Rare earth ions block the ion pores generated by the class II fusion proteins of alphaviruses and allow analysis of the biological functions of these pores.
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Recently, class II fusion proteins have been identified on the surface of alpha- and flaviviruses. These proteins have two functions besides membrane fusion: they generate an isometric lattice on the viral surface and they form ion-permeable pores at low pH. An attempt was made to identify inhibitors for the ion pores generated by the fusion proteins of the alphaviruses Semliki Forest virus and Sindbis virus. These pores can be detected and analysed in three situations: (i) in the target membrane during virus entry, by performing patch-clamp measurements of membrane currents; (ii) in the virus particle, by studying the entry of propidium iodide; and (iii) in the plasma membrane of infected cells, by Fura-2 fluorescence imaging of Ca2+ entry into infected cells. It is shown here that, at a concentration of 0.1 mM, rare earth ions block the ion permeability of alphavirus ion pores in all three situations. Even at a concentration of 0.5 mM, these ions do not block formation of the viral fusion pore, as they do not inhibit entry or multiplication of alphaviruses. The data indicate that ions flow through the ion pores into the virus particle in the endosome and from the endosome into the cytoplasm after fusion of the viral envelope with the endosomal membrane. These ion flows, however, are not necessary for productive infection. The possibility that the ability of class II fusion proteins to form ion-permeable pores reflects their origin from protein toxins that form ion-permeable pores, and that entry via class II fusion proteins may resemble the entry of non-enveloped viruses, is discussed. (+info)
Comparison of two cancer vaccines targeting tyrosinase: plasmid DNA and recombinant alphavirus replicon particles.
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PURPOSE: Immunization of mice with xenogeneic DNA encoding human tyrosinase-related proteins 1 and 2 breaks tolerance to these self-antigens and leads to tumor rejection. Viral vectors used alone or in heterologous DNA prime/viral boost combinations have shown improved responses to certain infectious diseases. The purpose of this study was to compare viral and plasmid DNA in combination vaccination strategies in the context of a tumor antigen. EXPERIMENTAL DESIGN: Using tyrosinase as a prototypical differentiation antigen, we determined the optimal regimen for immunization with plasmid DNA. Then, using propagation-incompetent alphavirus vectors (virus-like replicon particles, VRP) encoding tyrosinase, we tested different combinations of priming with DNA or VRP followed by boosting with VRP. We subsequently followed antibody production, T-cell response, and tumor rejection. RESULTS: T-cell responses to newly identified mouse tyrosinase epitopes were generated in mice immunized with plasmid DNA encoding human (xenogeneic) tyrosinase. In contrast, when VRP encoding either mouse or human tyrosinase were used as single agents, antibody and T-cell responses and a significant delay in tumor growth in vivo were observed. Similarly, a heterologous vaccine regimen using DNA prime and VRP boost showed a markedly stronger response than DNA vaccination alone. CONCLUSIONS: Alphavirus replicon particle vectors encoding the melanoma antigen tyrosinase (self or xenogeneic) induce immune responses and tumor protection when administered either alone or in the heterologous DNA prime/VRP boost approaches that are superior to the use of plasmid DNA alone. (+info)
Role for nsP2 proteins in the cessation of alphavirus minus-strand synthesis by host cells.
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In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and approximately 10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC+) that are active in plus-strand synthesis. Minus-strand synthesis and incorporation of [3H]uridine into replicative intermediates differed among PI replicons, depending on the location of the mutation in nsP2. Minus-strand synthesis by PI cells appeared normal; it was dependent on continuous P123 and P1234 polyprotein synthesis and ceased when protein synthesis was inhibited. The failure by the PI replicons to shut off minus-strand synthesis was not due to some defect in the PI cells but rather was due to the loss of some function in the mutated nsP2. This was demonstrated by showing that superinfection of PI cells with wt SFV triggered the shutdown of minus-strand synthesis, which we believe is a host response to infection with alphaviruses. Together, the results indicate alphavirus nsP2 functions to engage the host response to infection and activate a switch from the early-to-late phase. The loss of this function leads to continuous viral minus-strand synthesis and the production of unstable RC+. (+info)
Class II virus membrane fusion proteins.
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Enveloped animal viruses fuse their membrane with a host cell membrane, thus delivering the virus genetic material into the cytoplasm and initiating infection. This critical membrane fusion reaction is mediated by a virus transmembrane protein known as the fusion protein, which inserts its hydrophobic fusion peptide into the cell membrane and refolds to drive the fusion reaction. This review describes recent advances in our understanding of the structure and function of the class II fusion proteins of the alphaviruses and flaviviruses. Inhibition of the fusion protein refolding reaction confirms its importance in fusion and suggests new antiviral strategies for these medically important viruses. (+info)
Mechanisms of immunopathology in murine models of central nervous system demyelinating disease.
