY-TR were obtained from Invitrogen. HRP-conjugated antibodies were obtained from Amersham. A769662 was obtained from Santa Cruz. Other chemicals were obtained from Sigma.

Plaque Assay

[00075] Viruses were plaqued on MEFs as indicated. Confluent monolayers were treated with serial dilutions of virus for two hours, after which the viral inoculums were removed, and cells were overlayed with 0.75% agarose in MEM, and incubated at 37°C for 48 hours. Cells were fixed in 10% formaldehyde, and stained with crystal violet. Plaque number was determined manually, and plaque diameter was measured using MetaXpress software and used to calculate areas.

Viral Infections and Immunofluorescence

[00076] For all infections, washes and media changes were performed in the control untreated wells, as well as those infected with virus. Viral immunofluorescence experiments were performed in 96 well plates as previously described. Briefly, cells were grown overnight in 96 wells plates, media was removed and fresh media was added. When appropriate, drug was added at the indicated concentration in 5 μΐ PBS, and cells were incubated at 37 °C for 1 hour before addition of virus. Cells were infected with the indicated MOI of virus in complete media and spinoculated for 1 hour at 1200 RPM, and incubated at 37 °C. Cells were fixed and processed for immunofluorescence as previously described 10 hours post infection for RVFV, SINV, and VSV, and 24 hours post infection for KUNV unless otherwise indicated. Briefly, cells were fixed in 4% formaldehyde/PBS, washed twice in PBS/0.1% TritonX-100 (PBST), and blocked in 2% BSA/PBST. Primary antibodies were diluted in block, added to cells, and incubated overnight at 4°C. RVFV was stained with anti-RVFV ID8; KUNV was stained with anti-KUNV 9NS1. VSV and SINV expressed GFP, and did not require antibody staining. Cells were washed three times in PBST, and incubated in secondary antibody with Hoescht33342 (Sigma) counterstain for one hour at room temperature. Plates were imaged at 10X using an automated microscope (ImageXpress Micro), capturing four images per well per wavelength, and quantification was performed using MetaXpress image analysis software. Significance was determined using a Student's T-test. For immunofluorescence assays, a minimum of three wells per condition was imaged, with four images taken per well. To control for variability in baseline level of infection, a Student' s T-test was performed on both the raw percent infection data in each individual experiment, and across a minimum of three replicate experiments where the untreated control had been normalized. Significance was determined if p<0.05 in all tests.

One step Growth Curve

[00077] MEFs were infected with RVFV MOI 1 in 6 well dishes and incubated at 37 °C. Two hours post infection, inoculums was removed, and fresh medium was added. At indicated time point, medium was removed from infected cells and tittered on BHK cells by plaque assay.

RVFV Binding Assay

[00078] MEFs were grown overnight in a 6 well dish. Medium was replaced with 1 mL of fresh complete medium and cells were chilled to 4°C for 10 minutes. RVFV (MOI 10) was added on ice, and cells were incubated at 4°C for 1 hour to allow virus binding. Cells were washed in PBS, then treated with either PBS or 0.25% trypsin to remove bound virus as previously described. Cells were pelleted, then washed again, and lysed in Trizol to extract total RNA. Samples were then prepared for quantitative RT-PCR. cDNA was prepared from total RNA using M-MLV reverse transcriptase (Invitrogen) random primers, and transcripts were amplified by quantitative PCR. AACT was calculated for RVFV S segment using GAPDH as a cellular loading control.

Time of Addition Assay [00079] Time of addition experiments were performed as previously described. U20S cells were grown overnight, and the media was replaced. Cells were infected with RVFV (MOI 1), spun at 1200 rpm for 1 hour, and subsequently incubated at 37°C. 12mM 2DG, 200 μΜ A769662, or 12mM Ammonium Chloride were added either 1 hour prior to infection (-1), with infection (0), or 1, 2 or 4 hours after infection. 10 hours post infection cells were fixed in 4% formaldehyde in PBS and processed for immunofluorescence. Significance was determined using a Student's T test.

Immunoblotting and Northern blotting

[00080] MEFs were infected with RVFV MOI 1 in 6 well dishes (-50% infection) and incubated at 37 °C for indicated time point. For protein analysis, cells were washed briefly in cold PBS and lysed in NP40 lysis buffer supplemented with protease (Boehringer) and phosphatase (Sigma) inhibitor cocktails. Samples were separated by SDS-PAGE and blotted as described. HRP-conjugated secondary antibodies and Western Lightening Chemiluminescence Reagent were used for visualization. To analyze downstream effectors of AMPK, MEFs were treated with 12 mM 2DG, 10 μΜ oligomycin, or ΙΟΟμΜ A769662 for 4 hours, lysed and blotted as above.

