Host Antiviral Innate Immune Responses Research Articles

SARS-CoV-2 Nsp15 antagonizes the cGAS-STING-mediated antiviral innate immune responses.

Coronavirus (CoV) Nsp15 is a viral endoribonuclease (EndoU) with a preference for uridine residues. CoV Nsp15 is an innate immune antagonist which prevents dsRNA sensor recognition and stress granule formation by targeting viral and host RNAs. SARS-CoV-2 restricts and delays the host antiviral innate immune responses through multiple viral proteins, but the role of SARS-CoV-2 Nsp15 in innate immune evasion is not completely understood. Here, we generate an EndoU activity knockout rSARS-CoV-2Nsp15-H234A to elucidate the biological functions of Nsp15. Relative to wild-type rSARS-CoV-2, replication of rSARS-CoV-2Nsp15-H234A was significantly decreased in IFN-responsive A549-ACE2 cells but not in its STAT1 knockout counterpart. Transcriptomic analysis revealed upregulation of innate immune response genes in cells infected with rSARS-CoV-2Nsp15-H234A relative to wild-type virus, including cGAS-STING, cytosolic DNA sensors activated by both DNA and RNA viruses. Treatment with STING inhibitors H-151 and SN-011 rescued the attenuated phenotype of rSARS-CoV-2Nsp15-H234A. SARS-CoV-2 Nsp15 inhibited cGAS-STING-mediated IFN-β promoter and NF-κB reporter activity, as well as facilitated the replication of EV-D68 and NDV by diminishing cGAS and STING expression and downstream innate immune responses. Notably, the decline in cGAS and STING was also apparent during SARS-CoV-2 infection. The EndoU activity was essential for SARS-CoV-2 Nsp15-mediated cGAS and STING downregulation, but not all HCoV Nsp15 share the consistent substrate selectivity. In the hamster model, rSARS-CoV-2Nsp15-H234A replicated to lower titers in the nasal turbinates and lungs and induced higher innate immune responses. Collectively, our findings exhibit that SARS-CoV-2 Nsp15 serves as a host innate immune antagonist by targeting host cGAS and STING.

Molecular Mechanisms and Potential Antiviral Strategies of Liquid-Liquid Phase Separation during Coronavirus Infection.

Highly pathogenic coronaviruses have caused significant outbreaks in humans and animals, posing a serious threat to public health. The rapid global spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in millions of infections and deaths. However, the mechanisms through which coronaviruses evade a host's antiviral immune system are not well understood. Liquid-liquid phase separation (LLPS) is a recently discovered mechanism that can selectively isolate cellular components to regulate biological processes, including host antiviral innate immune signal transduction pathways. This review focuses on the mechanism of coronavirus-induced LLPS and strategies for utilizing LLPS to evade the host antiviral innate immune response, along with potential antiviral therapeutic drugs and methods. It aims to provide a more comprehensive understanding and novel insights for researchers studying LLPS induced by pandemic viruses.

Porcine deltacoronavirus nonstructural protein 2 inhibits type I and III IFN production by targeting STING for degradation

Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune response. The cGAS-STING signalling pathway plays an important role in antiviral innate immunity. However, it remains unclear whether PDCoV achieves immune evasion by regulating the cGAS-STING pathway. Here, we demonstrated that the nonstructural protein 2 (nsp2) encoded by PDCoV inhibits cGAS-STING-mediated type I and III interferon (IFN) responses via the regulation of porcine STING (pSTING) stability. Mechanistically, ectopically expressed PDCoV nsp2 was found to interact with the N-terminal region of pSTING. Consequently, pSTING was degraded through K48-linked ubiquitination and the proteasomal pathway, leading to the disruption of cGAS-STING signalling. Furthermore, K150 and K236 of pSTING were identified as crucial residues for nsp2-mediated ubiquitination and degradation. In summary, our findings provide a basis for elucidating the immune evasion mechanism of PDCoV and will contribute to the development of targets for anti-coronavirus drugs.

Tripartite Motif-Containing Protein 65 (TRIM65) Inhibits Hepatitis B Virus Transcription.

