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1.
DNA Cell Biol ; 41(4): 331-335, 2022 Apr.
Article in English | MEDLINE | ID: mdl-35325556

ABSTRACT

Sphingosine 1-phosphate lyase (SPL) is a critical component of sphingosine 1-phosphate (S1P) metabolism. SPL has been associated with several crucial cellular functions due to its role in S1P metabolism, but its role in viral infections is poorly understood. Studies show that SPL has an antiviral function against influenza A virus (IAV) by interacting with IKKɛ, promoting the type I interferon (IFN) innate immune response to IAV infection. However, a more recent study has revealed that IAV NS1 protein hampers this by triggering ubiquitination and subsequent degradation of SPL, which reduces the type I IFN innate immune response. In this study, we describe SPL, the type I IFN response, and known interactions between SPL and IAV.


Subject(s)
Influenza A virus , Influenza, Human , Host-Pathogen Interactions , Humans , Immunity, Innate , Lysophospholipids , Sphingosine/analogs & derivatives
2.
Virology ; 558: 67-75, 2021 06.
Article in English | MEDLINE | ID: mdl-33730651

ABSTRACT

The type I interferon (IFN)-mediated innate immune response is one of the central obstacles influenza A virus (IAV) must overcome in order to successfully replicate within the host. We have previously shown that sphingosine 1-phosphate (S1P) lyase (SPL) enhances IKKϵ-mediated type I IFN responses. Here, we demonstrate that the nonstructural protein 1 (NS1) of IAV counteracts the SPL-mediated antiviral response by inducing degradation of SPL. SPL was ubiquitinated and downregulated upon IAV infection or NS1 expression, whereas NS1-deficient IAV failed to elicit SPL ubiquitination or downregulation. Transiently overexpressed SPL increased phosphorylation of IKKϵ, resulting in enhanced expression of type I IFNs. However, this induction was markedly inhibited by IAV NS1. Collectively, this study reveals a novel strategy employed by IAV to subvert the type I IFN response, providing new insights into the interplay between IAV and host innate immunity.


Subject(s)
Aldehyde-Lyases/metabolism , Host Microbial Interactions/immunology , Immunity, Innate , Influenza A virus/immunology , Proteolysis , Viral Nonstructural Proteins/immunology , A549 Cells , Aldehyde-Lyases/genetics , Down-Regulation , HEK293 Cells , Host Microbial Interactions/genetics , Humans , Immune Evasion , Influenza, Human , Phosphorylation , Ubiquitination
3.
J Clin Invest ; 130(12): 6523-6538, 2020 12 01.
Article in English | MEDLINE | ID: mdl-32897877

ABSTRACT

Chronic viral infections are often established by the exploitation of immune-regulatory mechanisms that result in nonfunctional T cell responses. Viruses that establish persistent infections remain a serious threat to human health. Sphingosine kinase 2 (SphK2) generates sphingosine 1-phosphate, which is a molecule known to regulate multiple cellular processes. However, little is known about SphK2's role during the host immune responses to viral infection. Here, we demonstrate that SphK2 functions during lymphocytic choriomeningitis virus Cl 13 (LCMV Cl 13) infection to limit T cell immune pathology, which subsequently aids in the establishment of virus-induced immunosuppression and the resultant viral persistence. The infection of Sphk2-deficient (Sphk2-/-) mice with LCMV Cl 13 led to the development of nephropathy and mortality via T cell-mediated immunopathology. Following LCMV infection, Sphk2-/- CD4+ T cells displayed increased activity and proliferation, and these cells promoted overactive LCMV Cl 13-specific CD8+ T cell responses. Notably, oral instillation of an SphK2-selective inhibitor promoted protective T cell responses and accelerated the termination of LCMV Cl 13 persistence in mice. Thus, SphK2 is indicated as an immunotherapeutic target for the control of persistent viral infections.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Kidney Diseases/immunology , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/immunology , Phosphotransferases (Alcohol Group Acceptor)/immunology , Animals , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/pathology , Kidney Diseases/genetics , Kidney Diseases/pathology , Kidney Diseases/virology , Lymphocytic Choriomeningitis/genetics , Lymphocytic Choriomeningitis/pathology , Mice , Mice, Knockout , Phosphotransferases (Alcohol Group Acceptor)/genetics
4.
J Virol ; 94(7)2020 03 17.
Article in English | MEDLINE | ID: mdl-31915279

