ABSTRACT
Human Parainfluenza Virus Type 3 (HPIV3) is one of the main pathogens that cause acute lower respiratory tract infections in infants and young children. However, there are currently no effective antiviral drugs and vaccines. Herein, we found that a natural compound, curcumin, inhibits HPIV3 infection and has antiviral effects on entry and replication of the virus life cycle. Immunofluorescence and western blotting experiments revealed that curcumin disrupts F-actin and inhibits viral inclusion body (IB) formation, thus inhibiting virus replication. Curcumin can also downregulate cellular PI4KB and interrupt its colocalization in viral IBs. This study verified the antiviral ability of curcumin on HPIV3 infection and preliminarily elucidated its influence on viral replication, providing a theoretical basis for antiviral drug development of HPIV3 and other parainfluenza viruses.
Subject(s)
Curcumin/pharmacology , Inclusion Bodies, Viral/metabolism , Parainfluenza Virus 3, Human/physiology , Respirovirus Infections/metabolism , 1-Phosphatidylinositol 4-Kinase/genetics , 1-Phosphatidylinositol 4-Kinase/metabolism , A549 Cells , Actins/metabolism , Animals , Dogs , Down-Regulation , Drug Tapering , HeLa Cells , Humans , Inclusion Bodies, Viral/drug effects , Inclusion Bodies, Viral/genetics , Madin Darby Canine Kidney Cells , Parainfluenza Virus 3, Human/drug effects , Respirovirus Infections/drug therapy , Respirovirus Infections/genetics , Virus Internalization/drug effects , Virus Replication/drug effectsABSTRACT
Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antiviral Agents/pharmacology , Lung/virology , Parainfluenza Virus 3, Human/drug effects , Respirovirus Infections/prevention & control , Viral Fusion Proteins/antagonists & inhibitors , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibody Specificity , Antiviral Agents/immunology , B-Lymphocytes/immunology , B-Lymphocytes/virology , Cell Line , Disease Models, Animal , Epitopes , Host-Pathogen Interactions , Humans , Immunocompromised Host , Lung/immunology , Parainfluenza Virus 3, Human/immunology , Parainfluenza Virus 3, Human/pathogenicity , Respirovirus Infections/immunology , Respirovirus Infections/virology , Sigmodontinae , Viral Fusion Proteins/immunologyABSTRACT
The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of α-amino acid residues with ß-amino acid residues to generate α/ß-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional α-lipopeptide, our best α/ß-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals.
Subject(s)
Lipopeptides/pharmacology , Parainfluenza Virus 3, Human/drug effects , Respiratory Tract Infections/pathology , Amino Acid Sequence , Amino Acids/chemistry , Amino Acids/metabolism , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Cell Line , Cholesterol/chemistry , Drug Design , Humans , Lipopeptides/chemistry , Lipopeptides/metabolism , Parainfluenza Virus 3, Human/isolation & purification , Protein Multimerization , Rats , Respiratory Tract Infections/virology , Tissue Distribution , Transition Temperature , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/genetics , Viral Fusion Proteins/metabolism , Virus Internalization/drug effectsABSTRACT
The Caprine parainfluenza virus 3 (CPIV3) is a novel Paramyxovirus that is isolated from goats suffering from respiratory diseases. Presently, the pathogenesis of CPIV3 infection has not yet been fully characterized. The Type I interferon (IFN) is a key mediator of innate antiviral responses, as many viruses have developed strategies to circumvent IFN response, whether or how CPIV3 antagonizes type I IFN antiviral effects have not yet been characterized. This study observed that CPIV3 was resistant to IFN-α treatment and antagonized IFN-α antiviral responses on MDBK and goat tracheal epithelial (GTE) cell models. Western blot analysis showed that CPIV3 infection reduced STAT1 expression and phosphorylation, which inhibited IFN-α signal transduction on GTE cells. By screening and utilizing specific monoclonal antibodies (mAbs), three CPIV3 accessory proteins C, V and D were identified during the virus infection process on the GTE cell models. Accessory proteins C and V, but not protein D, was identified to antagonize IFN-α antiviral signaling. Furthermore, accessory protein C, but not protein V, reduced the level of IFN-α driven phosphorylated STAT1 (pSTAT1), and then inhibit STAT1 signaling. Genetic variation analysis to the PIV3 accessory protein C has found two highly variable regions (VR), with VR2 (31-70th aa) being involved in for the CPIV3 accessory protein C to hijack the STAT1 signaling activation. The above data indicated that CPIV3 is capable of inhibiting IFN-α signal transduction by reducing STAT1 expression and activation, and that the accessory protein C, plays vital roles in the immune escape process.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antiviral Agents/pharmacology , Immune Evasion , Interferon Type I/antagonists & inhibitors , Parainfluenza Virus 3, Human/drug effects , Parainfluenza Virus 3, Human/genetics , Paramyxoviridae Infections/veterinary , Animals , Antibodies, Monoclonal/pharmacology , Cell Line , Epithelial Cells/drug effects , Epithelial Cells/virology , Female , Goat Diseases/virology , Goats/virology , HEK293 Cells , High-Throughput Screening Assays , Humans , Interferon-alpha/pharmacology , Mice , Mice, Inbred BALB C , Parainfluenza Virus 3, Human/immunology , Paramyxoviridae Infections/drug therapy , Phosphorylation , STAT1 Transcription Factor/antagonists & inhibitors , STAT1 Transcription Factor/genetics , Signal Transduction/drug effectsABSTRACT