Tripartite motif (TRIM) proteins, comprising a family of over 100 members with conserved motifs, exhibit diverse biological functions. Several TRIM proteins influence viral infections through direct antiviral mechanisms or by regulating host antiviral innate immune responses. To identify TRIM proteins modulating hepatitis B virus (HBV) replication, we assessed 45 human TRIMs in HBV-transfected HepG2 cells. Our study revealed that ectopic expression of 12 TRIM proteins significantly reduced HBV RNA and subsequent capsid-associated DNA levels. Notably, TRIM65 uniquely downregulated viral pregenomic (pg) RNA in an HBV-promoter-specific manner, suggesting a targeted antiviral effect. Mechanistically, TRIM65 inhibited HBV replication primarily at the transcriptional level via its E3 ubiquitin ligase activity and intact B-box domain. Though HNF4α emerged as a potential TRIM65 substrate, disrupting its binding site on the HBV genome did not completely abolish TRIM65's antiviral effect. In addition, neither HBx expression nor cellular MAVS signaling was essential to TRIM65-mediated regulation of HBV transcription. Furthermore, CRISPR-mediated knock-out of TRIM65 in the HepG2-NTCP cells boosted HBV infection, validating its endogenous role. These findings underscore TRIM proteins' capacity to inhibit HBV transcription and highlight TRIM65's pivotal role in this process.

Viral RNA capping: Mechanisms and antiviral therapy.

RNA capping is an essential trigger for protein translation in eukaryotic cells. Many viruses have evolved various strategies for initiating the translation of viral genes and generating progeny virions in infected cells via synthesizing cap structure or stealing the RNA cap from nascent host messenger ribonucleotide acid(mRNA). In addition to protein translation, a new understanding of the role of the RNA cap in antiviral innate immunity has advanced the field of mRNA synthesis in vitro and therapeutic applications. Recent studies on these viral RNA capping systems have revealed startlingly diverse ways and molecular machinery. A comprehensive understanding of how viruses accomplish the RNA capping in infected cells is pivotal for designing effective broad-spectrum antiviral therapies. Here we systematically review the contemporary insights into the RNA-capping mechanisms employed by viruses causing human and animal infectious diseases, while also highlighting its impact on host antiviral innate immune response. The therapeutic applications of targeting RNA capping against viral infections and the development of RNA-capping inhibitors are also summarized.

RanBP2/Nup358 Mediates Sumoylation of STAT1 and Antagonizes Interferon-α-Mediated Antiviral Innate Immunity.

Type I interferon (IFN-I)-induced signaling plays a critical role in host antiviral innate immune responses. Despite this, the mechanisms that regulate this signaling pathway have yet to be fully elucidated. The nucleoporin Ran Binding Protein 2 (RanBP2) (also known as Nucleoporin 358 KDa, Nup358) has been implicated in a number of cellular processes, including host innate immune signaling pathways, and is known to influence viral infection. In this study, we documented that RanBP2 mediates the sumoylation of signal transducers and activators of transcription 1 (STAT1) and inhibits IFN-α-induced signaling. Specifically, we found that RanBP2-mediated sumoylation inhibits the interaction of STAT1 and Janus kinase 1 (JAK1), as well as the phosphorylation and nuclear accumulation of STAT1 after IFN-α stimulation, thereby antagonizing the IFN-α-mediated antiviral innate immune signaling pathway and promoting viral infection. Our findings not only provide insights into a novel function of RanBP2 in antiviral innate immunity but may also contribute to the development of new antiviral therapeutic strategies.

The Host Cytoskeleton Functions as a Pleiotropic Scaffold: Orchestrating Regulation of the Viral Life Cycle and Mediating Host Antiviral Innate Immune Responses.

Viruses are obligate intracellular parasites that critically depend on their hosts to initiate infection, complete replication cycles, and generate new progeny virions. To achieve these goals, viruses have evolved numerous elegant strategies to subvert and utilize different cellular machinery. The cytoskeleton is often one of the first components to be hijacked as it provides a convenient transport system for viruses to enter the cell and reach the site of replication. The cytoskeleton is an intricate network involved in controlling the cell shape, cargo transport, signal transduction, and cell division. The host cytoskeleton has complex interactions with viruses during the viral life cycle, as well as cell-to-cell transmission once the life cycle is completed. Additionally, the host also develops unique, cytoskeleton-mediated antiviral innate immune responses. These processes are also involved in pathological damages, although the comprehensive mechanisms remain elusive. In this review, we briefly summarize the functions of some prominent viruses in inducing or hijacking cytoskeletal structures and the related antiviral responses in order to provide new insights into the crosstalk between the cytoskeleton and viruses, which may contribute to the design of novel antivirals targeting the cytoskeleton.

SOCSs: important regulators of host cell susceptibility or resistance to viral infection.

