Influenza Virus Replication Research Articles

Cellular NS1-BP protein interacts with the mRNA export receptor NXF1 to mediate nuclear export of influenza virus M mRNAs

Influenza A viruses have eight genomic RNAs that are transcribed in the host cell nucleus. Two of the viral mRNAs undergo alternative splicing. The M1 mRNA encodes the matrix protein 1 (M1) and is also spliced into M2 mRNA, which encodes the proton channel matrix protein 2 (M2). Our previous studies have shown that the cellular NS1-binding protein (NS1-BP) interacts with the viral non-structural protein 1 (NS1) and M1 mRNA to promote M1 to M2 splicing. Another pool of NS1 protein binds the mRNA export receptor NXF1 (nuclear RNA export factor-1), leading to nuclear retention of cellular mRNAs. Here we show a series of biochemical and cell biological findings that suggest a model for nuclear export of M1 and M2 mRNAs despite the mRNA nuclear export inhibition imposed by the viral NS1 protein. NS1-BP competes with NS1 for NXF1 binding, allowing the recruitment of NXF1 to the M mRNAs after splicing. NXF1 then binds GANP (Germinal-center Associated Nuclear Protein), a member of the TRanscription and EXport complex (TREX)-2. Although both NS1 and NS1-BP remain in complex with GANP-NXF1, they dissociate once this complex docks at the nuclear pore complex (NPC), and the M mRNAs are translocated to the cytoplasm. Since this mRNA nuclear export pathway is key for expression of M1 and M2 proteins that function in viral intracellular trafficking and budding, these viral-host interactions are critical for influenza virus replication.

Candidate Genes Associated with Survival Following Highly Pathogenic Avian Influenza Infection in Chickens.

Highly pathogenic strains of avian influenza (HPAI) devastate poultry flocks and result in significant economic losses for farmers due to high mortality, reduced egg production, and mandated euthanization of infected flocks. Within recent years, HPAI outbreaks have affected egg production flocks across the world. The H5N2 outbreak in the US in 2015 resulted in over 99% mortality. Here, we analyze sequence data from chickens that survived (42 cases) along with uninfected controls (28 samples) to find genomic regions that differ between these two groups and that, therefore, may encompass prime candidates that are resistant to HPAI. Blood samples were obtained from survivors of the 2015 HPAI outbreak plus age and genetics-matched non-affected controls. A whole-genome sequence was obtained, and genetic variants were characterized and used in a genome-wide association study to identify regions showing significant association with survival. Regions associated with HPAI resistance were observed on chromosomes 1, 2, 5, 8, 10, 11, 15, 20, and 28, with a number of candidate genes identified. We did not detect a specific locus which could fully explain the difference between survivors and controls. Influenza virus replication depends on multiple components of the host cellular machinery, with many genes involved in the host response.

PB1-F2 of low pathogenicity H7N7 restricts apoptosis in avian cells

Avian influenza viruses (AIV) pose a continuous challenge to global health and economy. While countermeasures exist to control outbreaks in poultry, the persistent circulation of AIV in wild aquatic and shorebirds presents a significant challenge to effective disease prevention efforts. PB1-F2 is a non-structural protein expressed from a second open reading frame (+1) of the polymerase basic 1 (PB1) segment. The sequence and length of the PB1-F2 protein can vary depending on the host of origin. While avian isolates typically carry full-length PB1-F2, isolates from mammals, often express truncated forms. The selective advantage of the full-length PB1-F2 in avian isolates is not fully understood. Most research on the role of PB1-F2 in influenza virus replication has been conducted in mammalian systems, where PB1-F2 interfered with the host immune response and induced apoptosis. Here, we used Low Pathogenicity (LP) AIV H7N7 expressing full-length PB1-F2 as well as a knockout mutant. We found that the full-length PB1-F2 of LPAIV prolonged survival of infected cells by limiting apoptotic cell death. Furthermore, PB1-F2 knockout LPAIV significantly decreased MHC-I expression on fibroblasts, delayed tissue healing and increased phagocytic uptake of infected cells, whereas LPAIV expressing PB1-F2 has limited effects. These findings indicate that full-length PB1-F2 enables AIV to cause prolonged infections without severely harming the avian host. Our observations may explain maintenance of AIV in the natural bird reservoir in absence of severe clinical signs.