ABSTRACT

Influenza A virus (IAV) utilizes multiple strategies to confront or evade host type I interferon (IFN)-mediated antiviral responses in order to enhance its own propagation within the host. One such strategy is to induce the degradation of type I IFN receptor 1 (IFNAR1) by utilizing viral hemagglutinin (HA). However, the molecular mechanism behind this process is poorly understood. Here, we report that a cellular protein, poly(ADP-ribose) polymerase 1 (PARP1), plays a critical role in mediating IAV HA-induced degradation of IFNAR1. We identified PARP1 as an interacting partner for IAV HA through mass spectrometry analysis. This interaction was confirmed by coimmunoprecipitation analyses. Furthermore, confocal fluorescence microscopy showed altered localization of endogenous PARP1 upon transient IAV HA expression or during IAV infection. Knockdown or inhibition of PARP1 rescued IFNAR1 levels upon IAV infection or HA expression, exemplifying the importance of PARP1 for IAV-induced reduction of IFNAR1. Notably, PARP1 was crucial for the robust replication of IAV, which was associated with regulation of the type I IFN receptor signaling pathway. These results indicate that PARP1 promotes IAV replication by controlling viral HA-induced degradation of host type I IFN receptor. Altogether, these findings provide novel insight into interactions between influenza virus and the host innate immune response and reveal a new function for PARP1 during influenza virus infection.IMPORTANCE Influenza A virus (IAV) infections cause seasonal and pandemic influenza outbreaks, which pose a devastating global health concern. Despite the availability of antivirals against influenza, new IAV strains continue to persist by overcoming the therapeutics. Therefore, much emphasis in the field is placed on identifying new therapeutic targets that can more effectively control influenza. IAV utilizes several tactics to evade host innate immunity, which include the evasion of antiviral type I interferon (IFN) responses. Degradation of type I IFN receptor (IFNAR) is one known method of subversion, but the molecular mechanism for IFNAR downregulation during IAV infection remains unclear. Here, we have found that a host protein, poly(ADP-ribose) polymerase 1 (PARP1), facilitates IFNAR degradation and accelerates IAV replication. The findings reveal a novel cellular target for the potential development of antivirals against influenza, as well as expand our base of knowledge regarding interactions between influenza and the host innate immunity.


Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza, Human/virology , Poly (ADP-Ribose) Polymerase-1/metabolism , Receptor, Interferon alpha-beta/metabolism , A549 Cells , Animals , Antiviral Agents/pharmacology , Chlorocebus aethiops , Dogs , HEK293 Cells , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Humans , Immunity, Innate , Influenza, Human/immunology , Madin Darby Canine Kidney Cells , Mass Spectrometry , Microscopy, Confocal , RNA, Small Interfering/metabolism , Signal Transduction , Vero Cells
5.
Viruses ; 11(12)2019 11 27.
Article in English | MEDLINE | ID: mdl-31783527

ABSTRACT

The sphingosine 1-phosphate (S1P) metabolic pathway is a dynamic regulator of multiple cellular and disease processes. Identification of the immune regulatory role of the sphingosine analog FTY720 led to the development of the first oral therapy for the treatment of an autoimmune disease, multiple sclerosis. Furthermore, inhibitors of sphingosine kinase (SphK), which mediate S1P synthesis, are being evaluated as a therapeutic option for the treatment of cancer. In conjunction with these captivating discoveries, S1P and S1P-metabolizing enzymes have been revealed to display vital functions during virus infections. For example, S1P lyase, which is known for metabolizing S1P, inhibits influenza virus replication by promoting antiviral type I interferon innate immune responses. In addition, both isoforms of sphingosine kinase have been shown to regulate the replication or pathogenicity of many viruses. Pro- or antiviral activities of S1P-metabolizing enzymes appear to be dependent on diverse virus-host interactions and viral pathogenesis. This review places an emphasis on summarizing the functions of S1P-metabolizing enzymes during virus infections and discusses the opportunities for designing pioneering antiviral drugs by targeting these host enzymes.


