The pathway of West Nile flavivirus early internalization events was mapped in detail in this study. Overexpression of dominant-negative mutants of Eps15 strongly inhibits West Nile virus (WNV) internalization, and pharmacological drugs that blocks clathrin also caused a marked reduction in virus entry but not caveola-dependent endocytosis inhibitory agent, filipin. Using immunocryoelectron microscopy, WNV particles were seen within clathrin-coated pits after 2 min postinfection. Double-labeling immunofluorescence assays and immunoelectron microscopy performed with anti-WNV envelope or capsid proteins and cellular markers (EEA1 and LAMP1) revealed the trafficking pathway of internalized virus particles from early endosomes to lysosomes and finally the uncoating of the virus particles. Disruption of host cell cytoskeleton (actin filaments and microtubules) with cytochalasin D and nocodazole showed significant reduction in virus infectivity. Actin filaments are shown to be essential during the initial penetration of the virus across the plasma membrane, whereas microtubules are involved in the trafficking of internalized virus from early endosomes to lysosomes for uncoating. Cells treated with lysosomotropic agents were largely resistant to infection, indicating that a low-pH-dependent step is required for WNV infection. In situ hybridization of DNA probes specific for viral RNA demonstrated the trafficking of uncoated viral RNA genomes to the endoplasmic reticulum.The first step in the initiation of a successful virus infection cycle requires animal viruses binding to specific molecules on the cellular surface and followed by penetration into the host cells for the release of viral genome for replication. A number of different internalization and trafficking pathways are utilized by animal viruses to gain entry into host cells. These pathways include clathrin-mediated endocytosis, uptake via caveolae, macropinocytosis, phagocytosis, and other pathways that presently are poorly characterized (51). Semliki Forest virus (27), vesicular stomatitis virus (18), influenza virus (30), ecotropic murine leukemia virus (2, 34, 44), and Hantaan virus (28) gain entry into cells through a pH-dependent endocytic pathway. The acidification of endocytic vesicles trigger a conformational change in the viral envelope protein and subsequent release of viral genome at appropriate location of the cells for replication. In contrast, human immunodeficiency virus type 1 (33, 34), severe acute respiratory syndrome coronavirus (43), and amphotrophic murine leukemia virus (34) are observed to fuse at the plasma membrane and the nucleocapsids are released into the cytoplasm. This process is a pH-independent process. The entry event is often a major determinant of virus tropism and pathogenesis (50). Understanding the early event of virus replication cycle will provide opportunities to develop strategies to block this initial but crucial interaction.The family Flaviviridae are positive-sense, single-stranded RNA viruses that replicate in the ...

The functional receptor for the flavivirus West Nile (WNV) infection has been characterized in this study with a combination of biochemical and molecular approaches. A 105-kDa protease-sensitive glycoprotein that binds WNV was isolated from the plasma membrane of cells permissive to WNV infection. The protein was subjected to peptide sequencing, and this glycoprotein was identified as a member of the integrin superfamily. Infection of WNV was shown to be markedly inhibited in Vero cells pretreated with blocking antibodies against ␣ v ␤ 3 integrin and its subunits by receptor competition assay. It was also noted that cells pretreated with antibodies against ␣ v ␤ 3 integrin can effectively inhibit flavivirus Japanese encephalitis but to a lesser extent flavivirus dengue infections. West Nile virus entry is independent of divalent cations and is not highly blocked by arginine-glycine-aspartic acid (RGD) peptides, suggesting that the interaction between the virus and ␣ v ␤ 3 integrin is not highly dependent on the classical RGD binding motif. In addition, gene silencing of the ␤ 3 integrin subunit in cells has resulted in cells largely resistant to WNV infection. In contrast, expression of recombinant human ␤ 3 integrin substantially increased the permissiveness of CS-1 melanoma cells for WNV infection. Soluble ␣ v ␤ 3 integrin can also effectively block WNV infection in a dose-dependent manner. Furthermore, WNV infection also triggered the outside-in signaling pathway via the activation of integrin-associated focal adhesion kinase. The identification of ␣ v ␤ 3 integrin as a receptor for WNV provides insight into virusreceptor interaction, hence creating opportunities in the development of anti-viral strategies against WNV infection.

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

Immune sensor proteins are critical to the function of the human innate immune system. The full repertoire of cognate triggers for human immune sensors is not fully understood. Here, we report that human NLRP1 is activated by 3C proteases (3Cpros) of enteroviruses, such as human rhinovirus (HRV). 3Cpros directly cleave human NLRP1 at a single site between Glu130 and Gly131. This cleavage triggers N-glycine–mediated degradation of the autoinhibitory NLRP1 N-terminal fragment via the cullinZER1/ZYG11B complex, which liberates the activating C-terminal fragment. Infection of primary human airway epithelial cells by live human HRV triggers NLRP1-dependent inflammasome activation and IL-18 secretion. Our findings establish 3Cpros as a pathogen-derived trigger for the human NLRP1 inflammasome and suggest that NLRP1 may contribute to inflammatory diseases of the airway.

