FACI is a novel clathrin adaptor protein 2-binding protein that facilitates low-density lipoprotein endocytosis.

BACKGROUND: Cholesterol plays a vital role in multiple physiological processes. Cellular uptake of cholesterol is mediated primarily through endocytosis of low-density lipoprotein (LDL) receptor. New modifiers of this process remain to be characterized. Particularly, the role of fasting- and CREB-H-induced (FACI) protein in cholesterol homeostasis merits further investigation. METHODS: Interactome profiling by proximity labeling and affinity purification - mass spectrometry was performed. Total internal reflection fluorescence microscopy and confocal immunofluorescence microscopy were used to analyze protein co-localization and interaction. Mutational analysis was carried out to define the domain and residues required for FACI localization and function. Endocytosis was traced by fluorescent cargos. LDL uptake in cultured cells and diet-induced hypercholesterolemia in mice were assessed. RESULTS: FACI interacted with proteins critically involved in clathrin-mediated endocytosis, vesicle trafficking, and membrane cytoskeleton. FACI localized to clathrin-coated pits (CCP) on plasma membranes. FACI contains a conserved DxxxLI motif, which mediates its binding with the adaptor protein 2 (AP2) complex. Disruption of this motif of FACI abolished its CCP localization but didn't affect its association with plasma membrane. Cholesterol was found to facilitate FACI transport from plasma membrane to endocytic recycling compartment in a clathrin- and cytoskeleton-dependent manner. LDL endocytosis was enhanced in FACI-overexpressed AML12 cells but impaired in FACI-depleted HeLa cells. In vivo study indicated that hepatic FACI overexpression alleviated diet-induced hypercholesterolemia in mice. CONCLUSIONS: FACI facilitates LDL endocytosis through its interaction with the AP2 complex.

Cytoplasmic RNA sensors and their interplay with RNA-binding partners in innate antiviral response: theme and variations.

Sensing of pathogen-associated molecular patterns including viral RNA by innate immunity represents the first line of defense against viral infection. In addition to RIG-I-like receptors and NOD-like receptors, several other RNA sensors are known to mediate innate antiviral response in the cytoplasm. Double-stranded RNA-binding protein PACT interacts with prototypic RNA sensor RIG-I to facilitate its recognition of viral RNA and induction of host interferon response, but variations of this theme are seen when the functions of RNA sensors are modulated by other RNA-binding proteins to impinge on antiviral defense, proinflammatory cytokine production and cell death programs. Their discrete and coordinated actions are crucial to protect the host from infection. In this review, we will focus on cytoplasmic RNA sensors with an emphasis on their interplay with RNA-binding partners. Classical sensors such as RIG-I will be briefly reviewed. More attention will be brought to new insights on how RNA-binding partners of RNA sensors modulate innate RNA sensing and how viruses perturb the functions of RNA-binding partners.

FACI Is a Novel CREB-H-Induced Protein That Inhibits Intestinal Lipid Absorption and Reverses Diet-Induced Obesity.

BACKGROUND & AIMS: CREB-H is a key liver-enriched transcription factor governing lipid metabolism. Additional targets of CREB-H remain to be identified and characterized. Here, we identified a novel fasting- and CREB-H-induced (FACI) protein that inhibits intestinal lipid absorption and alleviates diet-induced obesity in mice. METHODS: FACI was identified by reanalysis of existing transcriptomic data. Faci-/- mice were generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-mediated genome engineering. RNA sequencing was performed to identify differentially expressed genes in Faci-/- mice. Lipid accumulation in the villi was assessed by triglyceride measurement and Oil red O staining. In vitro fatty acid uptake assay was performed to verify in vivo findings. RESULTS: FACI expression was enriched in liver and intestine. FACI is a phospholipid-binding protein that localizes to plasma membrane and recycling endosomes. Hepatic transcription of Faci was regulated by not only CREB-H, but also nutrient-responsive transcription factors sterol regulatory element-binding protein 1 (SREBP1), hepatocyte nuclear factor 4α (HNF4α), peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α), and CREB, as well as fasting-related cyclic adenosine monophosphate (cAMP) signaling. Genetic knockout of Faci in mice showed an increase in intestinal fat absorption. In accordance with this, Faci deficiency aggravated high-fat diet-induced obesity, hyperlipidemia, steatosis, and other obesity-related metabolic dysfunction in mice. CONCLUSIONS: FACI is a novel CREB-H-induced protein. Genetic disruption of Faci in mice showed its inhibitory effect on fat absorption and obesity. Our findings shed light on a new target of CREB-H implicated in lipid homeostasis.

Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is capable of inducing a storm of proinflammatory cytokines. In this study, we show that the SARS-CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). SARS-CoV and its ORF3a protein were found to be potent activators of pro-IL-1ß gene transcription and protein maturation, the 2 signals required for activation of the NLRP3 inflammasome. ORF3a induced pro-IL-1ß transcription through activation of NF-κB, which was mediated by TRAF3-dependent ubiquitination and processing of p105. ORF3a-induced elevation of IL-1ß secretion was independent of its ion channel activity or absent in melanoma 2 but required NLRP3, ASC, and TRAF3. ORF3a interacted with TRAF3 and ASC, colocalized with them in discrete punctate structures in the cytoplasm, and facilitated ASC speck formation. TRAF3-dependent K63-linked ubiquitination of ASC was more pronounced in SARS-CoV-infected cells or when ORF3a was expressed. Taken together, our findings reveal a new mechanism by which SARS-CoV ORF3a protein activates NF-κB and the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of p105 and ASC.-Siu, K.-L., Yuen, K.-S., Castaño-Rodriguez, C., Ye, Z.-W., Yeung, M.-L., Fung, S.-Y., Yuan, S., Chan, C.-P., Yuen, K.-Y., Enjuanes, L., Jin, D.-Y. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.

A novel transcript isoform of STING that sequesters cGAMP and dominantly inhibits innate nucleic acid sensing.

STING is a core adaptor in innate nucleic acid sensing in mammalian cells, on which different sensing pathways converge to induce type I interferon (IFN) production. Particularly, STING is activated by 2'3'-cGAMP, a cyclic dinucleotide containing mixed phosphodiester linkages and produced by cytoplasmic DNA sensor cGAS. Here, we reported on a novel transcript isoform of STING designated STING-ß that dominantly inhibits innate nucleic acid sensing. STING-ß without transmembrane domains was widely expressed at low levels in various human tissues and viral induction of STING-ß correlated inversely with IFN-ß production. The expression of STING-ß declined in patients with lupus, in which type I IFNs are commonly overproduced. STING-ß suppressed the induction of IFNs, IFN-stimulated genes and other cytokines by various immunostimulatory agents including cyclic dinucleotides, DNA, RNA and viruses, whereas depletion of STING-ß showed the opposite effect. STING-ß interacted with STING-α and antagonized its antiviral function. STING-ß also interacted with TBK1 and prevented it from binding with STING-α, TRIF or other transducers. In addition, STING-ß bound to 2'3'-cGAMP and impeded its binding with and activation of STING-α, leading to suppression of IFN-ß production. Taken together, STING-ß sequesters 2'3'-cGAMP second messenger and other transducer molecules to inhibit innate nucleic acid sensing dominantly.

Antiviral activity of double-stranded RNA-binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase.

PACT is a double-stranded RNA-binding protein that has been implicated in host-influenza A virus (IAV) interaction. PACT facilitates the action of RIG-I in the activation of the type I IFN response, which is suppressed by the viral nonstructural protein NS1. PACT is also known to interact with the IAV RNA polymerase subunit PA. Exactly how PACT exerts its antiviral activity during IAV infection remains to be elucidated. In the current study, we demonstrated the interplay between PACT and IAV polymerase. Induction of IFN-ß by the IAV RNP complex was most robust when both RIG-I and PACT were expressed. PACT-dependent activation of IFN-ß production was suppressed by the IAV polymerase subunits, polymerase acidic protein, polymerase basic protein 1 (PB1), and PB2. PACT associated with PA, PB1, and PB2. Compromising PACT in IAV-infected A549 cells resulted in the augmentation of viral RNA (vRNA) transcription and replication and IFN-ß production. Furthermore, vRNA replication was boosted by knockdown of PACT in both A549 cells and IFN-deficient Vero cells. Thus, the antiviral activity of PACT is mediated primarily via its interaction with and inhibition of IAV polymerase. Taken together, our findings reveal a new facet of the host-IAV interaction in which the interplay between PACT and IAV polymerase affects the outcome of viral infection and antiviral response.-Chan, C.-P., Yuen, C.-K., Cheung, P.-H. H., Fung, S.-Y., Lui, P.-Y., Chen, H., Kok, K.-H., Jin, D.-Y. Antiviral activity of double-stranded RNA-binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase.

