Recovery of African horse sickness virus from synthetic RNA.

African horse sickness virus (AHSV) is an insect-vectored emerging pathogen of equine species. AHSV (nine serotypes) is a member of the genus Orbivirus, with a morphology and coding strategy similar to that of the type member, bluetongue virus. However, these viruses are distinct at the genetic level, in the proteins they encode and in their pathobiology. AHSV infection of horses is highly virulent with a mortality rate of up to 90 %. AHSV is transmitted by Culicoides, a common European insect, and has the potential to emerge in Europe from endemic countries of Africa. As a result, a safe and effective vaccine is sought urgently. As part of a programme to generate a designed highly attenuated vaccine, we report here the recovery of AHSV from a complete set of RNA transcripts synthesized in vitro from cDNA clones. We have demonstrated the generation of mutant and reassortant AHSV genomes, their recovery, stable passage, and characterization. Our findings provide a new approach to investigate AHSV replication, to design AHSV vaccines and to aid diagnosis.

Baculovirus GP64-mediated entry into mammalian cells.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-ß-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

Involvement of ceramide in the propagation of Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus and one of the most important flaviviruses in the medical and veterinary fields. Although cholesterol has been shown to participate in both the entry and replication steps of JEV, the mechanisms of infection, including the cellular receptors of JEV, remain largely unknown. To clarify the infection mechanisms of JEV, we generated pseudotype (JEVpv) and recombinant (JEVrv) vesicular stomatitis viruses bearing the JEV envelope protein. Both JEVpv and JEVrv exhibited high infectivity for the target cells, and JEVrv was able to propagate and form foci as did authentic JEV. Anti-JEV envelope antibodies neutralized infection of the viruses. Treatment of cells with inhibitors for vacuolar ATPase and clathrin-mediated endocytosis reduced the infectivity of JEVpv, suggesting that JEVpv enters cells via pH- and clathrin-dependent endocytic pathways. Although treatment of the particles of JEVpv, JEVrv, and JEV with cholesterol drastically reduced the infectivity as previously reported, depletion of cholesterol from the particles by treatment with methyl beta-cyclodextrin enhanced infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and propagation of JEVrv, and these enhancements were inhibited by treatment with an SMase inhibitor or C(6)-ceramide. These results suggest that ceramide plays crucial roles in not only entry but also egress processes of JEV, and they should assist in the clarification of JEV propagation and the development of novel therapeutics against diseases caused by infection with flaviviruses.

Acquisition of complement resistance through incorporation of CD55/decay-accelerating factor into viral particles bearing baculovirus GP64.

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.

Baculovirus induces type I interferon production through toll-like receptor-dependent and -independent pathways in a cell-type-specific manner.

Autographa californica nuclear polyhedrosis virus (AcNPV) is a double-stranded-DNA virus that is pathogenic to insects. AcNPV was shown to induce an innate immune response in mammalian immune cells and to confer protection of mice from lethal viral infection. In this study, we have shown that production of type I interferon (IFN) by AcNPV in murine plasmacytoid dendritic cells (pDCs) and non-pDCs, such as peritoneal macrophages and splenic CD11c+ DCs, was mediated by Toll-like receptor (TLR)-dependent and -independent pathways, respectively. IFN regulatory factor 7 (IRF7) was shown to play a crucial role in the production of type I IFN by AcNPV not only in immune cells in vitro but also in vivo. In mouse embryonic fibroblasts (MEFs), AcNPV produced IFN-beta and IFN-inducible chemokines through TLR-independent and IRF3-dependent pathways, in contrast to the TLR-dependent and IRF3/IRF7-independent production of proinflammatory cytokines. Although production of IFN-beta and IFN-inducible chemokines was severely impaired in IFN promoter-stimulator 1 (IPS-1)-deficient MEFs upon infection with vesicular stomatitis virus, AcNPV produced substantial amounts of the cytokines in IPS-1-deficient MEFs. These results suggest that a novel signaling pathway(s) other than TLR- and IPS-1-dependent pathways participates in the production of type I IFN in response to AcNPV infection.

A single-amino-acid mutation in hepatitis C virus NS5A disrupting FKBP8 interaction impairs viral replication.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) regulates viral replication through its interaction with host and other viral proteins. We have previously shown that FK506-binding protein 8 (FKBP8) binds to NS5A and recruits Hsp90 to form a complex that participates in the replication of HCV. In this study, we examined the biochemical characteristics of the interaction and the intracellular localization of NS5A and FKBP8. Surface plasmon resonance analysis revealed that the dissociation constant of the interaction between the purified FKBP8 and NS5A expressed in bacteria was 82 nM. Mutational analyses of NS5A revealed that a single amino acid residue of Val or Ile at position 121, which is well conserved among all genotypes of HCV, is critical for the specific interaction with FKBP8. Substitution of the Val(121) to Ala drastically impaired the replication of HCV replicon cells, and the drug-resistant replicon cells emerging after drug selection were shown to have reverted to the original arrangement by replacing Ala(121) with Val. Examination of individual fields of the replicon cells by both fluorescence microscopy and electron microscopy (the correlative fluorescence microscopy-electron microscopy technique) revealed that FKBP8 is partially colocalized with NS5A in the cytoplasmic structure known as the membranous web. These results suggest that specific interaction of NS5A with FKBP8 in the cytoplasmic compartment plays a crucial role in the replication of HCV.

Hepatitis C virus nonstructural protein 5A modulates the toll-like receptor-MyD88-dependent signaling pathway in macrophage cell lines.

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries; however, the mechanism through which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) play a pivotal role in the recognition of viral infection and the induction of innate and adaptive immune responses. Several reports suggest that HCV infection induces the dysfunction of DCs in patients with chronic hepatitis C. Toll-like receptor (TLR) has been shown to play various roles in many viral infections; however, the involvement of HCV proteins in the TLR signaling pathway has not yet been precisely elucidated. In this study, we established mouse macrophage cell lines stably expressing HCV proteins and determined the effect of HCV proteins on the TLR signaling pathways. Immune cells expressing NS3, NS3/4A, NS4B, or NS5A were found to inhibit the activation of the TLR2, TLR4, TLR7, and TLR9 signaling pathways. Various genotypes of NS5A bound to MyD88, a major adaptor molecule in TLR, inhibited the recruitment of interleukin-1 receptor-associated kinase 1 to MyD88, and impaired cytokine production in response to TLR ligands. Amino acid residues 240 to 280, previously identified as the interferon sensitivity-determining region (ISDR) in NS5A, interacted with the death domain of MyD88, and the expression of a mutant NS5A lacking the ISDR partially restored cytokine production. These results suggest that the expression of HCV proteins modulates the TLR signaling pathway in immune cells.