Stimulator of interferon genes (STING), as a signaling hub in innate immunity, plays a central role for the effective initiation of host defense mechanisms against microbial infections. Upon binding of its ligand cyclic dinucleotides (CDNs) produced by the cyclic GMP-AMP synthase (cGAS) or invading bacteria, STING is activated, leading to the induction of both type I interferon responses and autophagy, which are critical for the control of certain microbial infections. RNA viruses, such as Parainfluenza virus (PIV) and Rhinovirus (HRV), are among the leading causes of respiratory infections that affect human health without effective treatments. Activation of STING pathway may provide a new therapeutic approach fighting against these viruses. However, the role of STING in the control of RNA virus infection remains largely unexplored. In this study, using dimeric amidobenzimidazole (diABZI), a newly discovered synthetic small molecule STING receptor agonist with much higher potency than CDNs, we found that activation of STING elicits potent antiviral effects against parainfluenza virus type 3 (PIV3) and human rhinovirus 16 (HRV16), two representative respiratory viral pathogens. Notably, while anti-PIV3 activity was depend on the induction of type I interferon responses through TANK-binding kinase 1 (TBK1), anti-HRV16 activity required the induction of autophagy-related gene 5 (ATG5)-dependent autophagy, indicating that two distinct antiviral mechanisms are engaged upon STING activation. Antiviral activity and individual specific pathway was further confirmed in infected primary bronchial epithelial cells. Our findings thus demonstrate the distinct antiviral mechanisms triggered by STING agonist and uncover the potential of therapeutic effect against different viruses.
Subject(s)
Antiviral Agents/pharmacology , Membrane Proteins/agonists , Parainfluenza Virus 3, Human/drug effects , Rhinovirus/drug effects , Signal Transduction/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/chemical synthesis , Autophagy , Cell Line , Cells, Cultured , HeLa Cells , Humans , Immunity, Innate , Mice , Parainfluenza Virus 3, Human/physiology , RAW 264.7 Cells , Rhinovirus/physiology , Signal Transduction/immunology , THP-1 CellsABSTRACT
Cholesterol-rich lipid rafts have been shown to play important roles in the life cycle of various non-enveloped and enveloped viruses. Deletion of cholesterol from lipid rafts could influence different steps of viral replication cycle including entry, infection, assembly and release. Caprine parainfluenza virus type3 (CPIV3) is a newly identified member of Paramyxoviridae family. CPIV3 is highly prevalence and threatened the goat industry in China. The infection mechanism of CPIV3 is under exploring and still not fully understood, the roles of cholesterol and lipid rafts for CPIV3 infection remains unclear. In this study, we investigated the association of cholesterol and lipid rafts with CPIV3 during the different viral replication stages (binding, entry and infection) in two cells [MDBK and goat bronchial epithelial (GBE) cells]. Methyl-ß- cyclodextrin (MßCD) was used to deplete cholesterol from cell and viral membranes. The results showed that MßCD treatment significantly inhibited CPIV3 entry and infection in these two cells with a dose-dependent manner, but didn't impair the binding of CPIV3. Addition of exogenous cholesterol to the cells after MßCD treatment restored the viral infection. In addition, treatment of MßCD only before virus-entry showed inhibitory effect in MDBK cells. Depletion of cholesterol from virion envelop also decreased the entry and infection of CPIV3 in the two cells. Furthermore, lipid rafts isolation test indicated that viral proteins (HN and N) co-localized with lipid rafts during infection in MDBK and GBE cells. Viral N protein co-localized with caveolin-1 (the marker of lipid rafts) in these two cells both at the entry and infection steps, as detected by con-focal laser scanning microscopy test. In conclusion, the results presented here demonstrated that cholesterol rich lipid rafts play an important role in CPIV3 life cycle. The findings give new insights on understanding of the mechanism of CPIV3 infection and provide a new anti-CPIV3 strategy.
