We describe an antiviral small molecule, LJ001, effective against numerous enveloped viruses including Influenza A, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses, and HIV-1. In sharp contrast, the compound had no effect on the infection of nonenveloped viruses. In vitro and in vivo assays showed no overt toxicity. LJ001 specifically intercalated into viral membranes, irreversibly inactivated virions while leaving functionally intact envelope proteins, and inhibited viral entry at a step after virus binding but before virus-cell fusion. LJ001 pretreatment also prevented virus-induced mortality from Ebola and Rift Valley fever viruses. Structure-activity relationship analyses of LJ001, a rhodanine derivative, implicated both the polar and nonpolar ends of LJ001 in its antiviral activity. LJ001 specifically inhibited virus-cell but not cell-cell fusion, and further studies with lipid biosynthesis inhibitors indicated that LJ001 exploits the therapeutic window that exists between static viral membranes and biogenic cellular membranes with reparative capacity. In sum, our data reveal a class of broad-spectrum antivirals effective against enveloped viruses that target the viral lipid membrane and compromises its ability to mediate virus-cell fusion.virology | viral entry | fusion inhibitor | small molecule | lipid membrane

The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Non-structural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70.Weconfirmed anNS5A/HSPinteraction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A.Atransient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion. Conclusion Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.

Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput, quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 3′ non-translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and presents new therapeutic targets as well as topological information for designing vaccine candidates.

Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. There is no effective therapy or vaccine available to HCV-infected patients other than interferon–ribavarin combination, which is effective in a relatively small percentage of infected patients. Our previous results have shown that a synthetic peptide (LAP) corresponding to the N-terminal 18 amino acids of the Lupus autoantigen (La) was a potent inhibitor of HCV IRES-mediated translation. We demonstrate here that LAP efficiently blocks HCV replication of infectious JFH1 virus in cell culture. Our data suggest that LAP forms complexes with IRES-transacting factors (ITAFs) PTB and PCBP2. LAP-mediated inhibition of HCV IRES-mediated translation in vitro could be fully rescued by recombinant PCB and PCBP2. Also transient expression of PTB/PCBP2 combination significantly restores HCV replication in LAP-inhibited cultures. These results suggest that ITAFs could be potential targets to block HCV replication.

Hepatitis C virus (HCV) causes chronic infection in humans leading to liver cirrhosis and hepatocellular carcinoma. rRNA transcription, catalyzed by RNA polymerase I (Pol I), plays a critical role in ribosome biogenesis, and changes in Pol I transcription rate are associated with profound alterations in the growth rate of the cell. Because rRNA synthesis is intimately linked to cell growth and frequently up-regulated in many cancers, we hypothesized that HCV might have the ability to activate rRNA synthesis in infected cells. We show here that rRNA promoter-mediated transcription is significantly (10-to 12-fold) activated in human liver-derived cells following infection with type 2 JFH-1 HCV or transfection with the subgenomic type 1 HCV replicon. Further analysis revealed that HCV nonstructural protein 5A (NS5A) was responsible for activation of rRNA transcription. Both the NH 2 -terminal amphipathic helix and the polyproline motifs of NS5A seem to be essential for rRNA transcription activation. The NS5A-dependent activation of rRNA transcription seems to be due to hyperphosphorylation and consequent activation of upstream binding factor (UBF), a Pol I DNA binding transcription factor. We further show that hyperphosphorylation of UBF occurs as a result of up-regulation of both cyclin D1 and cyclindependent kinase 4 by the HCV NS5A polypeptide. These results suggest that the endoplasmic reticulum-associated NS5A is able to transduce signals into the nucleoplasm via UBF hyperphosphorylation leading to rRNA transcription activation. These results could, at least in part, explain a mechanism by which HCV contributes to transformation of liver cells.