[00081] For RNA analysis, cells were lysed in Trizol buffer, and RNA was purified as previously described. To detect viral mRNA, total RNA from infected cells was separated on a 1% agarose/formaldehyde gel and blotted with the indicated probes as previously described. Samples were quantified and normalized against controls using ImageQuant software.

Cellular Lipid Staining

[00082] Cellular lipids were stained as previously described. MEFs were grown to confluence overnight, and then treated with PBS vehicle or ΙΟΟμΜ A769662 for 10 hours. Cells were fixed in 4% formaldehyde for 10 minutes and washed three times in PBS. Staining was performed with 10 μg/mL BODIPY-TR and counterstained with Hoescht33342 in 100 mM glycine in PBS overnight. Cells were washed three times in PBS and imaged using the ImageXpress Micro automated microscope. Integrated intensity of BODIPY signal per cell area was calculated using MetaXpress image analysis software. Significance was determined using a Student's T test.

Fatty Acid Synthesis Bypass Assay

[00083] Exogenous palmitate addition was performed as previously described. Delipidated Fetal Calf Serum and Albumin-bound palmitate were prepared as described and obtained as a kind gift from Robert Rawson. U20S cells were set up on day 0 in 96 well plates and grown over night in normal growth medium. On day 1 medium was removed and cells were washed briefly in PBS. Cells were treated with low glucose DMEM supplemented with 5% delipidated Fetal Calf Serum with or without 100 μΜ Albumin-bound palmitate, and incubated overnight. On day 2 cells were treated with 100 μΜ A769662 or PBS vehicle for 1 hour, and infected with RVFV for 10 hours. Cells were fixed, processed for immunofluorescence, and imaged at 10 X using the automated microscope ImageXpress Micro, as described above. Quantification was performed using MetaXpress image analysis software. Significance was determined using a Student's T-test.

ATP Assay

[00084] MEFs were infected with RVFV in white 96 well plates at MOI 2.5 or 12 (to infect 50 or 100% of cells, respectively), or treated with 12mM 2DG or 100 μΜ A769662, spun for 1 hour, and incubated at 37°C for 4 hours. ATP content was measured by luminescence with Cell Titer Glo reagent (Promega) according to the manufacturer's instructions.

IFN Assay

[00085] MEFs in 6 well dishes were treated with 10 U/mL interferon beta overnight, then infected with RVFV MOI 1 (sufficient for 50% infection) for 10 hours. Cells were lysed in Trizol and total RNA was extracted. Samples were then prepared for quantitative RT-PCR. cDNA was prepared from total RNA using M-MLV reverse transcriptase (Invitrogen) random primers, and transcripts were amplified by quantitative PCR. AACT was calculated for IFN and OAS 1 using GAPDH as a cellular loading control.

EXAMPLE 1

AMPK Restricts RVFV Infection

[00086] The cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. In particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. We found that the energy regulator AMPK, which potently inhibits fatty acid synthesis, restricts infection of the Bunyavirus, Rift Valley Fever Virus (RVFV), an important re- emerging arthropod-borne human pathogen for which there are no effective vaccines or therapeutics. We show restriction of RVFV both by AMPK and its upstream activator LKB1, indicating an antiviral role for this signaling pathway. Furthermore, we found that AMPK is activated during RVFV infection, leading to the phosphorylation and inhibition of acetyl-CoA carboxylase, the first rate-limiting enzyme in fatty acid synthesis. Activating AMPK pharmacologically both restricted infection and reduced lipid levels. This restriction could be bypassed by treatment with the fatty acid palmitate, demonstrating that AMPK restricts RVFV infection through its inhibition of fatty acid biosynthesis. Lastly, we found that this pathway plays a broad role in antiviral defense since additional viruses from disparate families were also restricted by AMPK and LKB l. Therefore, AMPK is an important component of the cell intrinsic immune response that restricts infection through a novel mechanism involving the inhibition of fatty acid metabolism.