Subject(s)
Cholesterol/metabolism , Membrane Microdomains/chemistry , Parainfluenza Virus 3, Human/physiology , Virus Internalization , Virus Replication , Animals , Bronchi/cytology , Bronchi/virology , Cattle , Cell Line , Epithelial Cells/virology , Gene Deletion , Goats , Kidney/cytology , Parainfluenza Virus 3, Human/drug effects , beta-Cyclodextrins/pharmacologyABSTRACT
The receptor binding protein of parainfluenza virus, hemagglutinin-neuraminidase (HN), is responsible for actively triggering the viral fusion protein (F) to undergo a conformational change leading to insertion into the target cell and fusion of the virus with the target cell membrane. For proper viral entry to occur, this process must occur when HN is engaged with host cell receptors at the cell surface. It is possible to interfere with this process through premature activation of the F protein, distant from the target cell receptor. Conformational changes in the F protein and adoption of the postfusion form of the protein prior to receptor engagement of HN at the host cell membrane inactivate the virus. We previously identified small molecules that interact with HN and induce it to activate F in an untimely fashion, validating a new antiviral strategy. To obtain highly active pretriggering candidate molecules we carried out a virtual modeling screen for molecules that interact with sialic acid binding site II on HN, which we propose to be the site responsible for activating F. To directly assess the mechanism of action of one such highly effective new premature activating compound, PAC-3066, we use cryo-electron tomography on authentic intact viral particles for the first time to examine the effects of PAC-3066 treatment on the conformation of the viral F protein. We present the first direct observation of the conformational rearrangement induced in the viral F protein.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), upon binding to its cell receptor, triggers conformational changes in the fusion protein (F). This action of HN activates F to reach its fusion-competent state. Using small molecules that interact with HN, we can induce the premature activation of F and inactivate the virus. To obtain highly active pretriggering compounds, we carried out a virtual modeling screen for molecules that interact with a sialic acid binding site on HN that we propose to be the site involved in activating F. We use cryo-electron tomography of authentic intact viral particles for the first time to directly assess the mechanism of action of this treatment on the conformation of the viral F protein and present the first direct observation of the induced conformational rearrangement in the viral F protein.
Subject(s)
Antiviral Agents/pharmacology , HN Protein/metabolism , Parainfluenza Virus 3, Human/drug effects , Viral Fusion Proteins/antagonists & inhibitors , Virus Internalization/drug effects , Antiviral Agents/isolation & purification , Cell Culture Techniques , Cell Line , Drug Discovery , Epithelial Cells/drug effects , Epithelial Cells/virology , HN Protein/genetics , High-Throughput Screening Assays , Humans , Molecular Docking Simulation , Parainfluenza Virus 3, Human/physiology , Paramyxoviridae Infections/drug therapy , Protein Binding/drug effects , Small Molecule Libraries/pharmacology , Viral Fusion Proteins/metabolismABSTRACT
Caprine parainfluenza virus type 3 (CPIV3) is the one of most common causative agents of caprine respiratory infection, resulting in significant economic losses in the goat and sheep industries. However, the molecular mechanisms and host genes involved in the pathogenesis of and immunity against CPIV3 infection remain poorly understood. In this study, we used RNA-Seq to understand the responses of madin-darby bovine kidney (MDBK) cells to CPIV3 infection. A total of 261 differentially-expressed genes (DEGs) were identified in CPIV3-infected compared with mock-infected MDBK cells at 24 h post-infection (hpi). The DEGs were mainly involved in immune system processes, metabolic processes, and signal transduction. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the most significantly enriched signaling pathways were MAPK, Wnt, PI3K-Akt, tumor necrosis factor, Toll-like receptor and ubiquitin-mediated proteolysis. STRING analysis revealed that seven interferon-stimulated genes (ISGs) were upregulated (IFI6, ISG15, OAS1Y, OAS1Z, MX1, MX2 and RSAD2) and may play a pivotal role during CPIV3 infection. Moreover, overexpression of these ISGs significantly reduced CPIV3 replication in vitro, while siRNA silencing markedly improved CPIV3 replication 24 and 48 hpi. Ours is the first study to profile the gene expression of CPIV3-infected MDBK cells. We identified seven ISGs that could be targeted in novel antiviral strategies against CPIV3.