Little is currently known about the infectious entry process of human enterovirus 71 (HEV71) into host cells, which may represent potential anti-viral targeting sites. In this study a targeted small-interfering RNA (siRNA) screening platform assay was established and validated to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics, and endosomal trafficking essential for HEV71 infection. Screen evaluation was conducted via the expression of well characterized dominant-negative mutants, bioimaging studies (double-labeled immunofluorescence assays, transmission electron microscopy analysis), secondary siRNA-based dosage dependence studies, and drug inhibition assays. The infectious entry of HEV71 into rhabdomyosarcoma cells was shown to be significantly inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis (CME) that include AP2A1, ARRB1, CLTC, CLTCL1, SYNJ1, ARPC5, PAK1, ROCK1, and WASF1. The functional role of CME was verified by the observation of strong co-localization between HEV71 particles and clathrin as well as dose-dependent inhibition of HEV71 infection upon siRNA knockdown of CME-associated genes. HEV71 entry by CME was further confirmed via inhibition by dominant-negative EPS15 mutants and treatment of CME drug inhibitors, with more than 80% inhibition observed at 20 M chlorpromazine. Furthermore, HEV71 infection was shown to be sensitive to the disruption of human genes in regulating early to late endosomal trafficking as well as endosomal acidic pH. The identification of clathrin-mediated endocytosis as the entry pathway for HEV71 infection of susceptible host cells contributes to a better understanding of HEV71 pathogenesis and enables future development of anti-viral strategies against HEV71 infection.

Chikungunya virus (CHIKV) is a mosquito-transmitted virus that has reemerged as a significant public health threat in the last decade. Since the 2005-2006 chikungunya fever epidemic in the Indian Ocean island of La Réunion, millions of people in more than 40 countries have been infected. Despite this, there is currently no antiviral treatment for chikungunya infection. In this study, an immunofluorescence-based screening platform was developed to identify potential inhibitors of CHIKV infection. A primary screen was performed using a highly purified natural product compound library, and 44 compounds exhibiting >70% inhibition of CHIKV infection were identified as positive hits. Among these, four were selected for dose-dependent inhibition assays to confirm their anti-CHIKV activity. Harringtonine, a cephalotaxine alkaloid, displayed potent inhibition of CHIKV infection (50% effective concentration [EC 50 ] ‫؍‬ 0.24 M) with minimal cytotoxicity and was selected for elucidation of its antiviral mechanism. Time-of-addition studies, cotreatment assays, and direct transfection of viral genomic RNA indicated that harringtonine inhibited an early stage of the CHIKV replication cycle which occurred after viral entry into cells. In addition, quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses indicated that harringtonine affects CHIKV RNA production as well as viral protein expression. Treatment of harringtonine against Sindbis virus, a related alphavirus, suggested that harringtonine could inhibit other alphaviruses. This study suggests for the first time that harringtonine exerts its antiviral effects by inhibiting CHIKV viral protein synthesis.

Dengue virus is a mosquito-borne flavivirus that represents an important emerging infectious disease and is an international health concern. Currently, there is no vaccine or effective antiviral therapy to prevent or to treat dengue virus infection. The slow progress in developing antiviral agents might be alleviated by the availability of efficient high-throughput anti-dengue virus screening assays. In this study, we report an immunofluorescence imagebased assay suitable for identification of small molecule inhibitors of dengue virus infection and replication. Using this assay, we have discovered that inhibitors of the c-Src protein kinase exhibit a potent inhibitory effect on dengue virus (serotypes 1-4) and murine flavivirus Modoc. Mechanism of action studies demonstrated that the c-Src protein kinase inhibitor dasatinib prevents the assembly of dengue virions within the virus-induced membranous replication complex. These results demonstrate that this cell-based screen may provide a powerful means to identify new potential targets for anti-dengue drug development while simultaneously providing pharmacological probes to investigate dengue virus-host cell interactions at the biochemical level. gen that causes dengue fever (DF) and a severe lifethreatening illness, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (1). DENV is a small, enveloped, positive-stranded RNA virus that belongs to the Flavivirus genus of the Flaviviridae family. Four distinct serotypes (DENV1 to -4) of dengue viruses are transmitted to humans through the bites of the mosquito species, Aedes aegypti and Aedes albopictus (2). It has been estimated that Ϸ50-100 million cases of DF, and Ϸ250,000-500,000 cases of DHF occur every year (3). Furthermore, 2.5 billion of people are at risk for infection in subtropical and tropical regions of the world (4) in the absence of effective intervention. The intracellular life cycle of DENV begins with receptor-mediated endocytosis of the virus into cells, followed by fusion of the viral envelope protein with the late endosomal membrane, which results in the release of the viral genome into the cytoplasm for replication. Replication of the viral RNA genome occurs within membrane-bound complexes formed from the endoplasmic reticulum membrane. Subsequently, virus particles are assembled and released via the host cell secretory machinery (5). Although replication of DENV involves complex interaction between viral proteins and cellular factors, many of these interactions remain unidentified and uncharacterized. Small molecules that specifically target different steps in the viral replication cycle could potentially be used as ''tool compounds'' to facilitate biochemical characterization of these host-virus interactions and might also be used to identify pharmacological intervention points for treatment of DENV infection. Although extensive studies have been carried out over the years to understand the pathogenicity of DENV infection, little progress has been made in the development of specific anti-DE...

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