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Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the heteroaryldihydropyrimidine (HAP) pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated more slowly in native agarose gel electrophoresis from A36V mutant than from the wild-type core protein. Moreover, we showed that the assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence, HBV core protein is a dominant antiviral target that may suppress the selection of drug-resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV. IMPORTANCE HBV core protein plays essential roles in many steps of the viral replication cycle. In addition to packaging viral pregenomic RNA (pgRNA) and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several different quaternary structures in infected hepatocytes, participate in and regulate HBV virion assembly, capsid uncoating, and covalently closed circular DNA (cccDNA) formation. It is anticipated that small molecular core protein assembly modulators may disrupt one or multiple steps of HBV replication, depending on their interaction with the distinct quaternary structures of core protein. The discovery of novel core protein-targeting antivirals, such as benzamide derivatives reported here and investigation of their antiviral mechanism may lead to the identification of antiviral therapeutics for the cure of chronic hepatitis B. [ABSTRACT FROM AUTHOR]

Although a highly effective vaccine is available, the number of yellow fever cases has increased over the past 2 decades, which highlights the pressing need for antiviral therapeutics. In a high-throughput screening campaign, we identified an acetic acid benzodiazepine (BDAA) compound which potently inhibits yellow fever virus (YFV). Interestingly, while treatment of YFV-infected cultures with 2 μMBDAA reduced the virion production by greater than 2 logs, the compound was not active against 21 other viruses from 14 different viral families. Selection and genetic analysis of drug-resistant viruses revealed that replacement of the proline at amino acid 219 (P219) of the nonstructural protein 4B (NS4B) with serine, threonine, or alanine conferred YFV with resistance to BDAA without apparent loss of replication fitness in cultured mammalian cells. However, replacement of P219 with glycine conferred BDAA resistance with significant loss of replication ability. Bioinformatics analysis predicts that the P219 amino acid is localized at the endoplasmic reticulum lumen side of the fifth putative transmembrane domain of NS4B, and the mutation may render the viral protein incapable of interacting with BDAA. Our studies thus revealed an important role and the structural basis for the NS4B protein in supporting YFV replication. Moreover, in YFV-infected hamsters, oral administration of BDAA protected 90% of the animals from death, significantly reduced viral load by greater than 2 logs, and attenuated virus infection-induced liver injury and body weight loss. The encouraging preclinical results thus warrant further development of BDAA or its derivatives as antiviral agents to treat yellow fever. [ABSTRACT FROM AUTHOR]

Removal of genome-bound viral DNA polymerase ought to be an essential step in the formation of hepadnavirus covalently closed circular DNA (cccDNA). We previously demonstrated that deproteinized (DP) relaxed circular DNA (rcDNA) of hepatitis B virus (HBV) existed in both the cytoplasm and nuclei of infected cells and the vast majority of cytoplasmic DP rcDNA was associated with DNase I-permeable nucleocapsids. In our efforts to investigate the role of the cytoplasmic DP rcDNA in cccDNA formation, we demonstrated that rcDNA deproteinization could occur in an endogenous DNA polymerase reaction with either virion-derived or intracellular nucleocapsids. As observed in the cytoplasm of virally infected cells, in vitro deproteinization requires the maturation of plus-strand DNA and results in changes in nucleocapsid structure that render the DP rcDNA susceptible to DNase I digestion. Remarkably, we found that the cytoplasmic DP rcDNA-containing nucleocapsids could be selectively immunoprecipitated with an antibody against the carboxyl-terminal peptide of HBV core protein and are associated with cellular nuclear transport receptors karyopherin-α and -β. Moreover, transfection of small interfering RNA targeting karyopherin-β1 mRNA or expression of a dominant-negative karyopherin-β1 in a stable cell line supporting HBV replication resulted in the accumulation of DP rcDNA in cytoplasm and reduction of nuclear DP rcDNA and cccDNA. Our results thus favor a hypothesis that completion of plus-strand DNA synthesis triggers the genomic DNA deproteinization and structural changes of nucleocapsids, which leads to the exposure of nuclear localization signals in the C terminus of core protein and mediates the nuclear transportation of DP rcDNA via interaction with karyopherin-α and -β. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFNstimulated 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. [ABSTRACT FROM AUTHOR]

n-(n-Nonyl)-deoxygalactonojirimycin (n,n-DGJ), an alkylated imino sugar, reduces the amount of HBV DNA produced within the stably transfected HBV-producing HepG2.2.15 line in culture and is under consideration for development as a human therapeutic, n,n-DGJ does not appear to inhibit HBV DNA polymerase activity or envelop antigen production (A. Mehta, S. Carrouee, B. Conyers, R. Jordan, T. Butters, R. A. Dwek, and T. M. Block, Hepatology 33:1488-1495, 2001), and the mechanism of antiviral action is unknown. In this study, the step in the virus life cycle affected by n,n-DGJ was explored. Using Northern analysis and immunoprecipitation with anti-HBc antibody, we found that, under conditions in which cell viability was not affected but viral DNA production was substantially reduced, neither the amount of HBV transcription products nor the core polypeptide was detectably reduced. However, the pregenomic RNA, endogenous polymerase activity, and core polypeptide sedimenting in sucrose gradients with a density consistent with that of assembled nucleocapsids were significantly less in the HepG2.2.15 cells incubated with n,n-DGJ. These data suggest that n,n-DGJ either prevents the maturation of HBV nucleocapsids or destabilizes the formed nucleocapsids. Although the cellular and viral mediators of this inhibition are not known, depletion of nucleocapsid has been attributed to some other compounds as well as interferon's mechanism of anti-HBV action. The similarities and differences between this alkylated imino sugar and these other mediators are discussed. [ABSTRACT FROM AUTHOR]

Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host innate and adaptive antiviral immunity against hepatitis B virus (HBV) infection in vivo. In an effort to elucidate the antiviral mechanism of these cytokines, 37 IFN-stimulated genes (ISGs), which are highly inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon overexpression in human hepatoma cells. One ISG candidate, indoleamine 2,3-dioxygenase (IDO), an IFN-γ-induced enzyme catalyzing tryptophan degradation, efficiently reduced the level of intracellular HBV DNA without altering the steady-state level of viral RNA. Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV replication, and tryptophan supplementation in culture completely restored HBV replication in IDO-expressing cells, indicating that the antiviral effect elicited by IDO is mediated by tryptophan deprivation. Interestingly, IDO-mediated tryptophan deprivation preferentially inhibited viral protein translation and genome replication but did not significantly alter global cellular protein synthesis. Finally, tryptophan supplementation was able to completely restore HBV replication in IFN-γ- but not IFN-α-treated cells, which strongly argues that IDO is the primary mediator of IFN-γ-elicited antiviral response against HBV in human hepatocyte-derived cells. [ABSTRACT FROM AUTHOR]

The liver-specific microRNA miR-122 has been shown to be required for the replication of hepatitis C virus (HCV) in the hepatoma cell line Huh7. The aim of this study was to test if HCV replication can be modulated by exogenously expressed miR-122 in human embryonic kidney epithelial cells (HEK-293). Our results demonstrate that miR-122 enhances the colony formation efficiency of the HCV replicon and increases the steady-state level of HCV RNA in HEK-293 cells. Therefore, we conclude that although miR-122 is not absolutely required, it greatly enhances HCV replication in nonhepatic cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway is one of the major oncogenic pathways and is activated in many types of human cancers, including hepatocellular carcinoma. It can also be activated by the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein. In the present study, we set out to determine the regulatory effects of this pathway on the replication of hepatitis B virus (HBV). Our results demonstrate that the expression of a constitutively active Akt1 profoundly inhibited HBV RNA transcription and consequently reduced HBV DNA replication in HepG2 cells. This suppression of HBV gene transcription was apparently mediated by the activation of mTOR, as it was abolished by the mTOR inhibitor rapamycin. Moreover, treatment of HBV-expressing HepG2.2.15 cells with inhibitors of PI3K, Akt, and mTOR increased the transcription of 3.5-kb and 2.4-kb viral RNA as well as the replication of HBV DNA. This observation implies that the basal level activation of this pathway in HepG2 cells regulated HBV replication. Consistent with previous reports showing that the HCV NS5A protein could bind to the p85 subunit of PI3K and activate the PI3K-Akt signal transduction pathway, our results showed that expression of this protein could inhibit HBV RNA transcription and reduce HBV DNA replication in HepG2 cells. Taken together, our results suggest that the activation of the PI3K-Akt pathway during liver oncogenesis may be at least partially responsible for the elimination of HBV replication from tumor cells and may also provide an explanation for the observed suppression of HBV replication by HCV coinfection. [ABSTRACT FROM AUTHOR]

Following infection, the physical state of linear herpes simplex virus (HSV) genomes may change into an "endless" or circular form. In this study, using Southern blot analysis of the HSV genome, we provide evidence that immediate-early protein ICP4 is involved in the process of converting the linear HSV-1 ICP4-deleted mutant strain d120 genome into its endless form. Under conditions where de novo viral DNA synthesis was inhibited, the genome of the ICP4 deletion mutant d120 failed to assume an endless conformation following infection of Vero cells (compared with the ability of wild-type strain KOS). This defect was reversed in the Vero-derived cell line E5, which produces the ICP4 protein, suggesting that ICP4 is necessary and sufficient to complement the d120 defect. When ICP4 protein was provided by the replication-defective DNA polymerase mutant HP66, the genomes of mutant d120 could assume an endless conformation in Vero cells. Western blot analysis using antibody specific to the ICP4 protein showed that although the d120 virions contained ICP4 protein, the majority of that ICP4 protein was in a 40-kDa truncated form, with only a small fraction present as a full-length 175-kDa protein. When expression of ICP4 protein from E5 cells was inhibited by cycloheximide, the d120 virion-associated ICP4 protein was unable to mediate endless formation after infection of E5 cells. Collectively, these data suggest that ICP4 protein has an important role in mediating the endless formation of the HSV-1 genome upon infection and that this function can be provided in trans. [ABSTRACT FROM AUTHOR]

Following infection, the physical state of linear herpes simplex virus (HSV) genomes may change into an "endless" or circular form. In this study, using Southern blot analysis of the HSV genome, we provide evidence that immediate-early protein ICP4 is involved in the process of converting the linear HSV-1 ICP4-deleted mutant strain d120 genome into its endless form. Under conditions where de novo viral DNA synthesis was inhibited, the genome of the ICP4 deletion mutant d120 failed to assume an endless conformation following infection of Vero cells (compared with the ability of wild-type strain KOS). This defect was reversed in the Vero-derived cell line E5, which produces the ICP4 protein, suggesting that ICP4 is necessary and sufficient to complement the d120 defect. When ICP4 protein was provided by the replication-defective DNA polymerase mutant HP66, the genomes of mutant d120 could assume an endless conformation in Vero cells. Western blot analysis using antibody specific to the ICP4 protein showed that although the d120 virions contained ICP4 protein, the majority of that ICP4 protein was in a 40-kDa truncated form, with only a small fraction present as a full-length 175-kDa protein. When expression of ICP4 protein from E5 cells was inhibited by cycloheximide, the d120 virion-associated ICP4 protein was unable to mediate endless formation after infection of E5 cells. Collectively, these data suggest that ICP4 protein has an important role in mediating the endless formation of the HSV-1 genome upon infection and that this function can be provided in trans.