Subject(s)
Aldehyde-Lyases/immunology , Immunity, Innate , Lysophospholipids/metabolism , Phosphotransferases (Alcohol Group Acceptor)/immunology , Sphingosine/analogs & derivatives , Virus Diseases/immunology , Viruses/enzymology , Aldehyde-Lyases/antagonists & inhibitors , Antiviral Agents/immunology , Antiviral Agents/therapeutic use , Host-Pathogen Interactions , Humans , Interferon Type I/therapeutic use , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Sphingosine/metabolism , Virus Diseases/virology , Virus Replication , Viruses/genetics , Viruses/immunology , Viruses/pathogenicity
6.
ACS Infect Dis ; 5(5): 750-758, 2019 05 10.
Article in English | MEDLINE | ID: mdl-30582687

ABSTRACT

An estimated 240 million are chronically infected with hepatitis B virus (HBV), which can lead to liver disease, cirrhosis, and hepatocellular carcinoma. Currently, HBV treatment options include only nucleoside reverse transcriptase inhibitors and the immunomodulatory agent interferon alpha, and these treatments are generally not curative. New treatments with novel mechanisms of action, therefore, are highly desired for HBV therapy. The viral core protein (Cp) has gained attention as a possible therapeutic target because of its vital roles in the HBV life cycle. Several classes of capsid assembly effectors (CAEs) have been described in detail, and these compounds all increase capsid assembly rate but inhibit HBV replication by different mechanisms. In this study, we have developed a thermal shift-based screening method for CAE discovery and characterization, filling a much-needed gap in high-throughput screening methods for capsid-targeting molecules. Using this approach followed by cell-based screening, we identified the compound HF9C6 as a CAE with low micromolar potency against HBV replication. HF9C6 caused large multicapsid aggregates when capsids were assembled in vitro and analyzed by transmission electron microscopy. Interestingly, when HBV-expressing cells were treated with HF9C6, Cp was excluded from cell nuclei, suggesting that this compound may inhibit nuclear entry of Cp and capsids. Furthermore, mutational scanning of Cp suggested that HF9C6 binds the known CAE binding pocket, indicating that key Cp-compound interactions within this pocket have a role in determining the CAE mechanism of action.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Viral Core Proteins/antagonists & inhibitors , Virus Internalization/drug effects , Hep G2 Cells , Hepatitis B virus/physiology , Hepatocytes/drug effects , Hepatocytes/virology , Humans , Virus Assembly/drug effects , Virus Replication/drug effects
7.
Eur J Med Chem ; 164: 179-192, 2019 Feb 15.
Article in English | MEDLINE | ID: mdl-30594676

ABSTRACT

Chronic hepatitis B virus (HBV) infection represents a major health threat. Current FDA-approved drugs do not cure HBV. Targeting HBV core protein (Cp) provides an attractive approach toward HBV inhibition and possibly infection cure. We have previously identified and characterized a 5-amino-3-methylthiophene-2,4-dicarboxamide (ATDC) compound as a structurally novel hit for capsid assembly effectors (CAEs). We report herein hit validation through studies on absorption, distribution, metabolism and excretion (ADME) properties and pharmacokinetics (PK), and hit optimization via analogue synthesis aiming to probe the structure-activity relationship (SAR) and structure-property relationship (SPR). In the end, these medicinal chemistry efforts led to the identification of multiple analogues strongly binding to Cp, potently inhibiting HBV replication in nanomolar range without cytotoxicity, and exhibiting good oral bioavailability (F). Two of our analogues, 19o (EC50 = 0.11 µM, CC50 > 100 µM, F = 25%) and 19k (EC50 = 0.31 µM, CC50 > 100 µM, F = 46%), displayed overall lead profiles superior to reported CAEs 7-10 used in our studies.


Subject(s)
Antiviral Agents/chemistry , Capsid/drug effects , Hepatitis B virus/ultrastructure , Thiophenes/pharmacology , Virus Assembly/drug effects , Antiviral Agents/chemical synthesis , Biological Availability , Capsid/metabolism , Hepatitis B virus/metabolism , Humans , Protein Binding , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/therapeutic use , Viral Core Proteins/metabolism , Virus Replication/drug effects
8.
Antiviral Res ; 158: 171-177, 2018 10.
Article in English | MEDLINE | ID: mdl-30125617

