Ju-Tao Guo

Effect of Alpha Interferon on the Hepatitis C Virus Replicon

ABSTRACT Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-α) and ribavirin combination therapy. The mechanism of the IFN-α response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-α reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-α independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-α. Finally, under our conditions, selection for IFN-α-resistant variants did not occur.

Replication of Hepatitis C Virus Subgenomes in Nonhepatic Epithelial and Mouse Hepatoma Cells

ABSTRACT The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known whether extrahepatic tissues are also infected by the virus and whether nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and in other species. We have tested this hypothesis and found that subgenomic HCV RNAs can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection.

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.

West Nile Virus Inhibits the Signal Transduction Pathway of Alpha Interferon

ABSTRACT West Nile virus (WNV) is a human pathogen that can cause neurological disorders, including meningoencephalitis. Experiments with mice and mammalian cell cultures revealed that WNV exhibited resistance to the innate immune program induced by alpha interferon (IFN-α). We have investigated the nature of this inhibition and have found that WNV replication inhibited the activation of many known IFN-inducible genes, because it prevented the phosphorylation and activation of the Janus kinases JAK1 and Tyk2. As a consequence, activation of the transcription factors STAT1 and STAT2 did not occur in WNV-infected cells. Moreover, we demonstrated that the viral nonstructural proteins are responsible for this effect. Thus, our results provided an explanation for the observed resistance of WNV to IFN-α in cells of vertebrate origin.

Liver-Specific MicroRNA miR-122 Enhances the Replication of Hepatitis C Virus in Nonhepatic Cells

ABSTRACT 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.

Interferon-Induced Cell Membrane Proteins, IFITM3 and Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct Mechanisms

ABSTRACT Tetherin and IFITM3 are recently identified interferon-induced cellular proteins that restrict infections by retroviruses and filoviruses and of influenza virus and flaviviruses, respectively. In our efforts to further explore their antiviral activities against other viruses and determine their antiviral mechanisms, we found that the two antiviral proteins potently inhibit the infection of vesicular stomatitis virus (VSV), a prototype member of the Rhabdoviridae family. Taking advantage of this well-studied virus infection system, we show that although both tetherin and IFITM3 are plasma membrane proteins, tetherin inhibits virion particle release from infected cells, while IFITM3 disrupts an early event after endocytosis of virion particles but before primary transcription of incoming viral genomes. Furthermore, we demonstrate that both the N-terminal 21 amino acid residues and C-terminal transmembrane region of IFITM3 are required for its antiviral activity. Collectively, our work sheds light on the mechanisms by which tetherin and IFITM3 restrict infection with rhabdoviruses and possibly other pathogenic viruses.

Apoptosis and Regeneration of Hepatocytes during Recovery from Transient Hepadnavirus Infections

ABSTRACT It is well known that hepatitis B virus infections can be transient or chronic, but the basis for this dichotomy is not known. To gain insight into the mechanism responsible for the clearance of hepadnavirus infections, we have performed a molecular and histologic analysis of liver tissues obtained from transiently infected woodchucks during the critical phase of the recovery period. We found as expected that clearance from transient infections occurred subsequent to the appearance of CD4+ and CD8+ T cells and the production of interferon gamma and tumor necrosis factor alpha in the infected liver. These events were accompanied by a significant increase in apoptosis and regeneration of hepatocytes. Surprisingly, however, accumulation of virus-free hepatocytes was delayed for several weeks following this initial influx of lymphocytes. In addition, we observed that chronically infected animals can exhibit levels of T-cell accumulation, cytokine expression, and apoptosis that are comparable with those observed during the initial phase of transient infections. Our results are most consistent with a model for recovery predicting replacement of infected hepatocytes with regenerated cells, which by unknown mechanisms remain protected from reinfection in animals that can be cured.

Characterization of the Intracellular Deproteinized Relaxed Circular DNA of Hepatitis B Virus: an Intermediate of Covalently Closed Circular DNA Formation

ABSTRACT Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is formed by conversion of capsid-associated relaxed circular DNA (rcDNA) via unknown mechanisms and exists in the nucleus of the infected hepatocyte as a minichromosome that serves as the transcription template for viral RNAs. To study the molecular pathway of cccDNA formation and its regulation by viral and cellular factors, we have established a cell line that supports the replication of an envelope protein-deficient HBV genome in a tetracycline-inducible manner. Following induction of HBV replication, the cells accumulate higher levels of cccDNA as well as larger amounts of deproteinized rcDNA (DP-rcDNA) than cells that replicate wild-type HBV genomes. These results indicate that HBV envelope proteins negatively regulate cccDNA formation, and conversion of DP-rcDNA into cccDNA is a rate-limiting step of cccDNA formation in HepG2 cells. Detailed analyses reveal the following: (i) DP-rcDNA exists in both cytoplasm and nucleus; (ii) while nuclear DP-rcDNA is sensitive to DNase I digestion, a small fraction of cytoplasmic DP-rcDNA is DNase I resistant; (iii) both DNase I-sensitive and -resistant cytoplasmic DP-rcDNAs cosediment with capsids and can be immunoprecipitated with HBV core antibody; and (iv) a primer extension assay maps the 5′ end of the minus strand of DP-rcDNA at the authentic end of virion rcDNA. Hence, our results favor a hypothesis that the removal of viral polymerase protein covalently linked to the 5′ end of the minus-strand DNA occurs inside the capsid in the cytoplasm and most possibly via a reaction that cleaves the phosphodiester bond between the tyrosine of the polymerase and the 5′ phosphoryl group of minus-strand DNA.

Identification of Disubstituted Sulfonamide Compounds as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation

ABSTRACT Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC50s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in the in vitro endogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection.

Activation of Pattern Recognition Receptor-Mediated Innate Immunity Inhibits the Replication of Hepatitis B Virus in Human Hepatocyte-Derived Cells

ABSTRACT Recognition of virus infections by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation associated gene 5 (MDA5), activates signaling pathways, leading to the induction of inflammatory cytokines that limit viral replication. To determine the effects of PRR-mediated innate immune response on hepatitis B virus (HBV) replication, a 1.3mer HBV genome was cotransfected into HepG2 or Huh7 cells with plasmid expressing TLR adaptors, myeloid differentiation primary response gene 88 (MyD88), and TIR-domain-containing adaptor-inducing beta interferon (TRIF), or RIG-I/MDA5 adaptor, interferon promoter stimulator 1 (IPS-1). The results showed that expressing each of the three adaptors dramatically reduced the levels of HBV mRNA and DNA in both HepG2 and Huh7 cells. However, HBV replication was not significantly affected by treatment of HBV genome-transfected cells with culture media harvested from cells transfected with each of the three adaptors, indicating that the adaptor-induced antiviral response was predominantly mediated by intracellular factors rather than by secreted cytokines. Analyses of involved signaling pathways revealed that activation of NF-κB is required for all three adaptors to elicit antiviral response in both HepG2 and Huh7 cells. However, activation of interferon regulatory factor 3 is only essential for induction of antiviral response by IPS-1 in Huh7 cells, but not in HepG2 cells. Furthermore, our results suggest that besides NF-κB, additional signaling pathway(s) are required for TRIF to induce a maximum antiviral response against HBV. Knowing the molecular mechanisms by which PRR-mediated innate defense responses control HBV infections could potentially lead to the development of novel therapeutics that evoke the host cellular innate antiviral response to control HBV infections.