key: cord-0795809-4ztedh24
authors: Aliyari, Saba R.; Ghaffari, Amir Ali; Pernet, Olivier; Parvatiyar, Kislay; Wang, Yao; Gerami, Hoda; Tong, Ann-Jay; Vergnes, Laurent; Takallou, Armin; Zhang, Adel; Wei, Xiaochao; Chilin, Linda D.; Wu, Yuntao; Semenkovich, Clay F.; Reue, Karen; Smale, Stephen T.; Lee, Benhur; Cheng, Genhong
title: Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2
date: 2022-02-28
journal: Acta Pharm Sin B
DOI: 10.1016/j.apsb.2022.02.019
sha: e9cbd47f9f081a577280e00d2b3851cc00f0c9b8
doc_id: 795809
cord_uid: 4ztedh24

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.

SARS-CoV-2 is an emerging, positive strand RNA virus from the Coronaviridae family 1 . As the etiological agent of the COVID-19 pandemic, SARS-CoV-2 has become one of the most challenging public health threats faced in decades, with a major sociological and economic impact 2 . Despite drastic global efforts to reduce the spread of infection and develop novel treatments and vaccines, the pandemic is to date still uncontrolled, with over 200 million cases and 4.5 million deaths by the end of August, 2021 3 . While the classic presentation of COVID-19 is a respiratory syndrome with a high case-fatality rate, particularly in seniors and those with certain pre-existing health conditions, SARS-CoV-2 infection can also trigger an autoimmune multi-systemic inflammatory disorder, particularly in children 4 . To combat novel viruses such as SARS-CoV-2 and future emerging viral infections to which we have no specific therapies, it is critical that we develop agents with broad antiviral effects. Investigating innate immune responses, which have evolved to protect the host against multiple types of viral infections, can help us to identify novel strategies to boost or replicate these broad defenses. The innate immune system provides a critical first line of defense against invading pathogens, including those of viral origin. Utilizing select germ line encoded pattern recognition receptors (PRRs), cells of the innate immune system detect pathogen-associated molecular patterns (PAMPs), which are highly invariable structures of microbial origin, elicits an antiviral gene program known as the host IFN-I response, characterized by the induction of IFN-I and ISGs. Viral RNA and DNA species generated during replicative cycles serve as PAMPs and are typically detected in the cytosol by nucleic acid sensing PRRs [5] [6] [7] [8] [9] . Recognition of viral RNA species by the RIG-I like family of RNA sensors, or viral DNA species by the cGAS, DDX41, or J o u r n a l P r e -p r o o f IFI16 DNA sensors, triggers the activation of the innate antiviral IFN-I response [10] [11] [12] . Microbial nucleic acids detected on the cell surface or in endosomal compartments can also activate the host IFN-I response via membrane bound PRRs, such as Toll-like receptors (TLRs) 3, 7, 8, and 9.

TLR4 10, 13, 14 which detects lipid A and lipopolysaccharide (LPS) derived from the cell walls of gram-negative bacteria, is also known to trigger the IFN-I response 15 .

A central paradigm of innate antiviral immunity is that IFN-I, namely IFNα and IFNβ, play essential roles in fostering an antiviral state 16, 17 . Operating primarily in a paracrine fashion, these cytokines bind to the IFNα/β receptor (IFNAR) on neighboring cells to instigate a Janus kinasesignal transducer and activator of transcription (JAK-STAT) signaling cascade, which culminates in the up-regulation of nearly 300 ISGs 18, 19 , which directly target viral components or orchestrate cellular processes that lead to the inhibition of virus replication and spread 20 .

Alternatively, certain genes are downregulated by IFNα, β, and γ 21 as a mechanism to control viral infections 22 . Modulation of enzymes involved in the fatty acid synthesis pathway plays a pivotal role in the regulation of infection for many viruses, including human cytomegalovirus (HCMV) 23 , Kaposi sarcoma-associated herpesvirus (KSHV) 24 , dengue virus (DENV) [25] [26] [27] , chikungunya virus 28 , Rotavirus 29 , and hepatitis C virus (HCV) [30] [31] [32] . Utilizing an RNA sequencing (RNAseq) approach, we comparatively evaluated gene expression profiles between wild type (WT) and Ifnar-deficient immune cells after PRR stimulation, and identified fatty acid synthase (Fasn) as an IFN-I suppressed gene that is required for optimal viral infectivity. Our results also reveal that FASN plays an essential role in mediating viral entry, and that inhibition of FASN by pharmacological compounds represents a novel broad antiviral strategy against a wild range of viruses, including SARS-CoV-2.