[00087] For our studies we used the lab adapted strain MP 12 that has 11 amino acid differences from the wild type strain, since the wild type strain must be used in high containment facilities. In order to test the role of AMPK in RVFV infection, we took advantage of mouse embryonic fibroblasts (MEF) that are genetically altered and null for both of the catalytic a subunits, AMPKal and ΑΜΡΚα2 (ΑΜΡΚα1/ΑΜΡΚα2^{" _}). We challenged either the ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs or their sibling control wild type MEFs with RVFV and measured infection by plaque assay (Figure 1A). We found an increase in titer from 5X10⁵ pfu/mL to 3xl0⁶ pfu/mL, indicating a 6-fold increase in the number of plaques formed in ΑΜΡΚα1/ΑΜΡΚα2^{_ ~} MEFs compared to wild type (Figure IB), concomitant with a 4-fold increase in average plaque area in ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs (Figure 1C). Moreover, RVFV infection was also increased in ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs as measured by an immunofluorescence assay that detects production of the RVFV N protein produced during viral replication (Figure ID, quantified in Figure IE), indicating that RVFV is able to infect and spread more efficiently in the absence of AMPK. Consistent with a role for AMPK both in early events during viral replication and in spread as measured by plaque assay (Figure 1A), we observed an increase in viral infection at early time points before virus spread, as well as increased spread in cells lacking AMPK by monitoring the production of RVFV N protein over time by microscopy (Figure 8A-B).

[00088] This increased spread, indicated by the increase in plaque size (Figure 1C), as well as the immunofluorescence assay (Figure 8A-B), could result from increased production of infectious virus or increased infectivity of the virions produced in cells lacking AMPK. We measured the amount of infectious virus produced in wild type and ΑΜΡΚα1/ΑΜΡΚα2^{_ ~} MEFs over time in a one-step growth curve. Medium from infected cells was collected at various times after infection, and virus was tittered on wild type BHK cells. Little virus (less than lxl0⁴ pfu/mL) was detected at 2-4 hpi, indicating that input virus was not detected in this assay (Figure IF). Virus release began at 8 hpi, where we already observed an 8-fold increase in titer in the AMPK deficient MEFs (1.6xl0⁵ pfu/mL versus 1.3xl0⁶) (Figure IF). This increase in titer was also observed at 12 hpi. Therefore, the increase in RVFV spread is due to increased virus production in ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs.

EXAMPLE 2

AMPK Activation Restricts RVFV

[00089] AMPK is activated through phosphorylation of a threonine residue on the catalytic alpha subunit. Since AMPK deficiency increased RVFV infection, we hypothesized that AMPK activation would inhibit infection. Therefore, we tested whether RVFV was sensitive to pharmacological treatments that activate AMPK. First, we tested drugs that activate AMPK by reducing the levels of cellular energy using an independent cell line, the human osteosarcoma cell line (U20S). We tested the glucose analog 2-deoxyglucose (2DG), and the ATP synthase inhibitor oligomycin, and found that both treatments significantly decreased infection by RVFV compared to vehicle controls (Figure 2A). In contrast, the AMPK inhibitor Compound C significantly, albeit modestly, increased RVFV infection (Figure 9A). Since 2DG and oligomycin activate AMPK indirectly by reducing cellular energy levels, and thus likely have other effects that may also contribute to viral infection, we tested whether these treatments affected vaccinia virus infection, which is not restricted by AMPK, but rather requires AMPK, independent of the energy sensing pathway for efficient viral infection. Vaccinia virus infection was not affected by these treatments (Figure 2B), indicating that the compound-treated cells remain healthy enough to support viral infection, and the reduced infection levels were specific to RVFV. Moreover, we found that none of these drug treatments reduced cell number by greater that 20%, and therefore were not cytotoxic (Figure 9B).

[00090] Next, we took advantage of a recently developed thienopyridone compound A769662 that activates AMPK directly, independently of the energy status of the cell. This drug mimics both allosteric activation of AMPK and inhibition of dephosphorylation without affecting binding of AMP to the gamma subunit. We found that RVFV infection of U20S cells was significantly reduced in the presence of this compound (Figure 2C), and that both 2DG and

A769662 inhibit RVFV in a dose-dependent manner (Figure 10A-B), indicating that AMPK activation restricts RVFV infection independently of the pleiotropic effects of reduced cellular energy levels. Moreover, we also found that the AMPK activating drugs 2DG and A769662 significantly inhibit RVFV infection in MEFs (Figure 2D). To determine if the effects of these drugs was specific for AMPK we treated ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs with the direct AMPK activator A769662. Treatment with this drug inhibited RVFV less than 2 fold in ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs and was not significant, whereas infection was inhibited greater than 5-fold in the wild type cells (Figure 11 A) with no toxicity in either cell type (Figure 11B), indicating that the major action of this drug was through AMPK as previously published. Taken together, these studies show that AMPK activation has antiviral activity against RVFV in multiple cell types.