Nasal symbiont Staphylococcus epidermidis restricts influenza A virus replication via the creation of a polyamine-deficient cellular environment

Studies on the immune-regulatory roles played by the commensal microbes residing in the nasal mucosa consider the contribution of antiviral immune responses. Here, we sought to identify the nasal microbiome, Staphylococcus epidermidis-regulated antiviral immune responses and the alteration of polyamine metabolites in nasal epithelium. We found that polyamines were required for the life cycle of influenza A virus (IAV) and depletion of polyamines disturbed IAV replication in normal human nasal epithelial (NHNE) cells. Inoculation of S. epidermidis also suppressed IAV infection and the concentration of polyamines including putrescine, spermidine, and spermine was completely attenuated in S. epidermidis-inoculated NHNE cells. S. epidermidis activated the enzyme involved in the production of ornithine from arginine and downregulated the activity of the enzyme involved in the production of putrescine from ornithine in nasal epithelium. S. epidermidis also induced the activation of enzymes that promote the extracellular export of spermine and spermidine in NHNE cells. Our findings demonstrate that S. epidermidis is shown to be able of creating an intracellular environment lacking polyamines in the nasal epithelium and promote the balance of cellular polyamines in favor of the host to restrict influenza virus replication.

Indole-core inhibitors of influenza a neuraminidase: iterative medicinal chemistry and molecular modeling

Influenza viruses that cause seasonal and pandemic flu are a permanent health threat. The surface glycoprotein, neuraminidase, is crucial for the infectivity of the virus and therefore an attractive target for flu drug discovery campaigns. We have designed and synthesized more than 40 3-indolinone derivatives. We mainly investigated the role of substituents at the 2 position of the core as well as the introduction of substituents or a nitrogen atom in the fused phenyl ring of the core for inhibition of influenza virus neuraminidase activity and replication in vitro and in vivo. After evaluating the compounds for their ability to inhibit the viral neuraminidase, six potent inhibitors 3c, 3e, 7c, 12o, 12v, 18d were progressed to evaluate for cytotoxicity and inhibition of influenza virus A/PR/8/34 replication in in MDCK cells. Two hit compounds 3e and 12o were tested in an animal model of influenza virus infection. Molecular mechanism of the 3-indolinone derivatives interactions with the neuraminidase was revealed in molecular dynamic simulations. Proposed inhibitors bind to the 430-cavity that is different from the conventional binding site of commercial compounds. The most promising 3-indolinone inhibitors demonstrate stronger interactions with the neuraminidase in molecular models that supports proposed binding site.

Inhibition of RAN attenuates influenza a virus replication and nucleoprotein nuclear export

ABSTRACT Nuclear export of the viral ribonucleoprotein (vRNP) is a critical step in the influenza A virus (IAV) life cycle and may be an effective target for the development of anti-IAV drugs. The host factor ras-related nuclear protein (RAN) is known to participate in the life cycle of several viruses, but its role in influenza virus replication remains unknown. In the present study, we aimed to determine the function of RAN in influenza virus replication using different cell lines and subtype strains. We found that RAN is essential for the nuclear export of vRNP, as it enhances the binding affinity of XPO1 toward the viral nuclear export protein NS2. Depletion of RAN constrained the vRNP complex in the nucleus and attenuated the replication of various subtypes of influenza virus. Using in silico compound screening, we identified that bepotastine could dissociate the RAN-XPO1-vRNP trimeric complex and exhibit potent antiviral activity against influenza virus both in vitro and in vivo. This study demonstrates the important role of RAN in IAV replication and suggests its potential use as an antiviral target.

A small protein encoded by PCBP1-AS1 is identified as a key regulator of influenza virus replication via enhancing autophagy.

Many annotated long noncoding RNAs (lncRNAs) contain small open reading frames (sORFs), some of which have been demonstrated to encode small proteins or micropeptides with fundamental biological importance. However, functions of lncRNAs-encoded small proteins or micropeptides in viral pathogenesis remain largely unexplored. Here, we identified a 110-amino acid small protein as a key regulator of influenza A virus (IAV) replication. This small protein that we call PESP was encoded by the putative lncRNA PCBP1-AS1. It was observed that both PCBP1-AS1 and PESP were significantly upregulated by IAV infection. Furthermore, they were markedly induced by treatment with either type I or type III interferon. Overexpression of either PCBP1-AS1 or PESP alone significantly enhanced IAV replication. In contrast, shRNA-mediated knockdown of PCBP1-AS1 or CRISPR/Cas9-mediated knockout of PESP markedly inhibited the viral production. Moreover, the targeted deletion or mutation of the sORF within the PCBP1-AS1 transcript, which resulted in the disruption of PESP expression, significantly diminished the capacity of PCBP1-AS1 to enhance IAV replication, underscoring the indispensable role of PESP in the facilitation of IAV replication by PCBP1-AS1. Interestingly, overexpression of PESP enhanced the IAV-induced autophagy by increasing the expression of ATG7, an essential autophagy effector enzyme. We also found that the 7-22 amino acids at the N-terminus of PESP were crucial for its functionality in modulating ATG7 expression and action as an enhancer of IAV replication. Additionally, HSP90AA1, a protein identified previously as a facilitator of autophagy, was found to interact with PESP, resulting in the stabilization of PESP and consequently an increase in the production of IAV. These data reveal a critical lncRNA-encoded small protein that is induced and exploited by IAV during its infection, and provide a significant insight into IAV-host interaction network.