The experiments were performed in accordance with the Institutional Animal Care and Use Committee guidelines from the University of California Los Angeles (CA, USA). Age and sex derived fibroblasts (MEF) were derived by skinning the tails of mice and incubating them directly in culture dishes in DMEM with 10% FBS. Cells were scraped and re-plated after 7 days.

Vero-E6 and Huh7.5 cell lines were purchased from ATCC, and Hela-ACE2 cells, a kind gift from Dr. Guangxiang Luo from UAB, were cultured DMEM supplemented with 10% FBS (HyClone), and 1% penicillin-streptomycin (Gibco) at 37 °C in a 5% CO2 humidified atmosphere.

5×10 5 BMDMs derived from wild type (C57BL/6) and Ifnar -/mice were stimulated with Lipid A (100ng/mL, Sigma) or saline control for 6 h. Cells were harvested in Trizol (Invitrogen) and RNA was isolated via Qiagen RNeasy Mini Kit (Qiagen) according to manufacturer's protocol.

Polyadenylated RNA was purified from 10 µg of total RNA using the Micro-polyA Purist kit (Ambion) according to manufacturer's protocol. Prior to cDNA library construction for RNA-Seq analyses, RNA was quantified and assessed for quality (RNA Integrity Value) using an Agilent 2100 Bioanalyzer (Agilent Technology, Santa Clara, CA, USA). cDNA libraries were constructed as previously described using 100 ng of PolyA+ purified RNA as input 37, 38 Reverse, 5′-CGAAGGTGTGACTTCCATG-3′.

RT-qPCR cycling conditions were 42 °C for 5 min, 95 °C for 10 s, and 40 cycles of 95 °C for 5 s, followed by 60 °C for 30 s.

All the SARS-CoV-2 based experiments were performed at the UCLA BSL3 facility.

VSV-G pseudotyped VSV-G luciferase pseudovirus (VSVΔG-Luc/G) was generated by methods were pre-treated with serially diluted C75 for 1 h, cells were infected with Ha-CoV-2(Luc) or its variants of concern for 18 h. Cells were lysed in Luciferase Assay Lysis Buffer (Promega) for luciferase assays using GloMax Discover Microplate Reader (Promega).

A previously described construct encoding Nipah-M1 fused with β-lactamase (βlaM) was used to package inside VSV-G 42 . HEK 293T cells were transfected with constructs encoding βlaM and VSV-G or βlaM alone (bald) at 3:1 ratio in 10 cm dishes by polyethylenimine (PEI) transfection reagent. The viral supernatants were collected, clarified, and concentrated by ultracentrifugation at >75,000×g on 20% sucrose cushion and the pellet was resuspended in NTE buffer.

Cells were collected in Trizol and RNA was isolated by standard isopropanol precipitation. RNA was quantified and 1 μg of RNA was reversed transcribed using iScript (BioRad) according to the manufacturer's instructions with random hexamer as primers. RT-qPCR analysis was done were imaged using the in vivo imaging system (IVIS, Xenogen). Briefly, mice were anesthetized with isoflurane and administered 3 mg D-luciferin/mouse by i.p. injection prior to imaging.

Grayscale photographs and color images of imaged mice were superimposed with LivingImage (Xenogen) and Igor (Wavemetrics) programs, similar to that previously described 20 . The mice were imaged on dorsal, ventral, right, and left side until the maximal luminescence has passed. 

Twenty hours prior to the cytotoxicity assay, 8×10 3 Hela-ACE-2, 1.2×10 4 Huh 7.5 and 1.2×10 4

Vero-E6 cells were seeded in 96 Well White/Clear Bottom Plate, TC Surface (Thermo Fisher).

Cells were treated with 2-fold serial dilutions (100-1.5 μmol/L) of C75, (160-2.5 μmol/L) of Cerulenin, and (160-2.5 μmol/L) of TVB-3166 for 20 h. The cell viability was determined by using Cell Titer-Glo® Luminescent cell viability assay (Promega). IC50 and CC50 values were calculated by non-linear regression analysis using GraphPad 5.

The data were analyzed with unpaired student t-test.by Prism software (GraphPad). All of the data are shown as Data presented as mean+standard deviation (SD) or mean±standard error of mean (SEM) from three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

To identify genes that are downregulated by IFN-I during PRR signaling, WT and Ifnar -/bone marrow-derived macrophages (BMDMs) were stimulated with Lipid A, a TLR4 agonist, and subjected to RNAseq analysis. Ingenuity software profiling of canonical pathways revealed that multiple genes in fatty acid biosynthesis and glycerophospholipid metabolism were downregulated in WT BMDMs, but upregulated in Ifnar -/-BMDMs ( Fig. 1A 

Our data indicate that FASN is suppressed during viral infections in an IFNAR1-dependent manner, suggesting a possibility that it may be involved in modulating viral infection. To to support viral infection in comparison to WT control cells (Fig. 2D and Fig. S2D ).