EXAMPLE 3

LKB1 Restricts RVFV Infection

[00091] Since pharmacological activation of AMPK restricted RVFV infection, we were interested in investigating which pathway upstream of AMPK was responsible for this restriction. The classic activator of AMPK is the tumor suppressor LKB1, which phosphorylates AMPK in response to a variety of stimuli that cause a reduction in cellular energy levels, such as glucose starvation or hypoxia. In order to determine if LKB1 signaling was important for AMPK-mediated RVFV restriction, we tested whether LKB 1 also restricted RVFV. We challenged MEFs that are null for LKB1 and complemented with either vector alone (LKBl^{" _}; Vec), or an LKB1 cDNA (LKBl^{" _}; LKB1) and found increased RVFV infection in MEFs lacking LKB1 by plaque assay (Figure 3A). Quantification revealed a 2- fold increase in the number of plaques (increase in average virus titer from 7.8xl0⁵ to 1.5xl0⁶ pfu/mL in LKB1 null MEFs) (Figure 3B) and a 5-fold increase in plaque area in LKBl^{" _}; Vec MEFs compared to MEFs complemented with LKB1 (Figure 3C). Moreover, we observed increased infection in the LKB l^"'^"; Vec MEFs compared to those complemented with LKB1 by immunofluorescence (Figure 3D, quantified in 3E). Finally, we measured RVFV infection over time in cells lacking LKB 1 and found increased infection in the absence of LKB 1 at early and late times after infection, indicating increased initial infection as well as spread (Figure 3F). Since AMPK activation downstream of LKB1 is dependent on a decrease in cellular energy, we measured cellular ATP levels during RVFV infection using a luciferase assay.

While 2DG significantly reduced cellular ATP levels, neither A769662 nor RVFV had any impact on ATP levels as measured by this assay (Figure 12). While infection with RVFV did not induce global changes in cellular ATP, this does not rule out localized changes in cellular energy that could influence AMPK.

[00092] In addition to LKB1 other upstream activators of AMPK have been identified. Notably, calcium-calmodulin kinase kinase (CaMKK) has been shown to activate AMPK in response to an increase in intercellular calcium. Since LKB1 did not restrict RVFV as strongly as AMPK did (Figure 3), we investigated if other upstream activators, such as CaMKK could also contribute to RVFV restriction. To this end, we treated U20S cells with the CaMKK inhibitor STO609 prior to infection, and found no increase in RVFV infection in response to this drug, although at very high concentrations there was a decrease in infection (Figure IOC). This decrease was likely due to additional kinases that are inhibited at these concentrations. This finding is consistent with previous reports that changes in intercellular calcium levels are not induced by RVFV infection. We next investigated if LKB1 and CaMKK function redundantly to restrict RVFV infection. We tested whether simultaneously inhibiting both LKB 1 and CaMKK would lead to a greater increase in RVFV infection than LKB 1 deficiency alone. To this end, prior to infection, we treated LKB1 null MEFs or those complemented with LKB1 with STO609 and monitored RVFV infection. Consistent with our previous findings, we observed a 3-fold increase in the percentage of infected cells in LKB1 null cells compared to those complemented with LKBl; however pretreatment with STO609 had no effect on infection level in either cell type (Figure 3G). In contrast, we found that pretreatment with the AMPK activating compound A769662 significantly inhibited RVFV in both LKBl null and complemented MEFs (Figure 3G). Taken together, these data show that LKBl is the major upstream activator responsible for AMPK-mediated restriction of RVFV.

EXAMPLE 4

AMPK restricts RVFV RNA replication

[00093] To dissect the mechanism by which AMPK restricts RVFV infection, we first determined which early step in the viral replication cycle is restricted by AMPK. We observed decreased protein production, as measured by immunofluorescence (Figure 1D-E and Figure 8) in addition to decreased production of infectious progeny virus (Figure IF) in the presence of AMPK. This indicates that AMPK may inhibit a step in the viral replication cycle at, or prior to, protein production. To determine if viral RNA replication was affected by AMPK, we monitored both viral genomic RNA replication and viral mRNA production in the presence or absence of AMPK. We found an increase in both viral mRNA (N) and genomic RNA (S segment) in AMPK deficient MEFs both early in infection and upon virus spread (Figure 4A-C). At 4 hpi, a time point prior to RVFV release, we observed a 3-fold increase in viral mRNA production in AMPK deficient MEFs compared to wild type, which continued to increase over time (Figure 4A-B). Likewise, genomic RNA production was increased prior to virus release and spread (Figure 4A and C). These data show that the increased N protein production observed by immunofluorescence at early time points (Figure 8A) may be due to increased N mRNA production.

