Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is formed by conversion of capsidassociated 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.Hepatitis B virus (HBV) causes transient and chronic infections of the liver. Transient infection frequently leads to acute hepatitis but, in rare cases, results in fatal, fulminant hepatitis. Chronic infection represents a major public health problem affecting an estimated 400 million individuals worldwide and carries a high risk for the development of chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma (20,27).HBV is the prototype virus of the Hepadnaviridae family and contains a relaxed circular partially double-stranded DNA (rcDNA; 3.2 kb in length) genome (43). A hallmark of HBV genomic DNA replication is protein-primed reverse transcription of an RNA intermediate called pregenomic RNA (pgRNA) (42, 49). The overall replication scheme of HBV is related to that of retroviruses but is mechanistically distinct (40). One of the most obvious differences is that the integration of viral genomic DNA into host cellular chromosomes is not an obligatory step in the HBV life cycle. Instead, upon the entry into hepatocytes, viral genomic DNA (rcDNA) in the nucleocapsid is transported into the nucleus and converted into an episomal covalently closed circular DNA (cccDNA), which serves as the template for the transcription of viral RNAs (3,31,40). As a key replication intermediate in HBV infection, a...

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-␣ 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-␣ is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause invasive neurological diseases and lethal hemorrhagic fever in humans, respectively (6, 32). Since its first incursion into New York City in 1999, WNV has rapidly spread throughout the continental United States and has recently reached South America (29, 34). In most cases, WNV infection of people resolves as an asymptomatic or a mild febrile illness. However, approximately 1% of infections result in severe neurological disorders, such as encephalitis and meningitis (27). Unlike WNV, for which people are only accidental hosts, DENV has fully adapted to humans (32). It has apparently lost the need for an enzootic cycle and causes a range of diseases in people, from acute febrile illness to life-threatening dengue hemorrhagic fever/dengue shock syndrome (6). Four distinct serotypes of DENV have spread throughout the tropical and subtropical parts of the world, with an estimated 50 to 100 million human cases annually and about 2.5 billion people worldwide being at risk of infection (32). Effective antiviral therapies and vaccines to treat or prevent WNV and DENV infections in humans are not yet available.Type I interferons (IFNs), represented by IFN-␣ and IFN-␤, have been demonstrated to play an essential role in defending against WNV and DENV infections. For example, mice with deficiencies in the induction of type I IFNs and the receptor or JAK-STAT signal transduction pathway of the cytokines are vulnerable to WNV and DENV infections (7,38,42,(49)(50)(51). In addition, a strain of WNV that fails to block the type I IFN signal transduction pathway is phenotypically attenuated in mice (23,50). Clinically, during acute DENV infection, innate immune responses play a key role in determining disease outcome (35), and resolution of WNV infection requires effective IFN-mediated innate host responses (23,43,53). Therefore, understanding how the IFN-mediated innate immune response functions is one of the critical frontiers in the molecular biology of WN...

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.The interferon (IFN) system is the first line of defense against virus infection in vertebrates. Infection of cells by viruses can be detected by host cellular pattern recognition receptors (PRRs), such as toll-like receptors and RIG-I-like receptors (1,33,42). Engagement of the PRRs with virusassociated molecular patterns, such as 5Ј-triphosphate and/or double-stranded viral RNA, triggers signaling cascades that lead to the synthesis and secretion of type I IFNs, represented by IFN-␣ and IFN-␤ (25). Type I IFNs bind to their cognate receptors on the cell surface and activate the receptor-associated Janus kinase 1 (JAK1) and tyrosine kinase 2 (Tyk2) through tyrosine phosphorylation, which in turn stimulates the tyrosine phosphorylation of STAT1 and STAT2. Phosphorylated STAT1 and STAT2, in combination with IRF9, form a trimeric ISGF3 transcription factor that translocates into the nucleus and activates the expression of IFN-stimulated genes (ISGs), whose products limit viral replication, regulate cell proliferation, and/or modulate host innate and adaptive immune responses (34,36,39).Although IFN treatment of cells induces the expression of hundreds of ISGs, only a few ISGs have thus far been demonstrated to instigate an antiviral state in cultured cells, and only four of them, including ISG15 (IFN-stimulated protein of 15 kDa), myxovirus resistance 1 (Mx1), RNase L, and doublestranded RNA-activated protein kinase (PKR), have been shown to play a role in mediating the IFN antiviral response in vivo with gene knockout mouse models (36). While it is most likely that inhibition of the infection of any given virus by IFNs is through induction of multiple ISGs that work cooperatively to disrupt multiple steps of viral replication, identification of individual antiviral ISGs and subsequent elucidation of their modes of action are essential to uncover the antiviral mechanism of IFNs and viral pathogenesis (2...

Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with alpha interferon (IFN-␣)-based therapies.However, the underlying mechanism of IFN-␣ therapy remains to be elucidated. To identify the cellular proteins that mediate the antiviral effects of IFN-␣, 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-␣ 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. Hepatitis C virus (HCV) is the sole member of the genusHepacivirus in the family Flaviviridae (43). It establishes persistent infections in the vast majority of infected individuals and is the only known positive-stranded RNA virus that causes persistent life-long infections in humans. Currently, HCV chronically infects more than 170 million people worldwide. Although the initial infection is largely asymptomatic, prolonged infection carries a high risk of chronic hepatitis, cirrhosis, and primary hepatocellular carcinoma (2).Although it has been elegantly demonstrated that HCV can evade the host cellular innate defense response through proteolytic cleavage of RIG-I/MDA5 adaptor protein MAVS and Tolllike receptor 3 adaptor protein TRIF (7,22,25,42,44,48,69), microarray studies performed with liver samples obtained from HCV transiently infected chimpanzees and chronically infected humans revealed that the induction of interferon (IFN)-stimulated genes (ISGs) in HCV-infected livers is a hallmark of the virus infection (5,6,33,39,58,61). These discoveries suggest that the HCV-infected liver is a constant battlefield between the virus and host innate immunity defense systems, and thus IFN-mediated innate responses induced by HCV may play an important role in shaping the pathogenesis and clini...

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