ABSTRACT
Early characterization of the immunostimulatory potential of therapeutic antisense oligonucleotides (ASOs) is crucial. At present, little is known about the toll-like receptor 9 (TLR9)-mediated immunostimulatory potential of third-generation locked nucleic acid (LNA)-modified ASOs. In this study, we have systematically investigated the TLR9-activating potential of LNA-modified oligonucleotides using different mouse and human cell culture systems. Although it has been reported that LNA modifications as well as cytosine methylation of 5'-cytosine-phosphate-guanine-3' (CpG) motifs can reduce TLR9 stimulation by phosphorothioate (PTO)-modified oligonucleotides, we identified CpG-containing LNA gapmers with substantial TLR9-stimulatory activity. We further identified immunostimulatory LNA gapmers without CpG motifs. Unexpectedly, methylation of cytosines only within the CpG motif did not necessarily reduce but could even increase TLR9 activation. In contrast, systematic methylation of all cytosines reduced or even abrogated TLR9 activation in most cases. Context dependently, the introduction of LNA-modifications into the flanks could either increase or decrease TLR9 stimulation. Overall, our results indicate that TLR9-dependent immunostimulatory potential is an individual feature of an oligonucleotide and needs to be investigated on a case-by-case basis.
ABSTRACT
BACKGROUND: The Spike protein mutation severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to decreased protective effect of various vaccines and mAbs, suggesting that blocking SARS-CoV-2 infection by targeting host factors would make the therapy more resilient against virus mutations. Angiotensin-converting enzyme 2 (ACE2) is the host receptor of SARS-CoV-2 and its variants, as well as many other coronaviruses. Downregulation of ACE2 expression in the respiratory tract may prevent viral infection. Antisense oligonucleotides (ASOs) can be rationally designed on the basis of sequence data, require no delivery system, and can be administered locally. OBJECTIVE: We sought to design ASOs that can block SARS-CoV-2 by downregulating ACE2 in human airway. METHODS: ACE2-targeting ASOs were designed using a bioinformatic method and screened in cell lines. Human primary nasal epithelial cells cultured at the air-liquid interface and humanized ACE2 mice were used to detect the ACE2 reduction levels and the safety of ASOs. ASO-pretreated nasal epithelial cells and mice were infected and then used to detect the viral infection levels. RESULTS: ASOs reduced ACE2 expression on mRNA and protein level in cell lines and in human nasal epithelial cells. Furthermore, they efficiently suppressed virus replication of 3 different SARS-CoV-2 variants in human nasal epithelial cells. In vivo, ASOs also downregulated human ACE2 in humanized ACE2 mice and thereby reduced viral load, histopathologic changes in lungs, and increased survival of mice. CONCLUSIONS: ACE2-targeting ASOs can effectively block SARS-CoV-2 infection. Our study provides a new approach for blocking SARS-CoV-2 and other ACE2-targeting virus in high-risk populations.
ABSTRACT
Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA's receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus-cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA's receptor binding preference does not necessarily reflect virus-cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.
Subject(s)
Influenza A virus , Influenza, Human , Humans , Influenza A virus/metabolism , Receptors, Virus/metabolism , Virus Attachment , Polysaccharides/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/chemistryABSTRACT
Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns.
Subject(s)
Glucose , Lymphadenopathy , Angiopoietin-Like Protein 4/genetics , Animals , Mice , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , TriglyceridesABSTRACT
Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular basis of this species barrier is incompletely understood. Here we combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain and observe striking differences in viral protein synthesis. Most importantly, the matrix protein M1 is inefficiently produced by the bird-adapted strain. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Strain-specific M segment splicing is controlled by the 3' splice site and functionally important for permissive infection. In silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, we identify M segment RNA splicing as a viral host range determinant.
Subject(s)
Influenza A virus/metabolism , Influenza, Human/metabolism , Orthomyxoviridae Infections/metabolism , Proteome/metabolism , Proteomics/methods , Animals , Birds , Dogs , HEK293 Cells , Host Specificity/genetics , Humans , Influenza A virus/genetics , Influenza A virus/physiology , Influenza, Human/virology , Madin Darby Canine Kidney Cells , Orthomyxoviridae Infections/virology , RNA Splicing , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolismABSTRACT
Ebola virus is the causative agent of Ebola virus disease, a severe, often fatal illness in humans. So far, there are no US Food and Drug Administration (FDA)-approved therapeutics directed against Ebola virus. Here, we selected the host factor Niemann-Pick C1 (NPC1), which has been shown to be essential for Ebola virus entry into host cytoplasm, as a therapeutic target for suppression by locked nucleic acid-modified antisense oligonucleotides. Screening of antisense oligonucleotides in human and murine cell lines led to identification of candidates with up to 94% knockdown efficiency and 50% inhibitory concentration (IC50) values in the submicromolar range. Selected candidate oligonucleotides led to efficient NPC1 protein knockdown in vitro without alteration of cell viability. Furthermore, they did not have immune stimulatory activity in cell-based assays. Treatment of Ebola-virus-infected HeLa cells with the most promising candidates resulted in significant (>99%) virus titer reduction, indicating that antisense oligonucleotides against NPC1 are a promising therapeutic approach for treatment of Ebola virus infection.