[00094] Next, we investigated whether entry, a step upstream of RNA replication, was inhibited by AMPK. First, we tested whether RVFV binding was more efficient in the absence of AMPK. To this end, MEFs were pre-bound with RVFV for an hour at 4°C, unbound virus was removed and RVFV binding was measured by quantitative RT-PCR to detect genomic RVFV S segment within virions. We observed no difference in virus binding in wild type or AMPK deficient cells (Figure 4D). Moreover, the majority of virus was removed by trypsin treatment in both wild type and AMPK deficient MEFs, indicating these virions had bound to the cell surface, but not entered (Figure 4D).

[00095] Since AMPK did not impede virus binding, we next performed a time of addition assay to test whether AMPK- activating drugs restricted entry. Since Bunyaviruses such as RVFV enter cells through a pH-dependent route of endocytosis, we used the lysosomotropic agent ammonium chloride, which raises the pH of lysosomal compartments, to define the timing of virus entry. Ammonium chloride inhibited infection strongly (to 20% of the 4 hpi addition) when added 1 hour prior to infection or with infection (t=0); however, by 1 hpi, more than 70% of infection had returned, indicating that the majority of RVFV had entered by this time point (Figure 4E). Thus we compared each treatment to the post entry level of RVFV infection (ammonium chloride added at 4 hpi). AMPK activating drugs 2DG, and A769662 significantly inhibited infection when added at post entry stages (Figure 4E); however, since one of the AMPK activating drugs, A769662, had a significantly greater impact on RVFV when added prior to or with infection, we cannot rule out that AMPK also inhibits RVFV entry. Taken together these data show that AMPK restricts RVFV during initial stages of replication post entry, likely at the step of RNA replication. This reduction in viral RNA and protein production likely leads to a reduction in release of infectious virus and spread observed at later stages of infection. EXAMPLE 5

The antiviral effects of AMPK are independent of type I interferon

[00096] The classical cell-mediated response to viral infection is the type I interferon system. Therefore, we investigated whether AMPK impacts the expression of interferon beta (IFN ) or its downstream effector 2'-5'-oligoadenylate synthetase 1 (OASl) by qRT-PCR. We found that RVFV infection induced both IFN and OAS l in both wild type and AMPK deficient cells although the basal levels and induction of these genes were higher in cells lacking AMPK (Figure 13A-B). This result was opposite to what would have been predicted, if IFN induction was responsible for the antiviral phenotype. In addition, we tested whether IFN treatment induced AMPK or ACC phosphorylation and found that it did not (Figure 13C, quantified in D). Altogether, these data show that AMPK has antiviral activity independent of the classical type I IFN response.

EXAMPLE 6

Acetyl-CoA Carboxylase Activity is Tightly Regulated by AMPK during RVFV

Infection

[00097] Since AMPK activation has antiviral activity against RVFV, we examined whether AMPK is activated by RVFV infection. To this end, we measured AMPK phosphorylation at Thrl72 by immunoblot. AMPK phosphorylation was increased at 4 and 8 hours after infection compared to uninfected controls (Figure 5A, quantified in Figure 14A), indicating that RVFV infection induced AMPK activation. Furthermore, we found that UV-irradiated virus, incapable of replication (Figure 15), also induced AMPK phosphorylation at 4 and 8 hours after treatment (Figure 5C), indicating that activation was triggered by incoming virus particles and viral replication was not required. Finally, we confirmed that LKB1 was required for RVFV-dependent activation of AMPK (Figure 16).

[00098] AMPK regulates several downstream pathways that could be important for viral infection, in particular protein translation and lipid synthesis. Thus, we examined the activation status of two classical downstream effectors of AMPK involved in translation and lipid biosynthesis which are inactivated by AMPK-mediated phosphorylation. Elongation Factor 2 (eEF2) is an important regulator of translation elongation, and Acetyl-CoA Carboxylase (ACC) consists of two enzymes involved in fatty acid metabolism (ACC1 and ACC2). Both eEF2 and ACC had increased levels of phosphorylation at 4 and 8 hours after infection with RVFV compared to uninfected controls, consistent with the activation status of AMPK (Figure 5A, quantified in Figure 14B-D). Little difference in total protein levels of AMPK, ACC or eEF2 was observed during infection. Taken together, these data show that RVFV infection leads to increased AMPK signaling.