J o u r n a l P r e -p r o o f

C75 (4-methylene-2-octyl-5-oxotetra-hydrofuran-3-carboxylic acid) is a synthetic FASN inhibitor that has been used extensively to study FASN function 43 . We determined the optimal dose for C75 that would not cause cellular toxicity (Fig. S2E ). Cellular treatment with C75 significantly reduced both RNA and DNA viruses in HEK 293T cells and human monocytederived macrophages (hPBMC) compared to untreated cells (Fig. 2E-H) . To determine whether inhibition of FASN could inhibit viral infection in vivo, we administered C75 to mice followed by viral infection and full body imaging analysis. Animals treated with C75 harbored lower viral loads after infection compared to untreated animals (Fig. 2I) . Epigallocatechin gallate (EGCG), a catechin found in tea leaves (Camellia sinensis) 44 , and cerulenin, an antifungal antibiotic and irreversible inhibitor of FASN 45 

In the setting of the COVID-19 pandemic, we investigated the potential role of FASN in SARS-CoV-2 infection. FASN mRNA levels in Hela-ACE-2 (Hela cells constitutively expressing ACE-2) were reduced after IFNα treatment and pI:C transfection. (Supporting Information Fig. S4A and S4B). The FASN expression was then measured in Hela-ACE-2 and Huh 7.5 cells infected with SARS-CoV-2. We used the recombinant SARS-CoV-2 expressing mNeonGreen protein Cells were then harvested and the viral RNA in the cell lysate was quantified by RT-qPCR.

Results showed a dose-dependent reduction of SARS-CoV-2 nucleocapsid protein (NP) (Fig.   4D -G). In parallel, infectious viral particles released in the supernatants of the treated cells were measured by standard plaque forming unit assay (PFU) by re-infecting Hela-ACE-2 cells. These results also showed a dose-dependent reduction of viral production in cells treated with the FASN inhibitors (Supporting Information Fig. S5A-S5D ).

Insert Fig. 4 In addition, we used the mNeonGreen recombinant SARS-CoV-2 to infect Vero-E6 and compared the number of GFP positive cells in the presence or absence of FASN inhibitors using flow cytometric analysis and fluorescent microscopy (Supporting Information Fig. S6 ). Results

SARS-CoV-2 when comparing cells without treatment (61% infected) to 4.5% infection after C75 and 6.6% after ECGC treatments.

As numerous SARS-CoV-2 variants have appeared since the initial outbreak, we wanted to test whether FASN inhibitors could also effectively suppress infection by these evolved strains, specifically the variants of concern. As shown in 

To determine the potential cytotoxic effect of the compounds used in our study, the 50% cytotoxic concentration (CC50) was evaluated by treating different cell types with 2-fold serial dilutions of C75, Cerulenin, and TVB-3166 for 18 h (Supporting Information Fig. S9A-S9I) .

We did not detect any apparent cytotoxicity with concentrations of the compounds used to inhibit viral infection in this study. The concentrations used to inhibit 50% of viral infection (IC50) was determined for C75, EGCG, cerulenin, and TVB-3166 by infecting cells with SARS-

Information Fig. S10A-S10E) Therefore, the effective dose for viral inhibition for the FASN inhibitors in this study was much smaller than the CC50. However, at higher concentrations there was an apparent toxicity for these inhibitors of FASN (Fig. S9A-S9I ).

Viruses 

Viruses reprogram the cellular metabolism of the host cell for optimal growth. The host innate immune signaling pathway counteracts viral invasion by inducing type-I-interferon (IFN-I).

Subsequently, IFN-I up-/downregulates expression of particular genes to block viral infection.

Fatty acid synthase (FASN), the sole enzyme involved in de novo fatty acid synthesis is downregulated by IFN-I upon viral infection. We have shown that FASN deficiency or suppression by pharmacological inhibitors including C75, cerulenin, EGCG, and TVB-3166 significantly blocks replication and spread of a wide variety of enveloped viruses including SARS-CoV-2 and its variants of concern. As viruses rely entirely on host cell's molecular machinery and metabolic products for adequate growth, suppressing host metabolic genes might be a general and effective strategy to fight against novel diseases caused by many different types of viruses. 

The authors declare no conflicts of interest. 

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