ABSTRACT
BACKGROUND: Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available. OBJECTIVE: We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study. METHODS: A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5'-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against in vitro-expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell-based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and ex vivo-cultured human sinonasal tissue. RESULTS: Screening phase-generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency. CONCLUSIONS: Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials.
Subject(s)
Antiviral Agents/pharmacology , DNA, Catalytic/pharmacology , RNA, Viral/drug effects , Rhinovirus , Virus Replication/drug effects , Common Cold/prevention & control , Drug Discovery , HumansABSTRACT
Virus infections induce sensitive antiviral responses within the host cell. The RNA helicase retinoic acid-inducible gene I (RIG-I) is a key sensor of influenza virus RNA that induces the expression of antiviral type I interferons. Recent evidence suggests a complex pattern of RIG-I regulation involving multiple interactions and cellular sites. In an approach employing affinity purification and quantitative mass spectrometry, we identified proteins with increased binding to RIG-I in response to influenza B virus infection. Among them was the RIG-I related RNA helicase DEAD box helicase 6 (DDX6), a known component of cytoplasmic mRNA-ribonucleoprotein (mRNP) granules like P-bodies and stress granules (SGs). RIG-I and DDX6 both localized to the cytosol and were detected in virus-induced SGs. Coimmunoprecipitation assays detected a basal level of complexes harboring RIG-I and DDX6 that increased after infection. Functionally, DDX6 augmented RIG-I mediated induction of interferon (IFN)-ß expression. Notably, DDX6 was found to bind viral RNA capable to stimulate RIG-I. These findings imply a novel function for DDX6 as an RNA co-sensor and signaling enhancer for RIG-I.
Subject(s)
Antiviral Agents/metabolism , DEAD Box Protein 58/metabolism , DEAD-box RNA Helicases/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction , A549 Cells , Cytoplasmic Granules/metabolism , DEAD Box Protein 58/chemistry , Gene Expression Regulation , HeLa Cells , Humans , Interferon-beta/genetics , Interferon-beta/metabolism , Protein Binding , Protein Domains , Protein Transport , RNA, Viral/metabolism , Receptors, ImmunologicABSTRACT
Influenza A virus (IAV) infections are a major cause for respiratory disease in humans, which affects all age groups and contributes substantially to global morbidity and mortality. IAV have a large natural host reservoir in avian species. However, many avian IAV strains lack adaptation to other hosts and hardly propagate in humans. While seasonal or pandemic IAV strains replicate efficiently in permissive human cells, many avian IAV cause abortive nonproductive infections in these hosts despite successful cell entry. However, the precise reasons for these differential outcomes are poorly defined. We hypothesized that the distinct course of an IAV infection with a given virus strain is determined by the differential interplay between specific host and viral factors. By using Spike-in SILAC mass spectrometry-based quantitative proteomics we characterized sets of cellular factors whose abundance is specifically up- or downregulated in the course of permissive versus nonpermissive IAV infection, respectively. This approach allowed for the definition and quantitative comparison of about 3500 proteins in human lung epithelial cells in response to seasonal or low-pathogenic avian H3N2 IAV. Many identified proteins were similarly regulated by both virus strains, but also 16 candidates with distinct changes in permissive versus nonpermissive infection were found. RNAi-mediated knockdown of these differentially regulated host factors identified Vpr binding protein (VprBP) as proviral host factor because its downregulation inhibited efficient propagation of seasonal IAV whereas overexpression increased viral replication of both seasonal and avian IAV. These results not only show that there are similar differences in the overall changes during permissive and nonpermissive influenza virus infections, but also provide a basis to evaluate VprBP as novel anti-IAV drug target.
Subject(s)
Carrier Proteins/metabolism , Epithelial Cells/metabolism , Epithelial Cells/virology , Influenza A Virus, H3N2 Subtype/physiology , Influenza, Human/virology , Proteomics/methods , A549 Cells , Cluster Analysis , Endocytosis , Epithelial Cells/pathology , HEK293 Cells , Humans , Isotope Labeling , Lung/pathology , Mass Spectrometry , Protein Serine-Threonine Kinases , Proteome/metabolism , RNA, Small Interfering/metabolism , Ubiquitin-Protein Ligases , Virus ReplicationABSTRACT
The influenza virus non-structural protein 1 (NS1) is a multifunctional virulence factor that plays a crucial role during infection by blocking the innate antiviral immune response of infected cells. In contrast to the well-studied NS1 protein of influenza A virus, knowledge about structure and functions of the influenza B virus homologue B/NS1, which shares less than 25 % sequence identity, is still limited. Here, we report on a reverse genetic analysis to study the role of a highly conserved class II Src homology 3 domain-binding motif matching the consensus PxxPx(K/R) that we identified at positions 122-127 of the B/NS1 protein. Surprisingly, glycine substitutions in the Src homology 3 domain-binding motif increased virus replication up to three orders of magnitude in human lung cells. Enhanced mutant virus propagation was accompanied by increased gene expression and apoptosis induction linking this motif to the control of programmed cell death. A MS-based interactome study revealed that the glycine substitutions facilitate binding of B/NS1 to heat shock protein 90-beta (HSP90ß). Moreover, recruitment of the viral polymerase basic protein 2 to the B/NS1-HSP90ß complex was observed. Pharmacological inhibition of HSP90 reduced mutant virus propagation suggesting that the mutation-induced involvement of HSP90ß enhanced viral replication. This study not only functionally characterizes a conserved motif within the B/NS1 protein, but also illustrates a rare example in which mutation of a highly conserved sequence within a viral protein does not result in high fitness costs, but rather increases viral replication via recruitment of a host factor.
