Jinhong Chang

Identification of Five Interferon-Induced Cellular Proteins That Inhibit West Nile Virus and Dengue Virus Infections

ABSTRACT Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-α for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-α is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.

Identification of Three Interferon-Inducible Cellular Enzymes That Inhibit the Replication of Hepatitis C Virus

ABSTRACT Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with alpha interferon (IFN-α)-based therapies. However, the underlying mechanism of IFN-α therapy remains to be elucidated. To identify the cellular proteins that mediate the antiviral effects of IFN-α, we created a HEK293-based cell culture system to inducibly express individual interferon-stimulated genes (ISGs) and determined their antiviral effects against HCV. By screening 29 ISGs that are induced in Huh7 cells by IFN-α and/or up-regulated in HCV-infected livers, we discovered that viperin, ISG20, and double-stranded RNA-dependent protein kinase (PKR) noncytolytically inhibited the replication of HCV replicons. Mechanistically, inhibition of HCV replication by ISG20 and PKR depends on their 3′-5′ exonuclease and protein kinase activities, respectively. Moreover, our work, for the first time, provides strong evidence suggesting that viperin is a putative radical S-adenosyl-l-methionine (SAM) enzyme. In addition to demonstrating that the antiviral activity of viperin depends on its radical SAM domain, which contains conserved motifs to coordinate [4Fe-4S] cluster and cofactor SAM and is essential for its enzymatic activity, mutagenesis studies also revealed that viperin requires an aromatic amino acid residue at its C terminus for proper antiviral function. Furthermore, although the N-terminal 70 amino acid residues of viperin are not absolutely required, deletion of this region significantly compromises its antiviral activity against HCV. Our findings suggest that viperin represents a novel antiviral pathway that works together with other antiviral proteins, such as ISG20 and PKR, to mediate the IFN response against HCV infection.

Alpha-Interferon Suppresses Hepadnavirus Transcription by Altering Epigenetic Modification of cccDNA Minichromosomes

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV) replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg) RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9) and 27 (H3K27) in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection.

Interferons Accelerate Decay of Replication-Competent Nucleocapsids of Hepatitis B Virus

ABSTRACT Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host antiviral immunity against hepatitis B virus (HBV) infection in vivo. However, the molecular mechanism by which these antiviral cytokines suppress HBV replication remains elusive. Using an immortalized murine hepatocyte (AML12)-derived cell line supporting tetracycline-inducible HBV replication, we show in this report that both IFN-α and IFN-γ efficiently reduce the amount of intracellular HBV nucleocapsids. Furthermore, we provide evidence suggesting that the IFN-induced cellular antiviral response is able to distinguish and selectively accelerate the decay of HBV replication-competent nucleocapsids but not empty capsids in a proteasome-dependent manner. Our findings thus reveal a novel antiviral mechanism of IFNs and provide a basis for a better understanding of HBV pathobiology.

Novel Imino Sugar Derivatives Demonstrate Potent Antiviral Activity against Flaviviruses

ABSTRACT Imino sugars, such as N-butyl-deoxynojirimycin and N-nonyl-deoxynojirimycin (NNDNJ), are glucose analogues that selectively inhibit cellular α-glucosidase I and II in the endoplasmic reticulum and exhibit antiviral activities against many types of enveloped viruses. Although these molecules have broad-spectrum antiviral activity, their development has been limited by a lack of efficacy and/or selectivity. We have previously reported that a DNJ derivative with a hydroxylated cyclohexyl side chain, called OSL-95II, has an antiviral efficacy similar to that of NNDNJ but significantly less toxicity. Building upon this observation, a family of imino sugar derivatives containing an oxygenated side chain and terminally restricted ring structures were synthesized and shown to have low cytotoxicity and superior antiviral activity against members of the Flaviviridae family, including bovine viral diarrhea virus, dengue virus (DENV), and West Nile virus. Of particular interest is that several of these novel imino sugar derivatives, such as PBDNJ0801, PBDNJ0803, and PBDNJ0804, potently inhibit DENV infection in vitro, with 90% effective concentration values at submicromolar concentrations and selectivity indices greater than 800. Therefore, these compounds represent the best in their class and may offer realistic candidates for the development of antiviral therapeutics against human DENV infections.

