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The emergence of West Nile virus (WNV) in the WesternWest Nile virus (WNV) is a positive-sense single-stranded RNA virus in the family Flaviviridae. Isolates of WNV are subdivided into two lineages: lineage I viruses are represented by emergent strains distributed throughout the world and have been associated with outbreaks of encephalitis and meningitis in Europe, the Middle East, and, most recently, in North America, whereas lineage II isolates are largely nonemergent/ endemic strains that are confined to the African subcontinent and the island countries of Madagascar and Cyprus (5,7,25,26). In most cases, WNV infection of humans can be characterized as asymptomatic or as a mild, febrile illness termed West Nile fever. However, a significant increase in the global incidence of severe neurological disease associated with WNV lineage I infections arose in the mid-1990s, culminating in the U.S. outbreak in 2003, which included 9,862 reported cases and 264 deaths (CDC website, http://www.cdc.gov/ncidod/dvbid /westnile/index.htm). After its introduction in New York City in 1999, WNV rapidly spread across the continent and now appears to have firmly established itself in the ecology of North America. The rapid emergence of WNV and its virulence within a naïve population suggest that epidemic forms of the virus may encode mechanisms to evade host immunity.Infection with WNV triggers a delayed host response that includes the activation of interferon regulatory factor-3 (IRF-3) and the subsequent production of alpha/beta interferon (IFN-␣/␤) (14,15,38). IFNs are a family of immunomodulatory cytokines that are produced in response to virus infection and serve as integral signal initiators of host intracellular defenses (40,46). Binding of IFN to the cognate IFN-␣/␤ receptor (IFNAR) on target cells results in the activation of the JAK-STAT pathway, which includes the receptor-associated kinases JAK1 and Tyk2 that in turn phosphorylate and activate their downstream effectors, STAT1 and STAT2. Activated phospho-STAT1/STAT2 heterodimers translocate to the nucleus to form a heterotrimeric complex with IRF-9 and induce the transcription of hundreds of interferon-stimulated genes (ISGs), whose products can direct antiviral and antiproliferative actions that limit virus replication and spread. Many viruses encode proteins that direct mechanisms to disrupt innate antiviral defenses and IFN-induced JAK-STAT signaling, and these processes have been linked to viral emergence in new host populations and species (16,23,44) and to pathogenic outcomes of infection (reviewed in references 11, 40, and 46). Importantly, virulent isolates of WNV have been shown to be capable of attenuating IFN actions by preventing STAT1 and STAT2 activation, although the mechanisms of this regulation and its influence in vivo were not defined (17, 29). Here we describe in vitro and in vivo studies comparing the genetic and phenotypic properties of a lineage I/emergent strain and a lineage II/nonemergent strain of WNV. Our data show that viral control of I...

An amphipathic ␣-helical peptide (C5A) derived from the membrane anchor domain of the hepatitis C virus (HCV) NS5A protein is virocidal for HCV at submicromolar concentrations in vitro. C5A prevents de novo HCV infection and suppresses ongoing infection by inactivating both extra-and intracellular infectious particles, and it is nontoxic in vitro and in vivo at doses at least 100-fold higher than required for antiviral activity. Mutational analysis indicates that C5A's amphipathic ␣-helical structure is necessary but not sufficient for its virocidal activity, which depends on its amino acid composition but not its primary sequence or chirality. In addition to HCV, C5A inhibits infection by selected flaviviruses, paramyxoviruses, and HIV. These results suggest a model in which C5A destabilizes viral membranes based on their lipid composition, offering a unique therapeutic approach to HCV and other viral infections.HCV ͉ amphipathic peptide ͉ antiviral peptide ͉ NS5A ͉ HIV H epatitis C virus (HCV), a member of the Flaviviridae family (1), is a single-stranded positive-sense RNA virus that causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (2, 3). HCV infects Ͼ170 million people worldwide and is the most common cause of liver transplantation in the United States (3). There is no vaccine available for HCV, and the only currently approved treatment (combination therapy with IFN and ribavirin) has limited efficacy and serious side effects (4, 5). Thus, development of new classes of antiviral compounds with improved efficacy and toxicity profiles is urgently needed.The development of HCV replicon technology several years ago (6) greatly accelerated the pace of antiviral drug discovery, leading to the development of HCV protease and polymerase inhibitors that are currently under clinical evaluation (7,8). The landscape for drug discovery improved further with the establishment of a cell culture model of HCV infection in 2005 (9-11), making it possible to search for inhibitors of every step in the HCV life cycle and agents that target the virus itself. We now report the discovery of several HCV-derived synthetic peptides that inhibit HCV infection in the cell culture infection system. One of those inhibitory peptides, an amphipathic ␣-helical 18-mer derived from the membrane anchor domain of the HCV nonstructural protein NS5A that was particularly potent against HCV and selected other virus infections, serves as the basis of this report. Results Identification of Antiviral Peptides.A peptide library of 441 overlapping peptides (18-mers offset by 11 amino acids) covering the entire HCV polyprotein (H77 strain, genotype 1a) was screened (20 M) for the ability to inhibit HCV infection (JFH-1) in a focus reduction assay using Huh-7.5.1 cells (Fig. 1). Thirteen peptides were shown to inhibit HCV focus formation by Ͼ90%. Validation of the antiviral activity of the 13 inhibitory peptides was performed by comparing the ability of each peptide (20 M) to inhibit the expansion of HCV RNA in Huh-7.5.1 cells ...

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The ability of viruses to control and/or evade the host antiviral response is critical to the establishment of a productive infection. We have previously shown that West Nile virus NY (WNV-NY) delays activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical to the initiation of the antiviral response. Here we demonstrate that the delayed activation of IRF-3 is essential for WNV-NY to achieve maximum virus production. Furthermore, WNV-NY utilizes a unique mechanism to control activation of IRF-3. In contrast to many other viruses that impose a nonspecific block to the IRF-3 pathway, WNV-NY eludes detection by the host cell at early times postinfection. To better understand this process, we assessed the role of the pathogen recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I) in sensing WNV-NY infection. RIG-I null mouse embryo fibroblasts (MEFs) retained the ability to respond to WNV-NY infection; however, the onset of the host response was delayed compared to wild-type (WT) MEFs. This suggests that RIG-I is involved in initially sensing WNV-NY infection, while other PRRs sustain and/or amplify the host response later in infection. The delayed initiation of the host response correlated with an increase in WNV-NY replication in RIG-I null MEFs compared to WT MEFs. Our data suggest that activation of the host response by RIG-I early in infection is important for controlling replication of WNV-NY. Furthermore, pathogenic strains of WNV may have evolved to circumvent stimulation of the host response until after replication is well under way.