Suppression of gluconeogenic gene transcription by SIK1-induced ubiquitination and degradation of CRTC1.

CRTCs are a group of three transcriptional coactivators required for CREB-dependent transcription. CREB and CRTCs are critically involved in the regulation of various biological processes such as cell proliferation, metabolism, learning and memory. However, whether CRTC1 efficiently induces gluconeogenic gene expression and how CRTC1 is regulated by upstream kinase SIK1 remain to be understood. In this work, we demonstrated SIK1-induced phosphorylation, ubiquitination and degradation of CRTC1 in the context of the regulation of gluconeogenesis. CRTC1 protein was destabilized by SIK1 but not SIK2 or SIK3. This effect was likely mediated by phosphorylation at S155, S167, S188 and S346 residues of CRTC1 followed by K48-linked polyubiquitination and proteasomal degradation. Expression of gluconeogenic genes such as that coding for phosphoenolpyruvate carboxykinase was stimulated by CRTC1, but suppressed by SIK1. Depletion of CRTC1 protein also blocked forskolin-induced gluconeogenic gene expression, knockdown or pharmaceutical inhibition of SIK1 had the opposite effect. Finally, SIK1-induced ubiquitination of CRTC1 was mediated by RFWD2 ubiquitin ligase at a site not equivalent to K628 in CRTC2. Taken together, our work reveals a regulatory circuit in which SIK1 suppresses gluconeogenic gene transcription by inducing ubiquitination and degradation of CRTC1. Our findings have implications in the development of new antihyperglycemic agents.

Suppression of Epstein-Barr virus DNA load in latently infected nasopharyngeal carcinoma cells by CRISPR/Cas9.

Epstein-Barr virus (EBV) infects more than 90% of the world's adult population. Once established, latent infection of nasopharyngeal epithelial cells with EBV is difficult to eradicate and might lead to the development of nasopharyngeal carcinoma (NPC) in a small subset of individuals. In this study we explored the anti-EBV potential of CRISPR/Cas9 targeting of EBV genome in infected NPC cells. We designed gRNAs to target different regions of the EBV genome and transfected them into C666-1 cells. The levels of EBV DNA in transfected cells were decreased by about 50%. The suppressive effect on EBV DNA load lasted for weeks but could not be further enhanced by re-transfection of gRNA. Suppression of EBV by CRISPR/Cas9 did not affect survival of C666-1 cells but sensitized them to chemotherapeutic killing by cisplatin and 5-fluorouracil. Our work provides the proof-of-principle for suppressing EBV DNA load with CRISPR/Cas9 and a potential new strategy to sensitize EBV-infected NPC cells to chemotherapy.

Human T-Cell Leukemia Virus Type 1 Infection and Adult T-Cell Leukemia.

Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus discovered to cause adult T-cell leukemia (ATL), a highly aggressive blood cancer. HTLV-1 research in the past 35 years has been most revealing in the mechanisms of viral oncogenesis. HTLV-1 establishes a lifelong persistent infection in CD4+ T lymphocytes. The infection outcome is governed by host immunity. ATL develops in 2-5% of infected individuals 30-50 years after initial exposure. HTLV-1 encodes two oncoproteins Tax and HBZ, which are required for initiation of cellular transformation and maintenance of cell proliferation, respectively. HTLV-1 oncogenesis is driven by a clonal selection and expansion process during which both host and viral factors cooperate to impair genome stability, immune surveillance, and other mechanisms of tumor suppression. A better understanding of HTLV-1 biology and leukemogenesis will reveal new strategies and modalities for ATL prevention and treatment.

PACT Facilitates RNA-Induced Activation of MDA5 by Promoting MDA5 Oligomerization.