Subject(s)
Interferons/pharmacology , Parainfluenza Virus 3, Human/physiology , Virus Replication , Animals , Cattle , Cell Line , Dogs , Fluorescent Antibody Technique, Indirect/veterinary , Gene Expression Regulation, Viral , Gene Knockdown Techniques/veterinary , Goats , Microspheres , Parainfluenza Virus 3, Human/drug effects , Parainfluenza Virus 3, Human/genetics , Parainfluenza Virus 3, Human/immunology , RNA, Viral/chemistry , RNA, Viral/isolation & purification , Radioimmunoprecipitation Assay/veterinary , Real-Time Polymerase Chain Reaction/veterinary , Transcriptome , Virus Replication/drug effects , Virus Replication/immunologyABSTRACT
Human parainfluenza virus 3 (HPIV3) and respiratory syncytial virus (RSV) are leading causes of lower respiratory tract infections. There are currently no vaccines or antiviral therapeutics to treat HPIV3 or RSV infections. We recently reported a peptide (VIQKI), derived from the C-terminal heptad repeat (HRC) domain of the HPIV3 fusion (F) glycoprotein that inhibits infection by both HPIV3 and RSV. The dual inhibitory activity of VIQKI is due to its unique ability to bind to the N-terminal heptad repeat (HRN) domains of both HPIV3 and RSV F, thereby preventing the native HRN-HRC interactions required for viral entry. Here we describe the structure-guided design of dual inhibitors of HPIV3 and RSV fusion with improved efficacy. We show that VIQKI derivatives possessing one (I456F) or two (I454F/I456F) phenylalanine substitutions near the N-terminus exhibit more stable assemblies with the RSV-HRN domain and enhanced antiviral efficacy against both HPIV3 and RSV infection. Cocrystal structures of the new Phe-substituted inhibitors coassembled with HPIV3 or RSV-HRN domains reveal that the I456F substitution makes intimate hydrophobic contact with the core trimers of both HPIV3 and RSV F.
Subject(s)
Antiviral Agents/pharmacology , Oligopeptides/pharmacology , Parainfluenza Virus 3, Human/drug effects , Respiratory Syncytial Virus, Human/drug effects , Virus Internalization/drug effects , Amino Acid Sequence , Antiviral Agents/chemistry , Crystallography, X-Ray , Hydrophobic and Hydrophilic Interactions , Molecular Structure , Oligopeptides/chemistry , Parainfluenza Virus 3, Human/physiology , Protein Conformation , Respiratory Syncytial Virus, Human/physiologyABSTRACT
Human parainfluenza virus 3 (HPIV3) and respiratory syncytial virus (RSV) cause lower respiratory infection in infants and young children. There are no vaccines for these pathogens, and existing treatments have limited or questionable efficacy. Infection by HPIV3 or RSV requires fusion of the viral and cell membranes, a process mediated by a trimeric fusion glycoprotein (F) displayed on the viral envelope. Once triggered, the pre-fusion form of F undergoes a series of conformational changes that first extend the molecule to allow for insertion of the hydrophobic fusion peptide into the target cell membrane and then refold the trimeric assembly into an energetically stable post-fusion state, a process that drives the merger of the viral and host cell membranes. Peptides derived from defined regions of HPIV3 F inhibit infection by HPIV3 by interfering with the structural transitions of the trimeric F assembly. Here we describe lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F that potently inhibit infection by both HPIV3 and RSV. The lead peptide inhibits RSV infection as effectively as does a peptide corresponding to the RSV HRC domain itself. We show that the inhibitors bind to the N-terminal heptad repeat (HRN) domains of both HPIV3 and RSV F with high affinity. Co-crystal structures of inhibitors bound to the HRN domains of HPIV3 or RSV F reveal remarkably different modes of binding in the N-terminal segment of the inhibitor.
