SUMMARY Interferons (IFN) are essential antiviral cytokines that establish the cellular antiviral state through upregulation of hundreds of interferon-stimulated genes (ISGs), most of which have uncharacterized functions and mechanisms. We identified Cholesterol-25-hydroxylase (Ch25h) as an antiviral ISG that can convert cholesterol to a soluble antiviral factor, 25-hydroxycholesterol (25HC). Ch25h expression or 25HC treatment in cultured cells broadly inhibits enveloped viruses including VSV, HSV, HIV, and MHV68 as well as acutely pathogenic EBOV, RVFV, RSSEV, and Nipah viruses under BSL4 conditions. As a soluble oxysterol, 25HC inhibits viral entry by blocking membrane fusion between virus and cell. In animal models, Ch25h-knockout mice were more susceptible to MHV68 lytic infection. Moreover, administration of 25HC in humanized mice suppressed HIV replication and rescued T-cell depletion. Thus, our studies demonstrate a unique mechanism by which IFN achieves its antiviral state through the production of a natural oxysterol to inhibit viral entry and implicate membrane-modifying oxysterols as potential antiviral therapeutics.

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...

Chronic hepatitis B virus (HBV) infection is a global public health challenge on the same scale as tuberculosis, HIV, and malaria. The International Coalition to Eliminate HBV (ICE-HBV) is a coalition of experts dedicated to accelerating the discovery of a cure for chronic hepatitis B. Following extensive consultation with more than 50 scientists from across the globe, as well as key stakeholders including people affected by HBV, we have identified gaps in our current knowledge and new strategies and tools that are required to achieve HBV cure. We believe that research must focus on the discovery of interventional strategies that will permanently reduce the number of productively infected cells or permanently silence the covalently closed circular DNA in those cells, and that will stimulate HBV-specific host immune responses which mimic spontaneous resolution of HBV infection. There is also a pressing need for the establishment of repositories of standardised HBV reagents and protocols that can be accessed by all HBV researchers throughout the world. The HBV cure research agenda outlined in this position paper will contribute markedly to the goal of eliminating HBV infection worldwide.

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...