Suppressors of cytokine signaling (SOCSs) are implicated in viral infection and host antiviral innate immune response. Recent studies demonstrate that viruses can hijack SOCSs to inhibit Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway, block the production and signaling of interferons (IFNs). At the same time, viruses can hijack SOCS to regulate non-IFN factors to evade antiviral response. Host cells can also regulate SOCSs to resist viral infection. The competition of the control of SOCSs may largely determine the fate of viral infection and the susceptibility or resistance of host cells, which is of significance for development of novel antiviral therapies targeting SOCSs. Accumulating evidence reveal that the regulation and function of SOCSs by viruses and hostcells are very complicated, which is determined by characteristics of both viruses and host cell types. This report presents a systematic review to evaluate the roles of SOCSs in viral infection and host antiviral responses. One of messages worth attention is that all eight SOCS members should beinvestigated to accurately characterize their roles and relative contribution in each viral infection, which mayhelp identify the most effective SOCS to be used in "individualized" antiviral therapy.

African Swine Fever Virus H240R Protein Inhibits the Production of Type I Interferon through Disrupting the Oligomerization of STING.

African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease in domestic pigs and wild boars. Domestic pigs infected with virulent African swine fever virus (ASFV) isolates have a high mortality, approaching 100%. Identification of ASFV genes related to virulence/pathogenicity and deletion of them are considered to be key steps in the development of live attenuated vaccines, because the ability of ASFV to escape host innate immune responses is related to viral pathogenicity. However, the relationship between the host antiviral innate immune responses and the pathogenic genes of ASFV has not been fully understood. In this study, the ASFV H240R protein (pH240R), a capsid protein of ASFV, was found to inhibit type I interferon (IFN) production. Mechanistically, pH240R interacted with the N-terminal transmembrane domain of stimulator of interferon genes (STING) and inhibited its oligomerization and translocation from the endoplasmic reticulum to the Golgi apparatus. Additionally, pH240R inhibited the phosphorylation of interferon regulatory factor 3 (IRF3) and TANK binding kinase 1 (TBK1), leading to reduced production of type I IFN. Consistent with these results, infection with H240R-deficient ASFV (ASFV-ΔH240R) induced more type I IFN than infection with its parental strain, ASFV HLJ/18. We also found that pH240R may enhance viral replication via inhibition of type I IFN production and the antiviral effect of interferon alpha (IFN-α). Taken together, our findings provide a new explanation for the reduction of ASFV's replication ability by knockout of the H240R gene and a clue for the development of live attenuated ASFV vaccines. IMPORTANCE African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and acute hemorrhagic viral disease with a high mortality, approaching 100% in domestic pigs. However, the relationship between viral pathogenicity and immune evasion of ASFV is not fully understood, which limits the development of safe and effective ASF vaccines, specifically, live attenuated vaccines. In this study, we found that pH240R, as a potent antagonist, inhibited type I IFN production by targeting STING and inhibiting its oligomerization and translocation from the endoplasmic reticulum to the Golgi apparatus. Furthermore, we also found that deletion of the H240R gene reduced viral pathogenicity by enhancing type I IFN production, which decreases ASFV replication. Taken together, our findings provide a clue for the development of an ASFV live attenuated vaccine via deleting the H240R gene.

Porcine reproductive and respiratory syndrome virus infection inhibits NF-κB signaling pathway through cleavage of IKKβ by Nsp4

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious porcine pathogen that causes serious economic losses to the world swine industry. The inhibitor kappa B kinase β (IKKβ), a catalytic subunit of the IKK complex, plays multiple roles in regulating the nuclear transcription factor kappa B (NF-κB) activity and a variety of cytokines transcription involved in immune responses. Here, we reported that the nonstructural protein 4 (Nsp4) of PRRSV cleaved IKKβ at the E378 site to inhibit the activation of NF-κB signaling pathway. Additionally, we clearly showed that cleavage of IKKβ by PRRSV Nsp4 depends on the 3 C-like serine protease activity of Nsp4 because the catalytically inactivate mutants of Nsp4 lost the function to cleave IKKβ. Furthermore, we found that hydrophobic patch at the KD-ULD junction of IKKβ could be disrupted by PRRSV Nsp4 via the cleavage of the E378 site, resulting in disruption of NF-κB activity. Of note, the two cleavage fragments of IKKβ lose their function to phosphorylate IκBα and activate NF-κB signaling pathway. Our findings provide a clue to better understand the pathogenic mechanism of PRRSV involved in PRRSV evasion of host antiviral innate immune responses.