[00099] To explore the mechanism by which AMPK restricts RVFV replication, we examined the impact of AMPK on translation and lipid biogenesis, both of which contribute to important steps in virus infection. In particular, AMPK inhibits translation initiation by inactivating mTORCl, and translation elongation by inactivating eEF2. Inactivation of mTORCl by AMPK leads to decreased translation initiation as well as increased autophagy, both of which could have anti-viral effects. Since AMPK activation inhibits mTORCl activity, we hypothesized that mTORCl, and thus protein synthesis, would be overactive in AMPK deficient cells, perhaps allowing for increased viral protein production and replication. We tested the requirement for mTORCl signaling in RVFV infection using the mTORCl inhibitor Rapamycin, and found no significant difference in RVFV infection in cells treated with Rapamycin compared to vehicle controls in either wild type or ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs (Figure 17A). This finding shows that the antiviral activity of AMPK is independent of mTORCl signaling. Furthermore, since AMPK activation can increase autophagy, which has been shown to have antiviral effects in some models, we tested whether inhibition of autophagy impacted RVFV infection by plaque assay, and found no significant difference in MEFs expressing a ATG5 hairpin, which knocks down ATG5, compared to control MEFs (Figure 17B-C).

[000100] Next we investigated whether reduced translation elongation through eEF2 inactivation could be responsible for AMPK's antiviral activity against RVFV (Figure 5 A). Since eEF2 is regulated by multiple upstream pathways in addition to AMPK, we first determined the sensitivity of eEF2 to AMPK regulation. In wild type MEFs, treatment with the AMPK activating drugs 2DG, oligomycin, or A769662 led to increased phosphorylation of AMPK, as well as downstream effectors eEF2 and ACC (Figure 5B, quantified in Figure 14E-H). As a control, we found that AMPK deficient MEFs did not express phosphorylated AMPK or total AMPK under any treatment condition. Interestingly, we observed an increase in phosphorylated eEF2 in response to all three drugs in ΑΜΡΚα1/ΑΜΡΚα2^{_ "} MEFs (Figure 5B, quantified in Figure 14H). In contrast, while we observed an increase in ACC phosphorylation in response to drug treatments in wild type MEFs, phosphorylated ACC was undetectable in AMPK deficient MEFs both basally and in response to treatment with AMPK activating compounds (Figure 5B). These phenotypes were not due to changes in total protein levels as they remained unchanged under all treatment conditions; although the AMPK deficient MEFs had a slightly lower basal level of ACC (Figure 5B). These findings indicate signaling pathways other than AMPK are important in regulating eEF2 phosphorylation, while ACC phosphorylation is exquisitely regulated by AMPK.

[000101] Given this observation, we pursued ACC as a potential regulator of antiviral defense. ACC is the first rate-limiting enzyme and master regulator of fatty acid metabolism, both by inhibiting fatty acid biosynthesis and activating fatty acid catabolism through beta- oxidation. Fatty acid biosynthesis is an important component of viral infection since numerous RNA viruses, including Bunyaviruses, proliferate cellular membrane structures for proper formation of the viral replication complex, in addition to using cellular membranes for their lipid coats. In order to assess the importance of fatty acid synthesis in RVFV infection, we tested the ability of RVFV to replicate within cells pretreated with the fatty acid synthase inhibitors. Fatty acid synthase is the next enzyme in fatty acid metabolism, using the product of ACC to generate palmitate, and thus is required for all fatty acid biosynthesis. We observed a 5 -fold decrease in RVFV infection in the presence of fatty acid synthase inhibitors cerulenin and C75 by immunofluorescence, similar to the decrease observed in cells pretreated with the AMPK activator A769662 (Figure 5D), indicating that de novo fatty acid synthesis is an important step early in RVFV infection.

[000102] ACC is the enzyme that converts acetyl-CoA into malonyl-CoA, a precursor in the synthesis of palmitate, the first product of de novo fatty acid biosynthesis. Since AMPK activation inhibits de novo fatty acid synthesis by inactivating ACC, we tested whether altered levels of AMPK activation or expression affected cellular lipid levels. To this end, we stained MEFs with the lipophilic BODIPY fluorescent dye. We found that treatment with the AMPK activator A769662 led to a decrease in BODIPY staining compared to untreated MEFs (Figure

5E, quantified in F), consistent with decreased fatty acid synthesis during AMPK activation. In contrast, MEFs lacking AMPK had increased BODIPY staining compared to wild type cells (Figure 5G, quantified in H). These findings show that the absence of AMPK leads to overproduction of cellular lipids, while AMPK activation globally reduces cellular lipid levels. EXAMPLE 7