Subject(s)
Lipopeptides/pharmacology , Parainfluenza Virus 3, Human/drug effects , Peptide Fragments/pharmacology , Respiratory Syncytial Viruses/drug effects , Viral Fusion Protein Inhibitors/pharmacology , Viral Fusion Proteins/pharmacology , Amino Acid Sequence , Crystallography, X-Ray , Humans , Lipopeptides/metabolism , Microbial Sensitivity Tests , Parainfluenza Virus 3, Human/chemistry , Peptide Fragments/metabolism , Protein Binding , Respiratory Mucosa/virology , Respiratory Syncytial Viruses/chemistry , Viral Fusion Protein Inhibitors/metabolism , Viral Fusion Proteins/metabolism , Virus Internalization/drug effectsABSTRACT
Human parainfluenza viruses cause acute respiratory tract infections and disease predominantly in young children and immunocompromised individuals. Currently, there are no vaccines to prevent hPIV infections, nor licensed anti-hPIV drugs. There is therefore a need for specific antiviral therapies to decrease the morbidity and mortality associated with hPIV diseases. Haemagglutinin-neuraminidase (HN) is one of two hPIV surface proteins with critical roles in host receptor recognition, binding and cleavage; it has been explored as a key drug development target for the past few decades with variable success. Recent advancements in computational modelling and the availability of the X-ray crystal structure of hPIV3 HN have improved our understanding of the structural and mechanistic features of HN. This review explores structural features of the HN protein that are being exploited for structure-guided inhibitor design. We describe past and present hPIV HN inhibition strategies based on sialic acid scaffolds, together with other novel approaches that decrease hPIV infectivity. Although many HN inhibitors have been developed and evaluated as anti-hPIV agents, currently only a host-directed therapy (DAS181) has succeeded in phase II clinical drug trials. Hence, the review concludes with future considerations for targeting the specific function(s) of hPIV HN and suggestions for antiviral drug design.
Subject(s)
Enzyme Inhibitors/pharmacology , HN Protein , N-Acetylneuraminic Acid/analogs & derivatives , Neuraminidase/antagonists & inhibitors , Paramyxoviridae Infections/drug therapy , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Child , Child, Preschool , Drug Delivery Systems/methods , Drug Design , Drug Resistance, Viral/drug effects , Enzyme Inhibitors/chemical synthesis , Genome, Viral , HN Protein/chemistry , HN Protein/genetics , HN Protein/metabolism , Humans , Immunocompromised Host , N-Acetylneuraminic Acid/chemical synthesis , N-Acetylneuraminic Acid/pharmacology , Parainfluenza Virus 1, Human/drug effects , Parainfluenza Virus 1, Human/genetics , Parainfluenza Virus 3, Human/drug effects , Parainfluenza Virus 3, Human/genetics , Paramyxoviridae Infections/pathology , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Internalization/drug effectsABSTRACT
Paramyxoviruses, specifically, the childhood pathogen human parainfluenza virus type 3, are internalized into host cells following fusion between the viral and target cell membranes. The receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into the cell through a coordinated process involving HN activation by receptor binding, which triggers conformational changes in the F protein to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein has been shown to be an effective antiviral strategy in vitro. Conformational changes in the F protein leading to adoption of the postfusion form of the protein-prior to receptor engagement of HN at the host cell membrane-render the virus noninfectious. We previously identified a small compound (CSC11) that implements this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely process. To assess the functionality of such compounds, it is necessary to verify that the postfusion state of F has been achieved. As demonstrated by Melero and colleagues, soluble forms of the recombinant postfusion pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to generate postfusion-specific antibodies. We produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. In this study, using systematic chemical modifications of CSC11, we synthesized a more potent derivative of this compound, CM9. Much like CSC11, CM9 causes premature triggering of the F protein through an interaction with HN prior to receptor engagement, thereby preventing fusion and subsequent infection. In addition to validating the potency of CM9 using plaque reduction, fusion inhibition, and binding avidity assays, we confirmed the transition to a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3 conformation-specific antibodies. We present both CM9 and these newly characterized postfusion antibodies as novel tools to explore and develop antiviral approaches. In turn, these advances in both our molecular toolset and our understanding of HN-F interaction will support development of more-effective antivirals. Combining the findings described here with our recently described physiologically relevant ex vivo system, we have the potential to inform the development of therapeutics to block viral infection.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into cells through a process involving HN activation by receptor binding, which triggers conformational changes in F to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein may be an effective antiviral strategy in vitro We identified and optimized small compounds that implement this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely fashion. To address that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. Both the novel antiviral compounds that we present and these newly characterized postfusion antibodies are novel tools for the exploration and development of antiviral approaches.