Deletion of African Swine Fever Virus (ASFV) H240R Gene Attenuates the Virulence of ASFV by Enhancing NLRP3-Mediated Inflammatory Responses.

African swine fever (ASF) is a highly contagious infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV), with a mortality rate of up to 100%. In order to replicate efficiently in macrophages and monocytes, ASFV has evolved multiple strategies to evade host antiviral responses. However, the underlying molecular mechanisms by which ASFV-encoded proteins execute immune evasion are not fully understood. In this study, we found that ASFV pH240R strongly inhibits transcription, maturation, and secretion of interleukin-1β (IL-1β). Importantly, pH240R not only targeted NF-κB signaling but also impaired NLRP3 inflammasome activation. In this mechanism, pH240R interacted with NF-kappa-B essential modulator (NEMO), a component of inhibitor of kappa B kinase (IKK) complex and subsequently reduced phosphorylation of IκBα and p65. In addition, pH240R bonded to NLRP3 to inhibit NLRP3 inflammasome activation, resulting in reduced IL-1β production. As expected, infection with H240R-deficient ASFV (ASFV-ΔH240R) induced more inflammatory cytokine expression both in vitro and in vivo than its parental ASFV HLJ/18 strain. Consistently, H240R deficiency reduced the viral pathogenicity in pigs compared with its parental strain. These findings reveal that the H240R gene is an essential virulence factor, and deletion of the H240R gene affects the pathogenicity of ASFV HLJ/18 by enhancing antiviral inflammatory responses, which provides insights for ASFV immune evasion mechanisms and development of attenuated live vaccines and drugs for prevention and control of ASF. IMPORTANCE African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and acute hemorrhagic viral disease of domestic pigs, with a high mortality approaching 100%. ASFV has spread rapidly worldwide and caused huge economic losses and ecological consequences. However, the pathogenesis and immune evasion mechanisms of ASFV are not fully understood, which limits the development of safe and effective ASF attenuated live vaccines. Therefore, investigations are urgently needed to identify virulence factors that are responsible for escaping the host antiviral innate immune responses and provide a new target for development of ASFV live-attenuated vaccine. In this study, we determined that the H240R gene is an essential virulence factor, and its depletion affects the pathogenicity of ASFV by enhancing NLRP3-mediated inflammatory responses, which provides theoretical support for the development of an ASFV attenuated live vaccine.

KLF16 inhibits PEDV replication by activating the type I IFN signaling pathway

KLF16, a member of KLFs (Krüppel-like factors), contributes to the progression of a variety of cancer types. There is, however, still uncertain regarding the role of KLF16 in viral replication and the signaling mechanism of type I IFN. It was discovered that KLF16 inhibited the replication of porcine epidemic diarrhea virus (PEDV) through the type I IFN signaling pathway. Besides, it can also be found that the expression of KLF16 was down-regulated after PEDV infection of LLC-PK1 cells. Furthermore, overexpression of KLF16 inhibited the replication of PEDV in Vero cells as well as LLC-PK1 cells, whereas the replication of PEDV was promoted by the knockdown of KLF16. KLF16 up-regulated the expression of interferons (IFNs) via the TRAF6-pTBK1-pIRF3 pathway with the aim of promoting the host antiviral innate immune response. In addition, the obtained findings proved that KLF16 plays a novel role in antiviral action, thereby offering novel possibilities for preventing and controlling PEDV.

TOB1 attenuates IRF3-directed antiviral responses by recruiting HDAC8 to specifically suppress IFN-β expression

Interferon regulatory factor 3 (IRF3) is a key transcription factor required for the secretion of type I interferons (IFN-α/β) and initiation of antiviral immune response. However, the negative feedback regulator of IRF3-directed antiviral response remains unknown. In this study, we demonstrated that viral infection induced the interaction of the transducer of ERBB2.1 (TOB1) with IRF3, which bound to the promoter region of Ifnb1 in macrophages. TOB1 inhibited Ifnb1 transcription by disrupting IRF3 binding and recruiting histone deacetylase 8 (HDAC8) to the Ifnb1 promoter region. Consequently, TOB1 attenuated IRF3-directed IFN-β expression in virus-infected macrophages. Tob1 deficiency enhanced antiviral response and suppressed viral replication in vivo. Thus, we identified TOB1 as a feedback inhibitor of host antiviral innate immune response and revealed a mechanism underlying viral immune escape.