MDA5 is a RIG-I-like cytoplasmic sensor of dsRNA and certain RNA viruses, such as encephalomyocarditis virus, for the initiation of the IFN signaling cascade in the innate antiviral response. The affinity of MDA5 toward dsRNA is low, and its activity becomes optimal in the presence of unknown cellular coactivators. In this article, we report an essential coactivator function of dsRNA-binding protein PACT in mediating the MDA5-dependent type I IFN response. Virus-induced and polyinosinic-polycytidylic acid-induced activation of MDA5 were severely impaired in PACT-knockout cells and attenuated in PACT-knockdown cells, but they were potentiated when PACT was overexpressed. PACT augmented IRF3-dependent type I IFN production subsequent to dsRNA-induced activation of MDA5. In contrast, PACT had no influence on MDA5-mediated activation of NF-κB. PACT required dsRNA interaction for its action on MDA5 and promoted dsRNA-induced oligomerization of MDA5. PACT had little stimulatory effect on MDA5 mutants deficient for oligomerization and filament assembly. PACT colocalized with MDA5 in the cytoplasm and potentiated MDA5 recruitment to the dsRNA ligand. Taken together, these findings suggest that PACT functions as an essential cellular coactivator of RIG-I, as well as MDA5, and it facilitates RNA-induced formation of MDA5 oligomers.

Interplay between SIRT1 and hepatitis B virus X protein in the activation of viral transcription.

Hepatitis B virus (HBV) genome is organized into a minichromosome known as covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. SIRT1 is an NAD+-dependent protein deacetylase which activates HBV transcription by promoting the activity of cellular transcription factors and coactivators. How SIRT1 and viral transactivator X protein (HBx) might affect each other remains to be clarified. In this study we show synergy and mutual dependence between SIRT1 and HBx in the activation of HBV transcription. All human sirtuins SIRT1 through SIRT7 activated HBV gene expression. The steady-state levels of SIRT1 protein were elevated in HBV-infected liver tissues and HBV-replicating hepatoma cells. SIRT1 interacted with HBx and potentiated HBx transcriptional activity on precore promoter and covalently closed circular DNA (cccDNA) likely through a deacetylase-independent mechanism, leading to more robust production of cccDNA, pregenomic RNA and surface antigen. SIRT1 and HBx proteins were more abundant when both were expressed. SIRT1 promoted the recruitment of HBx as well as cellular transcriptional factors and coactivators such as PGC-1α and FXRα to cccDNA. Depletion of SIRT1 suppressed HBx recruitment. On the other hand, SIRT1 recruitment to cccDNA was compromised when HBx was deficient. Whereas pharmaceutical agonists of SIRT1 such as resveratrol activated HBV transcription, small-molecule inhibitors of SIRT1 including sirtinol and Ex527 exhibited anti-HBV activity. Taken together, our findings revealed not only the interplay between SIRT1 and HBx in the activation of HBV transcription but also new strategies and compounds for developing antivirals against HBV.

Mutagenesis and Genome Engineering of Epstein-Barr Virus in Cultured Human Cells by CRISPR/Cas9.

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein-Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.

ß-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H.

CREB-H is an endoplasmic reticulum-resident bZIP transcription factor which critically regulates lipid homeostasis and gluconeogenesis in the liver. CREB-H is proteolytically activated by regulated intramembrane proteolysis to generate a C-terminally truncated form known as CREB-H-ΔTC, which translocates to the nucleus to activate target gene expression. CREB-H-ΔTC is a fast turnover protein but the mechanism governing its destruction was not well understood. In this study, we report on ß-TrCP-dependent ubiquitination and proteasomal degradation of CREB-H-ΔTC. The degradation of CREB-H-ΔTC was mediated by lysine 48-linked polyubiquitination and could be inhibited by proteasome inhibitor. CREB-H-ΔTC physically interacted with ß-TrCP, a substrate recognition subunit of the SCF(ß-TrCP) E3 ubiquitin ligase. Forced expression of ß-TrCP increased the polyubiquitination and decreased the stability of CREB-H-ΔTC, whereas knockdown of ß-TrCP had the opposite effect. An evolutionarily conserved sequence, SDSGIS, was identified in CREB-H-ΔTC, which functioned as the ß-TrCP-binding motif. CREB-H-ΔTC lacking this motif was stabilized and resistant to ß-TrCP-induced polyubiquitination. This motif was a phosphodegron and its phosphorylation was required for ß-TrCP recognition. Furthermore, two inhibitory phosphorylation sites close to the phosphodegron were identified. Taken together, our work revealed a new intracellular signaling pathway that controls ubiquitination and degradation of the active form of CREB-H transcription factor.