Zanamivir and baloxavir combination to cure persistent influenza and coronavirus infections after hematopoietic stem cell transplant

ObjectivesImmunocompromised patients may experience prolonged shedding of influenza virus potentially leading to severe infections. Alternatives to monotherapy with neuraminidase inhibitors should be evaluated to entirely suppress viral replication and prevent drug-resistant mutations. MethodsWe investigated the clinical and virological evolution in a case of persistent influenza A and human coronavirus OC43 (HCoV-OC43) coinfection in a hematopoietic stem cell transplant recipient after different therapeutic strategies. ResultsSuccessive oseltamivir and zanamivir monotherapies failed to control both infections, with positive results persisting for over 110 days each. This led to the emergence of highly resistant oseltamivir strains due to neuraminidase mutations (E119V and R292K) followed by a deletion (del245-248), while maintaining sensitivity to zanamivir. The intra-host viral diversity data showed that the treatments impacted viral diversity of influenza virus, but not of HCoV-OC43. Considering the patient's underlying condition and the impact of prolonged viral shedding on pulmonary function, eradicating the influenza virus was necessary. A 10-day regimen combining zanamivir and baloxavir-marboxil effectively controlled influenza virus replication and was associated with the clearance of HCoV-OC43, finally resulting in comprehensive respiratory recovery. ConclusionThese observations underscore the importance of further investigating combination treatments as the primary approach to achieve influenza eradication in immunocompromised patients.

In turkeys, unlike chickens, the non-structural NS1 protein does not play a significant role in the replication and tissue tropism of the H7N1 avian influenza virus

ABSTRACT The economic losses caused by high pathogenicity (HP) avian influenza viruses (AIV) in the poultry industry worldwide are enormous. Although chickens and turkeys are closely related Galliformes, turkeys are thought to be a bridging host for the adaptation of AIV from wild birds to poultry because of their high susceptibility to AIV infections. HPAIV evolve from low pathogenicity (LP) AIV after circulation in poultry through mutations in different viral proteins, including the non-structural protein (NS1), a major interferon (IFN) antagonist of AIV. At present, it is largely unknown whether the virulence determinants of HPAIV are the same in turkeys and chickens. Previously, we showed that mutations in the NS1 of HPAIV H7N1 significantly reduced viral replication in chickens in vitro and in vivo. Here, we investigated the effect of NS1 on the replication and virulence of HPAIV H7N1 in turkeys after inoculation with recombinant H7N1 carrying a naturally truncated wild-type NS1 (with 224 amino-acid “aa” in length) or an extended NS1 with 230-aa similar to the LP H7N1 ancestor. There were no significant differences in multiple-cycle viral replication or in the efficiency of NS1 in blocking IFN induction in the cell culture. Similarly, all viruses were highly virulent in turkeys and replicated at similar levels in various organs and swabs collected from the inoculated turkeys. These results suggest that NS1 does not play a role in the virulence or replication of HPAIV H7N1 in turkeys and further indicate that the genetic determinants of HPAIV differ in these two closely related galliform species.

Anti-Influenza activity of compounds derived from medicinal plants (Part I)

This review (I part) presents an analysis of publications devoted to the ant-viral antiinfluenza activity of medicinal plants. It has shown that 63 species are found to be promising sources for obtaining anti-influenza drugs. Their roots, stems, leaves and fruits contain different chemical compounds inhibiting reproduction of influenza virus, both in vitro and in vivo. These compounds have different mechanisms of action. Some of them act upon components of the viral particle, others upon signaling pathways in the host cells. The first part of our review focuses on 28 species.