ABSTRACT

Influenza continues to pose a threat to public health by causing illness and mortality in humans. Discovering host factors that regulate influenza virus propagation is vital for the development of novel drugs. We have previously reported that sphingosine kinase (SphK) 1 promotes influenza A virus (IAV) replication in vitro. Here we demonstrate that the other isoform of SphK, SphK2 promotes the replication of influenza A virus (IAV) in cultured cells, and temporary inhibition of SphK1 or SphK2 enhances the host defense against influenza in mice. IAV infection led to an increased expression and phosphorylation of SphK2 in host cells. Furthermore, pharmacologic inhibition or siRNA-based knockdown of SphK2 attenuated IAV replication in vitro. Notably, oral administration of an SphK2-specific inhibitor substantially improved the viability of mice following IAV infection. In addition, the local instillation of an SphK1-specific inhibitor or an inhibitor that globally blocks SphK1 and SphK2 provided protection to IAV-infected mice. Collectively, our results indicate that both SphK1 and SphK2 function as proviral factors during IAV infection in vivo. Therefore, SphK1 and SphK2 represent potential host targets for therapeutics against influenza.


Subject(s)
Influenza A virus/drug effects , Orthomyxoviridae Infections/drug therapy , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , A549 Cells , Adamantane/analogs & derivatives , Adamantane/pharmacology , Administration, Oral , Amino Alcohols/pharmacology , Aminophenols/pharmacology , Animals , Cell Line , Disease Models, Animal , Female , Gene Knockdown Techniques , HEK293 Cells , Humans , Influenza A virus/pathogenicity , Mice , Mice, Inbred C57BL , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Isoforms , Pyridines/pharmacology , RNA, Small Interfering , Sphingosine/analogs & derivatives , Sphingosine/pharmacology , Thiazoles/pharmacology , Virus Replication
9.
mSphere ; 3(2)2018 04 25.
Article in English | MEDLINE | ID: mdl-29669885

ABSTRACT

Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly in vitro have been previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cp in vitro and causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs. Interestingly, immunofluorescence analysis showed Cp aggregating in nuclear foci of Bay 38-7690-treated infected cells in a time- and dose-dependent manner. We found these foci to be associated with promyelocytic leukemia (PML) nuclear bodies (NBs), which are structures that affect many cellular functions, including DNA damage response, transcription, apoptosis, and antiviral responses. Cp aggregation is not an artifact of the cell system used, as it is observed in HBV-expressing HepAD38 cells, in HepG2 cells transfected with an HBV-expressing plasmid, and in HepG2-NTCP cells infected with HBV. Use of a Cp overexpression vector without HBV sequences shows that aggregation is independent of viral replication, and use of an HBV-expressing plasmid harboring a HAP resistance mutation in Cp abrogated the aggregation, demonstrating that the effect is due to direct compound-Cp interactions. These studies provide novel insight into the effects of HAP-based treatment at a single-cell level.IMPORTANCE Despite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and stability, capsid formation, and host immune modulators, with the ultimate goal of an HBV cure. Understanding the mechanisms by which novel antiviral agents act will be imperative for the development of curative HBV therapies.


Subject(s)
Antiviral Agents/pharmacology , Capsid Proteins/chemistry , Hepatitis B virus/drug effects , Inclusion Bodies, Viral/chemistry , Protein Aggregates/drug effects , Pyridines/pharmacology , Pyrimidines/pharmacology , Capsid/chemistry , Capsid/drug effects , Capsid Proteins/genetics , Fluorescent Antibody Technique , Hep G2 Cells , Hepatitis B/drug therapy , Hepatitis B virus/physiology , Humans , Recombinant Proteins/chemistry , Virus Assembly/drug effects , Virus Replication/drug effects
10.
J Virol ; 92(7)2018 04 01.
Article in English | MEDLINE | ID: mdl-29343571