Palmitate Rescues AMPK-Mediated Restriction of RVFV

[000103] If AMPK activation restricts RVFV infection by reducing levels of fatty acid synthesis, exogenous addition of fatty acids should restore infection. Therefore, we tested whether we could bypass the requirement for AMPK-regulated fatty acid synthesis by pretreating cells with palmitate, the first product of fatty acid biosynthesis. We treated U20S cells with palmitate overnight, and then added A769662 1 hour prior to infection with RVFV to activate AMPK. After 10 hours of infection, cells were fixed and stained for RVFV to measure percent infection in an immunofluorescence assay that monitors the initial round of infection. In cells treated with the AMPK activator A769662 alone, we found a 5-fold decrease in RVFV infection, consistent with our previous findings (Figure 6A, quantified in 6B). However, addition of palmitate prior to treatment with A769662 was able to restore infection to levels seen in untreated cells (Figure 6A, quantified in 6B). We observed a 5-fold increase in RVFV infection in cells treated with A769662 and palmitate compared to those treated with A769962 alone (Figure 6B), while addition of palmitate alone had little effect on infection (Figure 6A-B). Since chronic exposure to high concentrations of palmitate has previously been reported to inhibit AMPK activation, we confirmed by immunoblot that AMPK phosphorylation was not inhibited by the concentrations of palmitate used in our assay (Figure 18). Together, these data show that AMPK restricts RVFV infection primarily through inhibiting fatty acid biosynthesis.

EXAMPLE 8

AMPK Restricts Multiple Viruses That Depend on Lipids for Replication

[000104] A dependence on lipid biosynthesis and virally induced membrane modifications is not unique to Bunyaviruses; many RNA viruses require extensive membrane modifications and proliferations to support their replication complex. Therefore, we tested whether AMPK restricts additional arboviruses. To this end we tested the ability of the Flavivirus Kunjin virus (KUNV), the Togavirus Sindbis virus (SINV), and the Rhabdovirus Vesicular stomatitis virus (VSV) to grow in wild type and ΑΜΡΚα1/ΑΜΡΚα2^{_ ~} MEFs by immunofluorescence. KUNV (Figure 7A-B), SINV (Figure 7E-F) and VSV (Figure 71- J) had increased infections in ΑΜΡΚα1/ΑΜΡΚα2^{_ ~} MEFs compared to wild type MEFs. Moreover, KUNV (Figure 7C-D), SINV (Figure 7G-H), and VSV (Figure 7K-L) infections were also increased in LKB1^"; Vec compared to MEFs expressing LKB1, indicating that both AMPK and its canonical upstream activator LKBl restrict additional arboviruses. Moreover, we have found that KUNV is also sensitive to the AMPK activator A769662, and can be partially rescued by palmitate addition (Figure 19A-B), although palmitate treatment itself decreased KUNV infection (Figure 19C). These data show that AMPK may restrict multiple RNA viruses by limiting fatty acids. Taken together our data show that AMPK is broadly anti-viral across disparate virus families, and AMPK activator provide for anti-viral therapeutics.

[000105] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a viral disease in a subject, said disease caused by or associated with a virus that depends on fatty acid synthesis for its replication, the method comprising: administering to said subject a therapeutically effective amount of an AMP-Activated Kinase (AMPK) activator, a fatty acid synthesis inhibitor, or a combination thereof, thereby treating said viral disease in said subject.

2. The method of claim 1, wherein said viral disease is associated with a Bunyavirus.

3. The method of claim 1, wherein said viral disease is associated with a Rift Valley Fever Virus (RVFV).

4. The method of claim 1, wherein said viral disease is associated with a Sin Nombre, a Hantavirus, a Crimean-Congo hemorrhagic fever virus, a Flavivirus Kunjin virus (KUNV), a Togavirus Sindbis virus (SINV), or a Rhabdovirus Vesicular stomatitis virus (VSV).

5. The method claim 1, wherein said AMPK activator is Metformin.

6. The method claim 1, wherein said AMPK activator is Resveratrol, Thiazolidinedione, 5-aminoimidazole-4-carboxamide-l-beta-D-ribofuranoside (AICAR), Biguanide, Phenformin, a N-substituted-heterocycloalkyloxybenzamide compound, a Carboxamide compound, a Sulfonamide, a Thienopyridone derivative, an Imidazole derivative, a Thiazoles derivative, or a 3,4-substituted thiazole.

7. The method claim 1, wherein said AMPK activator is administered in conjunction with another agent.

8. The method claim 1, wherein said AMPK activator is administered in conjunction with said fatty acid inhibitor.

9. The method claim 1, wherein said AMPK activator is a direct AMPK activator.

10. The method claim 1, wherein said AMPK activator is an indirect AMPK activator.

11. The method claim 1, wherein said fatty acid synthesis inhibitor is a lipid lowering drug.

12. The method claim 1, wherein said subject is a human subject infected with said virus.

13. The method claim 1, wherein said subject is a domestic animal subject infected with said virus.

14. A composition comprising: an AMP-Activated Kinase (AMPK) activator, a fatty acid synthesis inhibitor, or a combination thereof, present in an amount effective to treat a viral disease caused by or associated with a virus that depends on fatty acid synthesis for its replication.