Sorbicillinoid HSL-2 inhibits the infection of influenza A virus via interaction with the PPAR-γ/NF-κB pathway

BackgroundDrug resistance is an important factor in the fight against influenza A virus (IAV). Natural products offer a rich source of lead compounds for the discovery of novel antiviral drugs. In a previous study, we isolated the sorbicillinoid polyketide HSL-2 from the mycelium of fungus Trichoderma sp. T-4-1. Here, we show that this compound exerts strong antiviral activity against a panel of IAVs. MethodsThe immunofluorescence and qRT-PCR assays were used to detect the inhibitory effect of HSL-2 toward the replication of influenza virus and IAV-induced expression of the pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. ResultsThe results indicated that HSL-2 inhibited influenza virus replication, and it significantly inhibited IAV-induced overexpression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β through modulating the PPAR-γ/NF-κB pathway. Notably, this effect was decreased when cells were transfected with PPAR-γ siRNA or treated with the PPAR-γ inhibitor T0070907. In addition, HSL-2 was able to attenuate lung inflammatory responses and to improve lung lesions in a mouse model of IAV infection. ConclusionsIn this paper, we identified a microbial secondary metabolite, HSL-2, with anti-influenza virus activity. This report is the first to describe the antiviral activity and mechanism of action of HSL-2, and it provides a new strategy for the development of novel anti-influenza virus drugs from natural sources.

The Identification and Function of Linc01615 on Influenza Virus Infection and Antiviral Response.

Influenza virus infection poses a great threat to human health globally each year. Non-coding RNAs (ncRNAs) in the human genome have been reported to participate in the replication process of the influenza virus, among which there are still many unknowns about Long Intergenic Non-Coding RNAs (LincRNAs) in the cell cycle of viral infections. Here, we observed an increased expression of Linc01615 in A549 cells upon influenza virus PR8 infection, accompanied by the successful activation of the intracellular immune system. The knockdown of Linc01615 using the shRNAs promoted the proliferation of the influenza A virus, and the intracellular immune system was inhibited, in which the expressions of IFN-β, IL-28A, IL-29, ISG-15, MX1, and MX2 were decreased. Predictions from the catRAPID website suggested a potential interaction between Linc01615 and DHX9. Also, knocking down Linc01615 promoted influenza virus proliferation. The subsequent transcriptome sequencing results indicated a decrease in Linc01615 expression after influenza virus infection when DHX9 was knocked down. Further analysis through cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) in HEK293 cells stably expressing DHX9 confirmed the interaction between DHX9 and Linc01615. We speculate that DHX9 may interact with Linc01615 to partake in influenza virus replication and that Linc01615 helps to activate the intracellular immune system. These findings suggest a deeper connection between DHX9 and Linc01615, which highlights the significant role of Linc01615 in the influenza virus replication process. This research provides valuable insights into understanding influenza virus replication and offers new targets for preventing influenza virus infections.

Lysophosphatidylcholines Promote Influenza Virus Reproduction through the MAPK/JNK Pathway in PMA-Differentiated THP-1 Macrophages.

Obesity and metabolic syndrome alter serum lipid profiles. They also increase vulnerability to viral infections and worsen the survival rate and symptoms after infection. How serum lipids affect influenza virus proliferation is unclear. Here, we investigated the effects of lysophosphatidylcholines on influenza A virus (IAV) proliferation. IAV particles in the culture medium were titrated using extraction-free quantitative PCR, and viral RNA and protein levels were assessed using real-time PCR and Western blot, respectively. RNA sequencing data were analyzed using PCA and heatmap analysis, and pathway analysis was performed using the KEGG mapper and PathIN tools. Statistical analysis was conducted using SPSS21.0. LPC treatment of THP-1 cells significantly increased IAV proliferation and IAV RNA and protein levels, and saturated LPC was more active in IAV RNA expression than unsaturated LPC was. The functional analysis of genes affected by LPCs showed that the expression of genes involved in IAV signaling, such as suppressor of cytokine signaling 3 (SOCS3), phosphoinositide-3-kinase regulatory subunit 3 (PI3K) and AKT serine/threonine kinase 3 (AKT3), Toll-like receptor 7 (TKR7), and interferon gamma receptor 1 (IFNGR1), was changed by LPC. Altered influenza A pathways were linked with MAPK and PI3K/AKT signaling. Treatment with inhibitors of MAPK or PI3K attenuated viral gene expression changes induced by LPCs. The present study shows that LPCs stimulated virus reproduction by modifying the cellular environment to one in which viruses proliferated better. This was mediated by the MAPK, JNK, and PI3K/AKT pathways. Further animal studies are needed to confirm the link between LPCs from serum or the respiratory system and IAV proliferation.

RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-β expression during virus infection, the expression level of IFN-β in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

Sodium Polyoxotungstate Inhibits the Replication of Influenza Virus by Blocking the Nuclear Import of vRNP.

Both pandemic and seasonal influenza are major health concerns, causing significant mortality and morbidity. Current influenza drugs primarily target viral neuraminidase and RNA polymerase, which are prone to drug resistance. Polyoxometalates (POMs) are metal cation clusters bridged by oxide anions. They have exhibited potent anti-tumor, antiviral, and antibacterial effects. They have remarkable activity against various DNA and RNA viruses, including human immunodeficiency virus, herpes simplex virus, hepatitis B and C viruses, dengue virus, and influenza virus. In this study, we have identified sodium polyoxotungstate (POM-1) from an ion channel inhibitor library. In vitro, POM-1 has been demonstrated to have potent antiviral activity against H1N1, H3N2, and oseltamivir-resistant H1N1 strains. POM-1 can cause virion aggregation during adsorption, as well as endocytosis. However, the aggregation is reversible; it does not interfere with virus adsorption and endocytosis. Our results suggest that POM-1 exerts its antiviral activity by inhibiting the nuclear import of viral ribonucleoprotein (vRNP). This distinct mechanism of action, combined with its wide range of efficacy, positions POM-1 as a promising therapeutic candidate for influenza treatment and warrants further investigation.

4'-Fluorouridine inhibits alphavirus replication and infection in vitro and in vivo.

Chikungunya virus (CHIKV) is an enveloped, positive-sense RNA virus that has re-emerged to cause millions of human infections worldwide. In humans, acute CHIKV infection causes fever and severe muscle and joint pain. Chronic and debilitating arthritis and joint pain can persist for months to years. To date, there are no approved antivirals against CHIKV. Recently, the ribonucleoside analog 4'-fluorouridine (4'-FlU) was reported as a highly potent orally available inhibitor of SARS-CoV-2, respiratory syncytial virus, and influenza virus replication. In this study, we assessed 4'-FlU's potency and breadth of inhibition against a panel of alphaviruses including CHIKV, and found that it broadly suppressed alphavirus production in cell culture. 4'-FlU acted on the viral RNA replication step, and the first 4 hours post-infection were the critical time for its antiviral effect. In vitro replication assays identified nsP4 as the target of inhibition. In vivo, treatment with 4'-FlU reduced disease signs, inflammatory responses, and viral tissue burden in mouse models of CHIKV and Mayaro virus infection. Treatment initiated at 2 hours post-infection was most effective; however, treatment initiated as late as 24-48 hours post-infection produced measurable antiviral effects in the CHIKV mouse model. 4'-FlU showed effective oral delivery in our mouse model and resulted in the accumulation of both 4'-FlU and its bioactive triphosphate form in tissues relevant to arthritogenic alphavirus pathogenesis. Together, our data indicate that 4'-FlU inhibits CHIKV infection in vitro and in vivo and is a promising oral therapeutic candidate against CHIKV infection.IMPORTANCEAlphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro, there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4'-fluorouridine (4'-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4'-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4'-FlU and accumulation of both 4'-FlU and its bioactive form in relevant tissues. In summary, 4'-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA.

Guanylate-binding protein 1 inhibits inflammatory factors produced by H5N1 virus through Its GTPase activity

The combination of inflammatory factors resulting from an influenza A virus infection is one of the main causes of death in host animals. Studies have shown that guinea pig guanosine monophosphate binding protein 1 (guanylate-binding protein 1, gGBP1) can downregulate cytokine production induced by the influenza virus. Therefore, exploring the innate immune defense mechanism of GBP1 in the process of H5N1 influenza virus infection has important implications for understanding the pathogenic mechanism, disease prevention, and the control of influenza A virus infections. We found that, in addition to inhibiting the early replication of influenza virus, gGBP1 also inhibited the production of CCL2 and CXCL10 cytokines induced by the influenza virus as well as the proliferation of mononuclear macrophages induced by these cytokines. These findings further confirmed that gGBP1 inhibited the production of cytokines through its GTPase activity and cell proliferation through its C-terminal α-helix structure. This study revealed the effect of gGBP1 on the production of cellular inflammatory factors during influenza virus infection and determined the key amino acid residues that assist in the inhibitory processes mediated by gGBP1.