Middle East respiratory syndrome coronavirus M protein suppresses type I interferon expression through the inhibition of TBK1-dependent phosphorylation of IRF3.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection has claimed hundreds of lives and has become a global threat since its emergence in Saudi Arabia in 2012. The ability of MERS-CoV to evade the host innate antiviral response may contribute to its severe pathogenesis. Many MERS-CoV-encoded proteins were identified to have interferon (IFN)-antagonizing properties, which correlates well with the reduced IFN levels observed in infected patients and ex vivo models. In this study, we fully characterized the IFN-antagonizing property of the MERS-CoV M protein. Expression of MERS-CoV M protein suppressed type I IFN expression in response to Sendai virus infection or poly(I:C) induction. This suppressive effect was found to be specific for the activation of IFN regulatory factor 3 (IRF3) but not nuclear factor-κB. MERS-CoV M protein interacted with TRAF3 and disrupted TRAF3-TBK1 association leading to reduced IRF3 activation. M proteins from MERS-CoV and SARS-CoV have three highly similar conserved N-terminal transmembrane domains and a C-terminal region. Using chimeric and truncation mutants, the N-terminal transmembrane domains of the MERS-CoV M protein were found to be sufficient for its inhibitory effect on IFN expression, whereas the C-terminal domain was unable to induce this suppression. Collectively, our findings suggest a common and conserved mechanism through which highly pathogenic MERS-CoV and SARS-CoV harness their M proteins to suppress type I IFN expression at the level of TBK1-dependent phosphorylation and activation of IRF3 resulting in evasion of the host innate antiviral response.

Suppression of Type I Interferon Production by Human T-Cell Leukemia Virus Type 1 Oncoprotein Tax through Inhibition of IRF3 Phosphorylation.

UNLABELLED: Infection with human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and tropical spastic paraparesis. Type I interferons (IFNs) are key effectors of the innate antiviral response, and IFN-α combined with the nucleoside reverse transcriptase inhibitor zidovudine is considered the standard first-line therapy for ATL. HTLV-1 oncoprotein Tax is known to suppress innate IFN production and response but the underlying mechanisms remain to be fully established. In this study, we report on the suppression of type I IFN production by HTLV-1 Tax through interaction with and inhibition of TBK1 kinase that phosphorylates IRF3. Induced transcription of IFN-ß was severely impaired in HTLV-1-transformed ATL cells and freshly infected T lymphocytes. The ability to suppress IRF3 activation was ascribed to Tax. The expression of Tax alone sufficiently repressed the induction of IFN production by RIG-I plus PACT, cGAMP synthase plus STING, TBK1, IKKε, IRF3, and IRF7, but not by IRF3-5D, a dominant-active phosphomimetic mutant. This suggests that Tax perturbs IFN production at the step of IRF3 phosphorylation. Tax mutants deficient for CREB or NF-κB activation were fully competent in the suppression of IFN production. Coimmunoprecipitation experiments confirmed the association of Tax with TBK1, IKKε, STING, and IRF3.In vitrokinase assay indicated an inhibitory effect of Tax on TBK1-mediated phosphorylation of IRF3. Taken together, our findings suggested a new mechanism by which HTLV-1 oncoprotein Tax circumvents the production of type I IFNs in infected cells. Our findings have implications in therapeutic intervention of ATL. IMPORTANCE: Human T-cell leukemia virus type 1 (HTLV-1) is the cause of adult T-cell leukemia (ATL), an aggressive and fatal blood cancer, as well as another chronic disabling disease of the spinal cord. Treatments are unsatisfactory, and options are limited. A combination of antiviral cellular protein alpha interferon and zidovudine, which is an inhibitor of a viral enzyme called reverse transcriptase, has been recommended as the standard first-line therapy for ATL. Exactly how HTLV-1 interacts with the cellular machinery for interferon production and action is not well understood. Our work sheds light on the mechanism of action for the inhibition of interferon production by an HTLV-1 oncogenic protein called Tax. Our findings might help to improve interferon-based anti-HTLV-1 and anti-ATL therapy.