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Many disorders of the CNS, such as multiple sclerosis (MS), are characterized by the loss of the myelin sheath surrounding nerve axons. MS is associated with infiltration of inflammatory cells into the brain and spinal cord, which may be the primary cause of demyelination or which may be induced secondary to axonal damage. Both the innate and adaptive arms of the immune system have been reported to play important roles in myelin destruction. Numerous murine demyelinating models, both virus-induced and/or autoimmune, are available, which reflect the clinical and pathological variability seen in human disease. This review will discuss the immunopathologic mechanisms involved in these demyelinating disease models. (+info)
Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses.
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A computer-assisted comparative analysis of the amino acid sequences of (putative) thiol proteases encoded by the genomes of several diverse groups of positive-stranded RNA viruses and distantly related to the family of cellular papain-like proteases is presented. A high level of similarity was detected between the leader protease of foot-and-mouth-disease virus and the protease of murine hepatitis coronavirus which cleaves the N-terminal p28 protein from the polyprotein. Statistically significant alignment of a portion of the rubella virus polyprotein with cellular papain-like proteases was obtained, leading to tentative identification of the papain-like protease as the enzyme mediating processing of the non-structural proteins of this virus. Specific grouping between the sequences of the proteases of alpha-viruses, and poty- and bymoviruses was revealed. It was noted that papain-like proteases of positive-stranded RNA viruses are much more variable both in their sequences and in genomic locations than chymotrypsin-related proteases found in the same virus class. A novel conserved domain of unknown function has also been identified which flanks the papain-like proteases of alpha-, rubi- and coronaviruses. (+info)
Recovery of a recombinant salmonid alphavirus fully attenuated and protective for rainbow trout.
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Sleeping disease virus (SDV) is a member of the new Salmonid alphavirus genus within the Togaviridae family. The single-stranded RNA genome of SDV is 11,894 nucleotides long, excluding the 3' poly(A) tail. A full-length cDNA has been generated; the cDNA was fused to a hammerhead ribozyme sequence at the 5' end and inserted into a transcription plasmid (pcDNA3) backbone, yielding pSDV. By transfection of pSDV into fish cells, recombinant SDV (rSDV) was successfully recovered. Demonstration of the recovery of rSDV was provided by immunofluorescence assay on rSDV-infected cells and by the presence of a genetic tag, a BlpI restriction enzyme site, introduced into the rSDV RNA genome. SDV infectious cDNA was used for two kinds of experiments (i) to evaluate the impact of various targeted mutations in nsP2 on viral replication and (ii) to study the virulence of rSDV in trout. For the latter aspect, when juvenile trout were infected by immersion in a water bath with the wild-type virus-like rSDV, no deaths or signs of disease appeared in fish, although they were readily infected. In contrast, cumulative mortality reached 80% in fish infected with the wild-type SDV (wtSDV). When rSDV-infected fish were challenged with wtSDV 3 and 5 months postinfection, a long-lasting protection was demonstrated. Interestingly, a variant rSDV (rSDV14) adapted to grow at a higher temperature, 14 degrees C instead of 10 degrees C, was shown to become pathogenic for trout. Comparison of the nucleotide sequences of wtSDV, rSDV, and rSDV14 genomes evidenced several amino acid changes, and some changes may be linked to the pathogenicity of SDV in trout. (+info)
Identification of a putative alphavirus receptor on mouse neural cells.
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Alphaviruses replicate in a wide variety of cells in vitro. The prototype alphavirus, Sindbis virus, causes an age-dependent encephalitis in mice and serves as an important model system for the study of alphavirus neurovirulence. To begin to understand the role of cellular virus receptors in the pathogenesis of Sindbis virus infection, we developed an anti-idiotypic antibody made in rabbits against a neutralizing monoclonal antibody specific for the E2 surface glycoprotein. The anti-idiotypic antibody (anti-Id 209) bound to N18 mouse neuroblastoma cells and inhibited adsorption of 35S-labeled virus by 50%. Binding of anti-Id 209 was inhibited by pretreatment of N18 cells with various proteases but not with neuraminidase or phospholipase, while virus binding was inhibited by pretreatment with phospholipase as well as protease. Anti-Id 209 precipitated proteins of 110 and 74 kDa from N18 cells intrinsically labeled with [35S]methionine. N18 cells grow with two phenotypes in culture, and immunoprecipitation of 125I-surface-labeled cells showed that the 74-kDa protein was present on loosely adherent cells growing in aggregates, while the 110-kDa protein was present in smaller amounts on firmly adherent cells growing as a monolayer. Analysis of brain cells from newborn mice by flow cytometry showed that all cells expressed the receptor protein at birth, but by 4 days after birth half of the cells had ceased receptor expression. A survey of other cell lines showed the protein to be present on murine fibroblastic and other rodent neuroblastoma cell lines but rarely on human neural or nonneural cell lines. These studies suggest that one of the receptors for Sindbis virus on mouse neural cells is a protein that is regulated during development of the nervous system. Developmental down-regulation of receptor protein expression may contribute to the age-dependent nature of susceptibility of mice to fatal alphavirus encephalitis. (+info)