Subject(s)
Antiviral Agents/pharmacology , HN Protein/metabolism , Parainfluenza Virus 3, Human/drug effects , Parainfluenza Virus 3, Human/physiology , Viral Fusion Proteins/metabolism , Virus Internalization/drug effects , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Viral/immunology , Antibodies, Viral/isolation & purification , Antiviral Agents/chemical synthesis , Cell Line , Chlorocebus aethiops , Humans , Protein Binding , Protein Conformation , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/immunology , Viral Plaque AssayABSTRACT
BACKGROUND: Human parainfluenza type 3 (HPIV3) is an important respiratory pathogen. Although a number of potential therapeutic candidates exist, there is currently no licensed therapy or vaccine. Ribavirin (RBV), favipiravir (FVP) and zanamivir (ZNV) are inhibitors with proven activity against influenza and with potential inhibitory activity against HPIV3 laboratory adapted strains in vitro. OBJECTIVES: To evaluate RBV, FVP and ZNV as inhibitors of minimally passaged UK clinical strains of HPIV3 as well as a laboratory adapted strain MK9 in vitro. STUDY DESIGN: The inhibitory action of RBV, FVP and ZNV was evaluated against nine minimally passaged clinical strains and a laboratory adapted strain MK9 using plaque reduction and growth curve inhibition in a cell culture model. RESULTS: Clinical isolates were found to be at least as susceptible as the laboratory adapted strains to RBV and FVP and significantly more susceptible to ZNV. However the inhibitory concentrations achieved by ZNV against clinical strains remain prohibitively high in vivo. CONCLUSIONS: RBV, FVP and ZNV were found to be effective inhibitors of HPIV3 in vitro. The lack of efficacy of RBV in vivo may be due to inability to reach required therapeutic levels. FVP, on the other hand, is a good potential therapeutic agent against HPIV3. Further studies using wild type clinical strains, as well as better formulation and delivery mechanisms may improve the utility of these three inhibitors.
Subject(s)
Amides/pharmacology , Antiviral Agents/pharmacology , Parainfluenza Virus 3, Human/drug effects , Pyrazines/pharmacology , Respirovirus Infections/virology , Ribavirin/pharmacology , Zanamivir/pharmacology , Cell Line, Tumor , Cytopathogenic Effect, Viral/drug effects , Humans , Parainfluenza Virus 3, Human/physiology , Virus Attachment/drug effects , Virus Replication/drug effectsABSTRACT
BACKGROUND: The prevention of bovine respiratory disease complex (BRD) in beef cattle is important to maintaining health and productivity of calves in feeding operations. OBJECTIVE: Determine whether BRD bacterial and viral pathogens are susceptible to the lactoperoxidase/hydrogen peroxide/iodide (LPO/H2 O2 /I- ) system in vitro and to determine whether the oral administration of sodium iodide (NaI) could achieve sufficient concentrations of iodine (I) in the respiratory secretions of weaned beef calves to inactivate these pathogens in vivo. ANIMALS: Sixteen weaned, apparently healthy, commercial beef calves from the University of Missouri, College of Veterinary Medicine teaching herd. METHODS: In vitro viral and bacterial assays were performed to determine susceptibility to the LPO/H2 O2 /I- system at varying concentrations of NaI. Sixteen randomly selected, healthy crossbred beef weanlings were administered 70 mg/kg NaI, or water, orally in a blinded, placebo-controlled trial. Blood and nasal secretions were collected for 72 hours and analyzed for I- concentration. RESULTS: Bovine herpesvirus-1, parainfluenza-3, Mannheimia haemolytica and Bibersteinia trehalosi were all inactivated or inhibited in vitro by the LPO/H2 O2 /I- reaction. Oral administration of NaI caused a marked increase in nasal fluid I concentration with a Cmax = 181 (1,420 µM I), T12 , a sufficient concentration to inactivate these pathogens in vitro. CONCLUSIONS AND CLINICAL IMPORTANCE: In vitro, the LPO/H2 O2 /I- system inactivates and inhibits common pathogens associated with BRD. The administration of oral NaI significantly increases the I concentration of nasal fluid indicating that this system might be useful in preventing bovine respiratory infections.
Subject(s)
Bovine Respiratory Disease Complex/prevention & control , Nasal Mucosa/chemistry , Sodium Iodide/pharmacology , Administration, Oral , Animals , Bovine Respiratory Disease Complex/microbiology , Bovine Respiratory Disease Complex/virology , Cattle , Herpesvirus 1, Bovine/drug effects , Hydrogen Peroxide/chemistry , Iodine/analysis , Lactoperoxidase/metabolism , Mannheimia haemolytica/drug effects , Nasal Mucosa/microbiology , Nasal Mucosa/virology , Parainfluenza Virus 3, Human/drug effects , Pasteurellaceae/drug effects , Sodium Iodide/administration & dosage , Sodium Iodide/analysisABSTRACT
Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC50 values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC50 ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).