ABSTRACT

Although influenza A virus (IAV) evades cellular defense systems to effectively propagate in the host, the viral immune-evasive mechanisms are incompletely understood. Our recent data showed that hemagglutinin (HA) of IAV induces degradation of type I IFN receptor 1 (IFNAR1). Here, we demonstrate that IAV HA induces degradation of type II IFN (IFN-γ) receptor 1 (IFNGR1), as well as IFNAR1, via casein kinase 1α (CK1α), resulting in the impairment of cellular responsiveness to both type I and II IFNs. IAV infection or transient HA expression induced degradation of both IFNGR1 and IFNAR1, whereas HA gene-deficient IAV failed to downregulate the receptors. IAV HA caused the phosphorylation and ubiquitination of IFNGR1, leading to the lysosome-dependent degradation of IFNGR1. Influenza viral HA strongly decreased cellular sensitivity to type II IFNs, as it suppressed the activation of STAT1 and the induction of IFN-γ-stimulated genes in response to exogenously supplied recombinant IFN-γ. Importantly, CK1α, but not p38 MAP kinase or protein kinase D2, was proven to be critical for HA-induced degradation of both IFNGR1 and IFNAR1. Pharmacologic inhibition of CK1α or small interfering RNA (siRNA)-based knockdown of CK1α repressed the degradation processes of both IFNGR1 and IFNAR1 triggered by IAV infection. Further, CK1α was shown to be pivotal for proficient replication of IAV. Collectively, the results suggest that IAV HA induces degradation of IFN receptors via CK1α, creating conditions favorable for viral propagation. Therefore, the study uncovers a new immune-evasive pathway of influenza virus.IMPORTANCE Influenza A virus (IAV) remains a grave threat to humans, causing seasonal and pandemic influenza. Upon infection, innate and adaptive immunity, such as the interferon (IFN) response, is induced to protect hosts against IAV infection. However, IAV seems to be equipped with tactics to evade the IFN-mediated antiviral responses, although the detailed mechanisms need to be elucidated. In the present study, we show that IAV HA induces the degradation of the type II IFN receptor IFNGR1 and thereby substantially attenuates cellular responses to IFN-γ. Of note, a cellular kinase, casein kinase 1α (CK1α), is crucial for IAV HA-induced degradation of both IFNGR1 and IFNAR1. Accordingly, CK1α is proven to positively regulate IAV propagation. Thus, this study unveils a novel strategy employed by IAV to evade IFN-mediated antiviral activities. These findings may provide new insights into the interplay between IAV and host immunity to impact influenza virus pathogenicity.


Subject(s)
Casein Kinase I/immunology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Immune Evasion , Influenza A Virus, H1N1 Subtype/immunology , Influenza, Human/immunology , Proteolysis , Receptor, Interferon alpha-beta/immunology , Receptors, Interferon/immunology , A549 Cells , Animals , Casein Kinase I/genetics , Chlorocebus aethiops , Dogs , HEK293 Cells , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza, Human/genetics , Influenza, Human/pathology , Madin Darby Canine Kidney Cells , Protein Kinase D2 , Protein Kinases/genetics , Protein Kinases/immunology , Receptor, Interferon alpha-beta/genetics , Receptors, Interferon/genetics , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , Vero Cells , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/immunology , Interferon gamma Receptor
11.
Nat Commun ; 8(1): 1882, 2017 12 01.
Article in English | MEDLINE | ID: mdl-29192235

ABSTRACT

Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we develop a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV (a) spliced and unspliced RNA, (b) cytoplasmic and nuclear DNA, and (c) Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We also report transcriptional "bursts" of nascent RNA from integrated proviral DNA, and concomitant HIV-1, HIV-2 transcription in co-infected cells. MICDDRP can be used to simultaneously detect multiple viral nucleic acid intermediates, characterize the effects of host factors or drugs on steps of the HIV life cycle, or its reactivation from the latent state, thus facilitating the development of antivirals and latency reactivating agents.


Subject(s)
DNA, Viral/chemistry , DNA, Viral/metabolism , Fluoroimmunoassay/methods , HIV-1/chemistry , RNA, Viral/chemistry , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/virology , DNA, Viral/genetics , HIV Infections/virology , HIV-1/genetics , HIV-1/metabolism , Humans , RNA Splicing , RNA, Viral/genetics , RNA, Viral/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism
12.
Article in English | MEDLINE | ID: mdl-28320718

ABSTRACT

Hepatitis B virus (HBV) RNase H (RNH) is an appealing therapeutic target due to its essential role in viral replication. RNH inhibitors (RNHIs) could help to more effectively control HBV infections. Here, we report 3-hydroxypyrimidine-2,4-diones as novel HBV RNHIs with antiviral activity. We synthesized and tested 52 analogs and found 4 that inhibit HBV RNH activity in infected cells. Importantly, 2 of these compounds inhibited HBV replication in the low micromolar range.


Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/genetics , Ribonuclease H/metabolism , Hepatitis B virus/drug effects , Humans , Ribonuclease H/genetics , Virus Replication/drug effects , Virus Replication/genetics
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