A New Long Noncoding RNA, MAHAT, Inhibits Replication of Porcine Reproductive and Respiratory Syndrome Virus by Recruiting DDX6 To Bind to ZNF34 and Promote an Innate Immune Response.

Long noncoding RNAs (lncRNAs) have increasingly been recognized as being integral to cellular processes, including the antiviral immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is costly to the global swine industry. To identify PRRSV-related lncRNAs, we performed RNA deep sequencing and compared the profiles of lncRNAs in PRRSV-infected and uninfected Marc-145 cells. We identified a novel lncRNA called MAHAT (maintaining cell morphology-associated and highly conserved antiviral transcript; LTCON_00080558) that inhibits PRRSV replication. MAHAT binds and negatively regulates ZNF34 expression by recruiting and binding DDX6, an RNA helicase forming a complex with ZNF34. Inhibition of ZNF34 expression results in increased type I interferon expression and decreased PRRSV replication. This finding reveals a novel mechanism by which PRRSV evades the host antiviral innate immune response by downregulating the MAHAT-DDX6-ZNF34 pathway. MAHAT could be a host factor target for antiviral therapies against PRRSV infection. IMPORTANCE Long noncoding RNAs (lncRNAs) play important roles in viral infection by regulating the transcription and expression of host genes, and interferon signaling pathways. Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic losses in the swine industry worldwide, but the mechanisms of its pathogenesis and immunology are not fully understood. Here, a new lncRNA, designated MAHAT, was identified as a regulator of host innate immune responses. MAHAT negatively regulates the expression of its target gene, ZNF34, by recruiting and binding DDX6, an RNA helicase, forming a complex with ZNF34. Inhibition of ZNF34 expression increases type I interferon expression and decreases PRRSV replication. This finding suggests that MAHAT has potential as a new target for developing antiviral drugs against PRRSV infection.

Hepatitis C Virus Nonstructural Protein 5A Interacts with Immunomodulatory Kinase IKKε to Negatively Regulate Innate Antiviral Immunity.

Hepatitis C virus (HCV) infection can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV employs diverse strategies to evade host antiviral innate immune responses to mediate a persistent infection. In the present study, we show that nonstructural protein 5A (NS5A) interacts with an NF-κB inhibitor immunomodulatory kinase, IKKε, and subsequently downregulats beta interferon (IFN-β) promoter activity. We further demonstrate that NS5A inhibits DDX3-mediated IKKε and interferon regulatory factor 3 (IRF3) phosphorylation. We also note that hyperphosphorylation of NS5A mediats protein interplay between NS5A and IKKε, thereby contributing to NS5A-mediated modulation of IFN-β signaling. Lastly, NS5A inhibits IKKε-dependent p65 phosphorylation and NF-κB activation. Based on these findings, we propose NS5A as a novel regulator of IFN signaling events, specifically by inhibiting IKKε downstream signaling cascades through its interaction with IKKε. Taken together, these data suggest an additional mechanistic means by which HCV modulates host antiviral innate immune responses to promote persistent viral infection.

Mechanisms of host type I interferon response modulation by the nucleocapsid proteins of alpha- and betacoronaviruses.

Coronaviruses can have a devastating impact on the health of humans and animals. Porcine epidemic diarrhea virus (PEDV) causes extremely high fatality rates in neonatal piglets, whereas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic in humans. As a critical component of the host antiviral innate immune response, type I interferon production and signaling play a very important role, especially in the initial phase of the antiviral responses. Coronaviruses have evolved multiple ways to counteract type I interferon responses. Although the primary functions of the nucleocapsid protein are to facilitate viral RNA replication and package viral genomic RNA into virions, recent studies have shown that the nucleocapsid protein is also involved in virus-host interactions. The aim of this review is to summarize our current understanding of how the nucleocapsid proteins of PEDV and SARS-CoV-2 modulate type I interferon responses. This knowledge will be useful for developing strategies to combat coronavirus infections.

HBx inhibits DNA sensing signaling pathway via ubiquitination and autophagy of cGAS

BackgroundCyclic GMP-AMP synthase (cGAS) is a crucial DNA sensor and plays an important role in host antiviral innate immune responses. During hepatitis B virus (HBV) infection, the cGAS signaling pathway can suppress HBV replication. As an important regulatory protein of HBV, hepatitis B virus X protein (HBx) may serve as an antagonistic character to the cGAS/STING signaling pathway. In this study, we aim to investigate the functional role of HBx in the cGAS/STING signaling pathway.MethodsThe effects of HBx on IFN-β promoter activity were measured by Dual-luciferase reporter assays. Ubiquitination and autophagy were analyzed by Western-blot and Co-immunoprecipitation assays.ResultsOur results show that HBx down-regulates IFN-I production by directly promoting ubiquitination and autophagy degradation of cGAS.ConclusionsHBV can antagonize host cGAS DNA sensing to promote HBV replication and provide novel insights to develop novel approaches against HBV infection.