Subject(s)
Influenza B virus/physiology , Influenza, Human/virology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Replication , Amino Acid Substitution , Cell Line , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Humans , Influenza B virus/chemistry , Influenza B virus/genetics , Influenza, Human/genetics , Influenza, Human/metabolism , Protein Binding , Viral Nonstructural Proteins/genetics , src Homology DomainsABSTRACT
UNLABELLED: Influenza A virus (IAV) infection provokes an antiviral response involving the expression of type I and III interferons (IFN) and IFN-stimulated genes (ISGs) in infected cell cultures. However, the spatiotemporal dynamics of the IFN reaction are incompletely understood, as previous studies investigated mainly the population responses of virus-infected cultures, although substantial cell-to-cell variability has been documented. We devised a fluorescence-activated cell sorting-based assay to simultaneously quantify expression of viral antigens and ISGs, such as ISG15, MxA, and IFIT1, in IAV-infected cell cultures at the single-cell level. This approach revealed that seasonal IAV triggers an unexpected asymmetric response, as the major cell populations expressed either viral antigen or ISG, but rarely both. Further investigations identified a role of the viral NS1 protein in blocking ISG expression in infected cells, which surprisingly did not reduce paracrine IFN signaling to noninfected cells. Interestingly, viral ISG control was impaired in cultures infected with avian-origin IAV, including the H7N9 virus from eastern China. This phenotype was traced back to polymorphic NS1 amino acids known to be important for stable binding of the polyadenylation factor CPSF30 and concomitant suppression of host cell gene expression. Most significantly, mutation of two amino acids within the CPSF30 attachment site of NS1 from seasonal IAV diminished the strict control of ISG expression in infected cells and substantially attenuated virus replication. In conclusion, our approach revealed an asymmetric, NS1-dependent ISG induction in cultures infected with seasonal IAV, which appears to be essential for efficient virus propagation. IMPORTANCE: Interferons are expressed by infected cells in response to IAV infection and play important roles in the antiviral immune response by inducing hundreds of interferon-stimulated genes (ISGs). Unlike many previous studies, we investigated the ISG response at the single-cell level, enabling novel insights into this virus-host interaction. Hence, cell cultures infected with seasonal IAV displayed an asymmetric ISG induction that was confined almost exclusively to noninfected cells. In comparison, ISG expression was observed in larger cell populations infected with avian-origin IAV, suggesting a more resolute antiviral response to these strains. Strict control of ISG expression by seasonal IAV was explained by the binding of the viral NS1 protein to the polyadenylation factor CPSF30, which reduces host cell gene expression. Mutational disruption of CPSF30 binding within NS1 concomitantly attenuated ISG control and replication of seasonal IAV, illustrating the importance of maintaining an asymmetric ISG response for efficient virus propagation.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation , Host-Pathogen Interactions , Influenza A virus/immunology , Interferons/metabolism , China , Cleavage And Polyadenylation Specificity Factor/metabolism , Flow Cytometry/methods , Humans , Mutant Proteins/genetics , Mutant Proteins/metabolism , Single-Cell Analysis , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
UNLABELLED: During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE: Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.
Subject(s)
Annexin A5/genetics , Influenza A virus/genetics , Signal Transduction/genetics , Virion/genetics , Virus Replication , Animals , Annexin A5/metabolism , Cell Line, Tumor , Chemokine CXCL10/genetics , Chemokine CXCL10/metabolism , Dogs , Epithelial Cells/metabolism , Epithelial Cells/virology , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Influenza A virus/metabolism , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/metabolism , Interferon-gamma/pharmacology , Madin Darby Canine Kidney Cells , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Mice , Monocytes/metabolism , Monocytes/virology , Protein Transport , STAT Transcription Factors/genetics , STAT Transcription Factors/metabolism , Viral Load , Virion/chemistry , Virion/metabolism , Virus ReleaseABSTRACT
UNLABELLED: A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. IMPORTANCE: Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation of JNK in the course of an IAV infection.