Tanreqing injection inhibits influenza virus replication by promoting the fusion of autophagosomes with lysosomes: An integrated pharmacological study

Ethnopharmacological relevanceTanreqing injection (TRQ) is widely used, traditional Chinese medicine (TCM) injection used in China to treat respiratory infections. Modern pharmacological studies have confirmed that TRQ can protect against influenza viruses. However, the mechanism by which TRQ inhibits influenza viruses remains unclear. Aim of the studyTo explore the therapeutic effects and possible mechanisms of TRQ inhibition by the influenza virus. Materials and methodsUltra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was used to determine the chemical composition of TRQ. Isobaric tags for relative and absolute quantification (iTRAQ) were used to define differential proteins related to TRQ inhibition of viruses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for functional annotation. For experimental validation, we established an in vitro model of the influenza virus infection by infecting A549 cells with the virus. The detection of the signaling pathway was carried out through qPCR, western blotting,and immunofluorescence. ResultsFifty one components were identified using UPLC/Q-TOF MS. We confirmed the inhibitory effect of TRQ on influenza virus replication in vitro. Ninety nine differentially expressed proteins related to the inhibitory effect of TRQ were identified using iTRAQ. KEGG functional enrichment analysis showed that the TRQ may inhibit influenza virus replication by affecting autophagy. Through network analysis, 29 targets were selected as major targets, and three key targets, HSPA5, PARP1, and GAPDH, may be the TRQ targets affecting autophagy. In vitro experiments showed that TRQ inhibits influenza virus replication by interfering with the expression and localization of STX17 and VAMP8 proteins, thereby promoting the fusion of autophagosomes with lysosomes. ConclusionTRQ inhibits influenza virus replication by promoting the fusion of autophagosomes with lysosomes. We additionally established potential gene and protein targets which are affected by TRQ. Therefore, our findings provide new therapeutic targets and a foundation further studies on influenza treatment with TRQ.

Chicken UFL1 Restricts Avian Influenza Virus Replication by Disrupting the Viral Polymerase Complex and Facilitating Type I IFN Production.

During avian influenza virus (AIV) infection, host defensive proteins promote antiviral innate immunity or antagonize viral components to limit viral replication. UFM1-specific ligase 1 (UFL1) is involved in regulating innate immunity and DNA virus replication in mammals, but the molecular mechanism by which chicken (ch)UFL1 regulates AIV replication is unclear. In this study, we first identified chUFL1 as a negative regulator of AIV replication by enhancing innate immunity and disrupting the assembly of the viral polymerase complex. Mechanistically, chUFL1 interacted with chicken stimulator of IFN genes (chSTING) and contributed to chSTING dimerization and the formation of the STING-TBK1-IRF7 complex. We further demonstrated that chUFL1 promoted K63-linked polyubiquitination of chSTING at K308 to facilitate chSTING-mediated type I IFN production independent of UFMylation. Additionally, chUFL1 expression was upregulated in response to AIV infection. Importantly, chUFL1 also interacted with the AIV PA protein to inhibit viral polymerase activity. Furthermore, chUFL1 impeded the nuclear import of the AIV PA protein and the assembly of the viral polymerase complex to suppress AIV replication. Collectively, these findings demonstrate that chUFL1 restricts AIV replication by disrupting the viral polymerase complex and facilitating type I IFN production, which provides new insights into the regulation of AIV replication in chickens.

Cryo-EM structure of Influenza A virus NS1 and antiviral protein kinase PKR complex

Influenza A virus is the cause of a widespread human disease with high morbidity and mortality rates. The influenza virus encodes non-structural protein 1 (NS1), an exceedingly multifunctional virulence component. NS1 plays essential roles in viral replication and evasion of the cellular innate immune system. Protein kinase RNA-activated also known as protein kinase R (PKR) phosphorylates translation initiation factor eIF-2α on serine 51 to inhibit protein synthesis in virus-infected mammalian cells. Consequently, PKR activation inhibits mRNA translation, which results in the assert of both viral protein synthesis and cellular and possibly apoptosis in response to virus infection. Host signaling pathways are important in the replication of influenza virus, but the mechanisms involved remain to be characterized. Herein, the structure of NS1 and PKR complex was determined using Cryo-EM. We found the N91, E94, and G95 residues of PKR bind directly with N188, D125, and K126, respectively, of NS1. Furthermore, the study shows that PKR peptide offers a potential treatment for Influenza A virus infections.