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

ABSTRACT Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.

Antiviral therapies targeting host ER alpha-glucosidases: Current status and future directions

Abstract Endoplasmic reticulum (ER)-resident α-glucosidases I and II sequentially trim the three terminal glucose moieties on N-linked glycans attached to nascent glycoproteins. These reactions are the first steps of N-linked glycan processing and are essential for proper folding and function of many glycoproteins. Because most viral envelope glycoproteins contain N-linked glycans, inhibition of ER α-glucosidases with derivatives of 1-deoxynojirimycin (DNJ) or castanospermine (CAST), two well-studied pharmacophores of α-glucosidase inhibitors, efficiently disrupts the morphogenesis of a broad spectrum of enveloped viruses. Moreover, both DNJ and CAST derivatives have been demonstrated to prevent the death of mice infected with several distinct flaviviruses and filoviruses and suppress the multiplication of several other species of viruses in infected animals. N-Butyl derivative of DNJ (NB-DNJ) and 6 O-bytanoyl prodrug of CAST (Bu-CAST) have been evaluated in human clinical trials for their antiviral activities against human immunodeficiency virus and hepatitis C virus, and there is an ongoing trial of treating dengue patients with Bu-CAST. This article summarizes the current status of ER α-glucosidase-targeted antiviral therapy and proposes strategies for development of more efficacious and specific ER α-glucosidase inhibitors as broad-spectrum, drug resistance-refractory antiviral therapeutics. These host function-targeted, broad-spectrum antiviral agents do not rely on time-consuming etiologic diagnosis, and should therefore be particularly promising in the management of viral hemorrhagic fever and respiratory tract viral infections, medical conditions that can be caused by many different enveloped RNA viruses, with a short window for medical intervention.

Combination of α-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection.

Inhibitors of Endoplasmic Reticulum α-Glucosidases Potently Suppress Hepatitis C Virus Virion Assembly and Release

ABSTRACT α-Glucosidases I and II are endoplasmic reticulum-resident enzymes that are essential for N-linked glycan processing and subsequent proper folding of glycoproteins. In this report, we first demonstrate that downregulation of the expression of α-glucosidase I, II, or both in Huh7.5 cells by small hairpin RNA technology inhibited the production of hepatitis C virus (HCV). In agreement with the essential role of α-glucosidases in HCV envelope glycoprotein processing and folding, treatment of HCV-infected cells with a panel of imino sugar derivatives, which are competitive inhibitors of α-glucosidases, did not affect intracellular HCV RNA replication and nonstructural protein expression but resulted in the inhibition of glycan processing and subsequent degradation of HCV E2 glycoprotein. As a consequence, HCV virion assembly and secretion were inhibited. In searching for imino sugars with better antiviral activity, we found that a novel imino sugar, PBDNJ0804, had a superior ability to inhibit HCV virion assembly and secretion. In summary, we demonstrated that glucosidases are important host factor-based antiviral targets for HCV infection. The low likelihood of drug-resistant virus emergence and potent antiviral efficacy of the novel glucosidase inhibitor hold promise for its development as a therapeutic agent for the treatment of chronic hepatitis C.

The innate immune response to hepatitis B virus infection: Implications for pathogenesis and therapy

Pattern recognition receptor (PRR)-mediated innate immune responses play an essential role in defending the host from viral infections. Intriguingly, hepatitis B virus (HBV) has been shown to induce negligible innate immune responses during the early phase of infection. Whether this is due to the failure of the virus to activate PRRs or suppression of PRR signaling pathways by the virus remains controversial. However, a plethora of evidence suggests that HBV is sensitive to PRR ligand-induced antiviral responses. This review summarizes current understanding of the interaction between HBV and PRR-mediated host innate immunity, antiviral mechanisms of PRR responses against HBV and strategies to combat chronic HBV infection via induction of host innate antiviral responses.