PACT- and RIG-I-Dependent Activation of Type I Interferon Production by a Defective Interfering RNA Derived from Measles Virus Vaccine.

UNLABELLED: The live attenuated measles virus vaccine is highly immunostimulatory. Identification and characterization of its components that activate the innate immune response might provide new strategies and agents for the rational design and development of chemically defined adjuvants. In this study, we report on the activation of type I interferon (IFN) production by a defective interfering (DI) RNA isolated from the Hu-191 vaccine strain of measles virus. We found that the Hu-191 virus induced IFN-ß much more potently than the Edmonston strain. In the search for IFN-inducing species in Hu-191, we identified a DI RNA specifically expressed by this strain. This DI RNA, which was of the copy-back type, was predicted to fold into a hairpin structure with a long double-stranded stem region of 206 bp, and it potently induced the expression of IFN-ß. Its IFN-ß-inducing activity was further enhanced when both cytoplasmic RNA sensor RIG-I and its partner, PACT, were overexpressed. On the contrary, this activity was abrogated in cells deficient in PACT or RIG-I. The DI RNA was found to be associated with PACT in infected cells. In addition, both the 5'-di/triphosphate end and the double-stranded stem region on the DI RNA were essential for its activation of PACT and RIG-I. Taken together, our findings support a model in which a viral DI RNA is sensed by PACT and RIG-I to initiate an innate antiviral response. Our work might also provide a foundation for identifying physiological PACT ligands and developing novel adjuvants or antivirals. IMPORTANCE: The live attenuated measles virus vaccine is one of the most successful human vaccines and has largely contained the devastating impact of a highly contagious virus. Identifying the components in this vaccine that stimulate the host immune response and understanding their mechanism of action might help to design and develop better adjuvants, vaccines, antivirals, and immunotherapeutic agents. We identified and characterized a defective interfering RNA from the Hu-191 vaccine strain of measles virus which has safely been used in millions of people for many years. We further demonstrated that this RNA potently induces an antiviral immune response through cellular sensors of viral RNA known as PACT and RIG-I. Similar types of viral RNA that bind with and activate PACT and RIG-I might retain the immunostimulatory property of measles virus vaccines but would not induce adaptive immunity. They are potentially useful as chemically defined vaccine adjuvants, antivirals, and immunostimulatory agents.

SIRT1 Suppresses Human T-Cell Leukemia Virus Type 1 Transcription.

UNLABELLED: Human T-cell leukemia virus type 1 (HTLV-1)-associated diseases are poorly treatable, and HTLV-1 vaccines are not available. High proviral load is one major risk factor for disease development. HTLV-1 encodes Tax oncoprotein, which activates transcription from viral long terminal repeats (LTR) and various types of cellular promoters. Counteracting Tax function might have prophylactic and therapeutic benefits. In this work, we report on the suppression of Tax activation of HTLV-1 LTR by SIRT1 deacetylase. The transcriptional activity of Tax on the LTR was largely ablated when SIRT1 was overexpressed, but Tax activation of NF-κB was unaffected. On the contrary, the activation of the LTR by Tax was boosted when SIRT1 was depleted. Treatment of cells with resveratrol shunted Tax activity in a SIRT1-dependent manner. The activation of SIRT1 in HTLV-1-transformed T cells by resveratrol potently inhibited HTLV-1 proviral transcription and Tax expression, whereas compromising SIRT1 by specific inhibitors augmented HTLV-1 mRNA expression. The administration of resveratrol also decreased the production of cell-free HTLV-1 virions from MT2 cells and the transmission of HTLV-1 from MT2 cells to uninfected Jurkat cells in coculture. SIRT1 associated with Tax in HTLV-1-transformed T cells. Treatment with resveratrol prevented the interaction of Tax with CREB and the recruitment of CREB, CRTC1, and p300 to Tax-responsive elements in the LTR. Our work demonstrates the negative regulatory function of SIRT1 in Tax activation of HTLV-1 transcription. Small-molecule activators of SIRT1 such as resveratrol might be considered new prophylactic and therapeutic agents in HTLV-1-associated diseases. IMPORTANCE: Human T-cell leukemia virus type 1 (HTLV-1) causes a highly lethal blood cancer or a chronic debilitating disease of the spinal cord. Treatments are unsatisfactory, and vaccines are not available. Disease progression is associated with robust expression of HTLV-1 genes. Suppressing HTLV-1 gene expression might have preventive and therapeutic benefits. It is therefore critical that host factors controlling HTLV-1 gene expression be identified and characterized. This work reveals a new host factor that suppresses HTLV-1 gene expression and a natural compound that activates this suppression. Our findings not only provide new knowledge of the host control of HTLV-1 gene expression but also suggest a new strategy of using natural compounds for prevention and treatment of HTLV-1-associated diseases.

CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

Requirement of CRTC1 coactivator for hepatitis B virus transcription.

Transcription of hepatitis B virus (HBV) from the covalently closed circular DNA (cccDNA) template is essential for its replication. Suppressing the level and transcriptional activity of cccDNA might have anti-HBV effect. Although cellular transcription factors, such as CREB, which mediate HBV transcription, have been well described, transcriptional coactivators that facilitate this process are incompletely understood. In this study we showed that CREB-regulated transcriptional coactivator 1 (CRTC1) is required for HBV transcription and replication. The steady-state levels of CRTC1 protein were elevated in HBV-positive hepatoma cells and liver tissues. Ectopic expression of CRTC1 or its homolog CRTC2 or CRTC3 in hepatoma cells stimulated the activity of the preS2/S promoter of HBV, whereas overexpression of a dominant inactive form of CRTC1 inhibited HBV transcription. CRTC1 interacts with CREB and they are mutually required for the recruitment to the preS2/S promoter on cccDNA and for the activation of HBV transcription. Accumulation of pregenomic RNA (pgRNA) and cccDNA was observed when CRTC1 or its homologs were overexpressed, whereas the levels of pgRNA, cccDNA and secreted HBsAg were diminished when CRTC1 was compromised. In addition, HBV transactivator protein HBx stabilized CRTC1 and promoted its activity on HBV transcription. Our work reveals an essential role of CRTC1 coactivator in facilitating and supporting HBV transcription and replication.

Middle east respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response.

UNLABELLED: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging pathogen that causes severe disease in human. MERS-CoV is closely related to bat coronaviruses HKU4 and HKU5. Evasion of the innate antiviral response might contribute significantly to MERS-CoV pathogenesis, but the mechanism is poorly understood. In this study, we characterized MERS-CoV 4a protein as a novel immunosuppressive factor that antagonizes type I interferon production. MERS-CoV 4a protein contains a double-stranded RNA-binding domain capable of interacting with poly(I · C). Expression of MERS-CoV 4a protein suppressed the interferon production induced by poly(I · C) or Sendai virus. RNA binding of MERS-CoV 4a protein was required for IFN antagonism, a property shared by 4a protein of bat coronavirus HKU5 but not by the counterpart in bat coronavirus HKU4. MERS-CoV 4a protein interacted with PACT in an RNA-dependent manner but not with RIG-I or MDA5. It inhibited PACT-induced activation of RIG-I and MDA5 but did not affect the activity of downstream effectors such as RIG-I, MDA5, MAVS, TBK1, and IRF3. Taken together, our findings suggest a new mechanism through which MERS-CoV employs a viral double-stranded RNA-binding protein to circumvent the innate antiviral response by perturbing the function of cellular double-stranded RNA-binding protein PACT. PACT targeting might be a common strategy used by different viruses, including Ebola virus and herpes simplex virus 1, to counteract innate immunity. IMPORTANCE: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging and highly lethal human pathogen. Why MERS-CoV causes severe disease in human is unclear, and one possibility is that MERS-CoV is particularly efficient in counteracting host immunity, including the sensing of virus invasion. It will therefore be critical to clarify how MERS-CoV cripples the host proteins that sense viruses and to compare MERS-CoV with its ancestral viruses in bats in the counteraction of virus sensing. This work not only provides a new understanding of the abilities of MERS-CoV and closely related bat viruses to subvert virus sensing but also might prove useful in revealing new strategies for the development of vaccines and antivirals.