A Tug of War: Pseudorabies Virus and Host Antiviral Innate Immunity.

The non-specific innate immunity can initiate host antiviral innate immune responses within minutes to hours after the invasion of pathogenic microorganisms. Therefore, the natural immune response is the first line of defense for the host to resist the invaders, including viruses, bacteria, fungi. Host pattern recognition receptors (PRRs) in the infected cells or bystander cells recognize pathogen-associated molecular patterns (PAMPs) of invading pathogens and initiate a series of signal cascades, resulting in the expression of type I interferons (IFN-I) and inflammatory cytokines to antagonize the infection of microorganisms. In contrast, the invading pathogens take a variety of mechanisms to inhibit the induction of IFN-I production from avoiding being cleared. Pseudorabies virus (PRV) belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. PRV is the causative agent of Aujeszky’s disease (AD, pseudorabies). Although the natural host of PRV is swine, it can infect a wide variety of mammals, such as cattle, sheep, cats, and dogs. The disease is usually fatal to these hosts. PRV mainly infects the peripheral nervous system (PNS) in swine. For other species, PRV mainly invades the PNS first and then progresses to the central nervous system (CNS), which leads to acute death of the host with serious clinical and neurological symptoms. In recent years, new PRV variant strains have appeared in some areas, and sporadic cases of PRV infection in humans have also been reported, suggesting that PRV is still an important emerging and re-emerging infectious disease. This review summarizes the strategies of PRV evading host innate immunity and new targets for inhibition of PRV replication, which will provide more information for the development of effective inactivated vaccines and drugs for PRV.

Knockout of HDAC9 Gene Enhances Foot-and-Mouth Disease Virus Replication.

Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease that mainly infects cloven-hoofed animals. Propagation of FMDV by cell culture is an important method to preserve viral biological and antigenic characteristics, which is crucial in FMD monitoring and vaccine production. However, only a few cell lines are sensitive to FMDV, and there is still a lot of room for improvement. Acetylation is an important post-translational modification, which is dynamically regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). However, the study of the relationship between FMDV and HDACs is still unclear. HDAC9 belongs to the class II of HDACs family; in this study, HDAC9 knockout (KO) BHK-21 cells were successfully established using CRISPR/cas9 technology. The results of karyotype analysis, growth curve analysis, and morphological observation showed that the HDAC9 knockout cell line was stable in growth and morphological characteristics. After infection with FMDV, the expression of viral RNA and protein, viral titers, and the copies of viral RNA in HDAC9-KO cells were significantly higher than those in NC cells. Meanwhile, RNA-seq technology was used to sequence HDAC9-KO cells and NC cells infected and uninfected with FMDV. It was found that the differentially expressed innate immune factors containing NFKBIA, SOD2, IL2RG, BCL2L1, CXCL1/2/3, and IL1RAP have significantly enriched in the Jak-STAT, NOD-like receptor, Toll-like receptor, NF-κB, and MAPK signaling pathway. RT-qPCR was performed to detect the expression level of differentially expressed genes and showed consistency with the RNA-seq data. These results preliminarily reveal the role of HDAC9 in host antiviral innate immune response, and the HDAC9-KO cell line could also serve as a useful tool for FMDV research.

Identification of SARS-CoV-2 Papain-like Protease (PLpro) Inhibitors Using Combined Computational Approach.

In the current pandemic, finding an effective drug to prevent or treat the infection is the highest priority. A rapid and safe approach to counteract COVID‐19 is in silico drug repurposing. The SARS‐CoV‐2 PLpro promotes viral replication and modulates the host immune system, resulting in inhibition of the host antiviral innate immune response, and therefore is an attractive drug target. In this study, we used a combined in silico virtual screening for candidates for SARS‐CoV‐2 PLpro protease inhibitors. We used the Informational spectrum method applied for Small Molecules for searching the Drugbank database followed by molecular docking. After in silico screening of drug space, we identified 44 drugs as potential SARS‐CoV‐2 PLpro inhibitors that we propose for further experimental testing.