Subject(s)
Antiviral Agents/pharmacology , Chelating Agents/pharmacology , Hydrazones/pharmacology , Simplexvirus/drug effects , Vaccinia virus/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Cell Line , Cell Line, Tumor , Chelating Agents/chemical synthesis , Chelating Agents/metabolism , Chlorocebus aethiops , Copper/metabolism , Epithelial Cells/drug effects , Epithelial Cells/virology , Fibroblasts/drug effects , Fibroblasts/virology , Humans , Hydrazones/chemical synthesis , Hydrazones/metabolism , Inhibitory Concentration 50 , Magnesium/metabolism , Manganese/metabolism , Orthoreovirus, Mammalian/drug effects , Orthoreovirus, Mammalian/growth & development , Orthoreovirus, Mammalian/metabolism , Parainfluenza Virus 3, Human/drug effects , Parainfluenza Virus 3, Human/growth & development , Parainfluenza Virus 3, Human/metabolism , Phlebovirus/drug effects , Phlebovirus/growth & development , Phlebovirus/metabolism , Respiratory Syncytial Viruses/drug effects , Respiratory Syncytial Viruses/growth & development , Respiratory Syncytial Viruses/metabolism , Simplexvirus/growth & development , Simplexvirus/metabolism , Vaccinia virus/growth & development , Vaccinia virus/metabolism , Vero Cells , Vesiculovirus/drug effects , Vesiculovirus/growth & development , Vesiculovirus/metabolismABSTRACT
Human parainfluenza type-3 virus (hPIV-3) is one of the principal aetiological agents of acute respiratory illness in infants worldwide and also shows high disease severity in the elderly and immunocompromised, but neither therapies nor vaccines are available to treat or prevent infection, respectively. Using a multidisciplinary approach we report herein that the approved drug suramin acts as a non-competitive in vitro inhibitor of the hPIV-3 haemagglutinin-neuraminidase (HN). Furthermore, the drug inhibits viral replication in mammalian epithelial cells with an IC50 of 30 µM, when applied post-adsorption. Significantly, we show in cell-based drug-combination studies using virus infection blockade assays, that suramin acts synergistically with the anti-influenza virus drug zanamivir. Our data suggests that lower concentrations of both drugs can be used to yield high levels of inhibition. Finally, using NMR spectroscopy and in silico docking simulations we confirmed that suramin binds HN simultaneously with zanamivir. This binding event occurs most likely in the vicinity of the protein primary binding site, resulting in an enhancement of the inhibitory potential of the N-acetylneuraminic acid-based inhibitor. This study offers a potentially exciting avenue for the treatment of parainfluenza infection by a combinatorial repurposing approach of well-established approved drugs.
Subject(s)
Epithelial Cells/drug effects , Parainfluenza Virus 3, Human/drug effects , Suramin/pharmacology , Zanamivir/pharmacology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Binding Sites , Cell Line , Drug Synergism , Epithelial Cells/virology , HN Protein/metabolism , Host-Pathogen Interactions/drug effects , Kidney/cytology , Kinetics , Macaca mulatta , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Parainfluenza Virus 3, Human/metabolism , Parainfluenza Virus 3, Human/physiology , Protein Binding , Suramin/metabolism , Viral Proteins/antagonists & inhibitors , Viral Proteins/metabolism , Virus Replication/drug effects , Zanamivir/metabolismABSTRACT
Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.
Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Fatty Acid Synthase, Type I/antagonists & inhibitors , Protein Processing, Post-Translational , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Viruses/drug effects , Virus Replication/drug effects , Administration, Oral , Animals , Antiviral Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Fatty Acid Synthase, Type I/genetics , Fatty Acid Synthase, Type I/metabolism , Gene Expression , HeLa Cells , Hep G2 Cells , Host-Pathogen Interactions , Humans , Lipoylation/drug effects , Mice , Mice, Inbred BALB C , Palmitic Acid/antagonists & inhibitors , Palmitic Acid/metabolism , Parainfluenza Virus 3, Human/drug effects , Parainfluenza Virus 3, Human/growth & development , Parainfluenza Virus 3, Human/metabolism , Respiratory Mucosa/drug effects , Respiratory Mucosa/enzymology , Respiratory Mucosa/virology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/growth & development , Respiratory Syncytial Viruses/metabolism , Rhinovirus/drug effects , Rhinovirus/growth & development , Rhinovirus/metabolism , Viral Proteins/antagonists & inhibitors , Viral Proteins/genetics , Viral Proteins/metabolism , Virion/drug effects , Virion/growth & development , Virion/metabolismABSTRACT
Most respiratory tract infections are self-limiting and caused by viruses, and do not warrant antibiotic treatment. Despite this, patients with respiratory tract infections often receive antibiotics, fuelling the rise of antibiotic resistance. Therefore, there is a need to encourage patients to try alternative non-antibiotic therapies, which ideally treat the symptoms and the cause. Lozenges containing amylmetacresol and 2,4-dichlorobenzyl alcohol (AMC/DCBA lozenges) as well as lozenges containing hexylresorcinol have been shown to provide effective symptomatic relief for sore throat. In this study, we investigated whether these lozenges also have virucidal effects in vitro against two viruses associated with respiratory tract infections, parainfluenza virus type 3 and cytomegalovirus. Both viruses were incubated with AMC/DCBA lozenge, placebo lozenge or the active ingredients (AMC/DCBA) as free substances, and parainfluenza virus type 3 was incubated with hexylresorcinol lozenge, placebo lozenge or hexylresorcinol as a free substance. Virucidal effects were observed with the active lozenges and the active ingredients as free substances against both parainfluenza virus type 3 and cytomegalovirus. Mean reductions in viral titre were significantly greater compared with placebo lozenge and peak effects were observed for the shortest incubation time, 1min. These findings suggest that AMC/DCBA lozenge and hexylresorcinol lozenge have the potential to have local antiviral effects in patients with sore throat due to viral respiratory tract infections. Use of such over-the-counter treatments for self-limiting respiratory tract infections may satisfy patients' desire for an anti-infective medication and reduce the demand for antibiotics.