15. The composition of claim 14, wherein said viral disease is associated with a Bunyavirus.

16. The composition of claim 14, wherein said viral disease is associated with a Rift Valley Fever Virus (RVFV).

17. The composition of claim 14, wherein said viral disease is associated with a Sin Nombre, a Hantavirus, a Crimean-Congo hemorrhagic fever virus, a Flavivirus Kunjin virus (KUNV), a Togavirus Sindbis virus (SINV), or a Rhabdovirus Vesicular stomatitis virus (VSV).

18. The composition claim 14, wherein said AMPK activator is Metformin.

19. The composition claim 14, wherein said AMPK activator is Resveratrol, Thiazolidinedione, 5-aminoimidazole-4-carboxamide-l-beta-D-ribofuranoside (AICAR), Biguanide, Phenformin, a N-substituted-heterocycloalkyloxybenzamide compound, a Carboxamide compound, a Sulfonamide, a Thienopyridone derivative, an Imidazole derivative, a Thiazoles derivative, or a 3,4-substituted thiazole.

20. The method claim 14, wherein said fatty acid synthesis inhibitor is a lipid lowering drug.

21. A method for treating a Rift Valley Fever Virus (RVFV) infection in a subject, the method comprising: administering to said subject a therapeutically effective amount of an AMP-Activated Kinase (AMPK) activator, a fatty acid synthesis inhibitor, or a combination thereof, thereby treating said Rift Valley Fever Virus (RVFV) infection in said subject.

22. The method claim 21, wherein said AMPK activator is Metformin.

23. The method claim 21, wherein said AMPK activator is Resveratrol, Thiazolidinedione, 5-aminoimidazole-4-carboxamide-l-beta-D-ribofuranoside (AICAR), Biguanide, Phenformin, a N-substituted-heterocycloalkyloxybenzamide compound, a Carboxamide compound, a Sulfonamide, a Thienopyridone derivative, an Imidazole derivative, a Thiazoles derivative, or a 3,4-substituted thiazole.

24. The method claim 21, wherein said AMPK activator is administered in conjunction with another agent.

25. The method claim 21, wherein said AMPK activator is administered in conjunction with said fatty acid inhibitor.

26. The method claim 21, wherein said AMPK activator is a direct AMPK activator.

27. The method claim 21, wherein said AMPK activator is an indirect AMPK activator.

28. The method claim 21, wherein said fatty acid synthesis inhibitor is a lipid lowering drug.

29. The method claim 21, wherein said subject is a human subject infected with said virus.

30. The method claim 21, wherein said subject is a domestic animal subject infected with said virus.

31. A composition comprising: an AMP-Activated Kinase (AMPK) activator, a fatty acid synthesis inhibitor, or a combination thereof, present in an amount effective to treat a Rift Valley Fever Virus (RVFV) infection.

32. The composition claim 31, wherein said AMPK activator is Metformin.

33. The composition claim 31, wherein said AMPK activator is Resveratrol, Thiazolidinedione, 5-aminoimidazole-4-carboxamide-l-beta-D-ribofuranoside (AICAR), Biguanide, Phenformin, a N-substituted-heterocycloalkyloxybenzamide compound, a Carboxamide compound, a Sulfonamide, a Thienopyridone derivative, an Imidazole derivative, a Thiazoles derivative, or a 3,4-substituted thiazole.

34. The composition claim 31, wherein said fatty acid synthesis inhibitor is a lipid lowering drug.

35. A method for identifying a molecule to effectively treat a viral disease, said disease caused by or associated with a virus that depends on fatty acid synthesis for its replication, the method comprising: screening a plurality of AMP-Activated Kinase (AMPK) activators or a plurality of fatty acid synthesis inhibitors to effectively treat said viral disease, thereby identifying a molecule to effectively treat said viral disease.

36. A method for identifying a molecule to effectively treat a viral disease, said disease caused by or associated with a virus that depends on fatty acid synthesis for its replication, the method comprising: testing a AMP-Activated Kinase (AMPK) activator or a fatty acid synthesis inhibitor; determining whether said AMPK activator or fatty acid synthesis inhibitor effectively treats said viral disease, thereby identifying a molecule to effectively treat said viral disease.