Subject(s)
Antiviral Agents/metabolism , Benzyl Alcohols/metabolism , Cresols/metabolism , Cytomegalovirus/drug effects , Hexylresorcinol/metabolism , Parainfluenza Virus 3, Human/drug effects , Tablets/metabolism , Cytomegalovirus/physiology , Humans , Microbial Sensitivity Tests , Microbial Viability/drug effects , Parainfluenza Virus 3, Human/physiology , Time Factors , Viral LoadABSTRACT
BACKGROUND: Parainfluenza virus (PIV), a common pediatric pathogen, is associated with significant morbidity in immunocompromised (IC) hosts. DAS181, a novel sialidase fusion protein inhibitor, seems to be effective against PIV in vitro and in vivo; its use in IC children has not been evaluated. METHODS: Patients were diagnosed with PIV infection using a quantitative reverse transcription-polymerase chain reaction. DAS181 was obtained under emergency investigational new drug applications and was administered via aerosol chamber or nebulizer. Patients were assessed daily for their clinical condition and adverse outcomes. RESULTS: Four pediatric hematopoietic cell transplantation (HCT) patients with PIV detected in respiratory specimens were identified and treated with DAS 181. Patients 1 and 2 were diagnosed with PIV lower respiratory tract infection (LRTI) by bronchoalveolar lavage at 9 months and 2 days after allogeneic transplantation, respectively. Patient 3 was on chemotherapy prior to planned autologous HCT at time of PIV diagnosis from a nasal swab. Patient 4 was diagnosed with PIV via nasal wash 2 days after HCT. Patients 1-3 had clinical symptoms and chest imaging consistent with LRTI. Inhaled DAS181 was administered for 5-10 days. All 4 patients tolerated therapy well. Clinical improvement in oxygen requirement and respiratory rate was observed in all patients who required oxygen at therapy initiation. Viral load decreased in all patients within 1 week of therapy and became undetectable by day 3 of therapy in patient 3. CONCLUSION: DAS181 was used to treat 4 severely IC pediatric patients with PIV disease. The drug was well tolerated. Improvement in both viral loads and symptoms after initiation of therapy was observed in all cases. This report supports prospective, randomized studies in IC patients with PIV infection.
Subject(s)
Immunocompromised Host/physiology , Parainfluenza Virus 2, Human/drug effects , Parainfluenza Virus 3, Human/drug effects , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , Respiratory Tract Infections/drug therapy , Respirovirus Infections/drug therapy , Rubulavirus Infections/drug therapy , Alanine Transaminase/blood , Alanine Transaminase/drug effects , Aspartate Aminotransferases/blood , Aspartate Aminotransferases/drug effects , Child , Child, Preschool , Humans , Infant , Leukemia, Myeloid, Acute/complications , Male , Neuroblastoma/complications , Parainfluenza Virus 2, Human/physiology , Parainfluenza Virus 3, Human/physiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/complications , Prospective Studies , Random Allocation , Recombinant Fusion Proteins/administration & dosage , Respiratory Tract Infections/diagnosis , Severe Combined Immunodeficiency/complications , Transplantation/adverse effects , Viral Load/drug effects , Viral Load/physiologyABSTRACT
Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a cellular attachment site for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as 'molecular sinks' and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we generated decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform.