RESUMO
UNLABELLED: Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are â¼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCE: It is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.
Assuntos
Interações Hospedeiro-Patógeno , Membranas Mitocondriais/virologia , Nodaviridae/fisiologia , Replicação Viral , Animais , Linhagem Celular , Microscopia Crioeletrônica , Drosophila , Membranas Mitocondriais/ultraestrutura , Nodaviridae/ultraestruturaRESUMO
Igs in vertebrates comprise equally sized H and L chains, with exceptions such as H chain-only Abs in camels or natural Ag receptors in sharks. In Reptilia, Igs are known as IgYs. Using immunoassays with isotype-specific mAbs, in this study we show that green turtles (Chelonia mydas) have a 5.7S 120-kDa IgY comprising two equally sized H/L chains with truncated Fc and a 7S 200-kDa IgY comprised of two differently sized H chains bound to L chains and apparently often noncovalently associated with an antigenically related 90-kDa moiety. Both the 200- and 90-kDa 7S molecules are made in response to specific Ag, although the 90-kDa molecule appears more prominent after chronic Ag stimulation. Despite no molecular evidence of a hinge, electron microscopy reveals marked flexibility of Fab arms of 7S and 5.7S IgY. Both IgY can be captured with protein G or melon gel, but less so with protein A. Thus, turtle IgY share some characteristics with mammalian IgG. However, the asymmetrical structure of some turtle Ig and the discovery of an Ig class indicative of chronic antigenic stimulation represent striking advances in our understanding of immunology.
Assuntos
Isotipos de Imunoglobulinas/imunologia , Imunoglobulinas/imunologia , Imunoglobulinas/ultraestrutura , Tartarugas/imunologia , Animais , Anticorpos/imunologia , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Processamento de Imagem Assistida por Computador , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Microscopia Eletrônica de Transmissão/veterinária , Dados de Sequência Molecular , Receptores Fc/imunologiaRESUMO
RNAi is an evolutionarily conserved gene regulatory process that operates in a wide variety of organisms. During RNAi, long double-stranded RNA precursors are processed by Dicer proteins into â¼21-nt siRNAs. Subsequently, siRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute-family proteins and guide RISC to target RNAs via complementary base pairing, leading to posttranscriptional gene silencing. Select pre-mRNA splicing factors have been implicated in RNAi in fission yeast, worms, and flies, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle implicated in splicing, is required for RNAi and antiviral immunity in cultured cells and in vivo. SmD1 interacts with both Dicer-2 and dsRNA precursors and is indispensable for optimal siRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the small interfering RISC without significantly affecting the expression of major canonical siRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the small interfering RISC, including Argonaute 2, both in flies and in humans. Notably, RNAi defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the RNAi and splicing machineries are physically and functionally distinct entities. Our results suggest that Drosophila SmD1 plays a direct role in RNAi-mediated gene silencing independently of its pre-mRNA splicing activity and indicate that the dual roles of splicing factors in posttranscriptional gene regulation may be evolutionarily widespread.
Assuntos
Drosophila/genética , Interferência de RNA/fisiologia , Proteínas Centrais de snRNP/genética , Proteínas Centrais de snRNP/metabolismo , Animais , Northern Blotting , Linhagem Celular , Drosophila/fisiologia , Imunoprecipitação , Precursores de RNA/genética , RNA Interferente Pequeno/genéticaRESUMO
Arthropod-borne pathogens account for millions of deaths each year. Understanding the genetic mechanisms controlling vector susceptibility to pathogens has profound implications for developing novel strategies for controlling insect-transmitted infectious diseases. The fact that many viruses carry genes that have anti-apoptotic activity has long led to the hypothesis that induction of apoptosis could be a fundamental innate immune response. However, the cellular mechanisms mediating the induction of apoptosis following viral infection remained enigmatic, which has prevented experimental verification of the functional significance of apoptosis in limiting viral infection in insects. In addition, studies with cultured insect cells have shown that there is sometimes a lack of apoptosis, or the pro-apoptotic response happens relatively late, thus casting doubt on the functional significance of apoptosis as an innate immunity. Using in vivo mosquito models and the native route of infection, we found that there is a rapid induction of reaper-like pro-apoptotic genes within a few hours following exposure to DNA or RNA viruses. Recapitulating a similar response in Drosophila, we found that this rapid induction of apoptosis requires the function of P53 and is mediated by a stress-responsive regulatory region upstream of reaper. More importantly, we showed that the rapid induction of apoptosis is responsible for preventing the expression of viral genes and blocking the infection. Genetic changes influencing this rapid induction of reaper-like pro-apoptotic genes led to significant differences in susceptibility to viral infection.
Assuntos
Baculoviridae/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/imunologia , Nodaviridae/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Aedes/imunologia , Aedes/virologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Células Cultivadas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Drosophila melanogaster/virologia , Epigênese Genética , Feminino , Regulação Viral da Expressão Gênica , Imunidade Inata , Insetos Vetores/imunologia , Insetos Vetores/virologia , Larva , Modelos Biológicos , Neuropeptídeos/genética , RNA Viral/genética , Deleção de Sequência , Spodoptera/imunologia , Spodoptera/virologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Flock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3' end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.
Assuntos
Grânulos Citoplasmáticos/virologia , Nodaviridae/metabolismo , Biossíntese de Proteínas , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/virologia , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Cricetinae , Drosophila melanogaster , Nodaviridae/genética , Infecções por Vírus de RNA/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Virais/genéticaRESUMO
The discovery of broadly neutralizing antibodies that recognize highly conserved epitopes in the membrane-proximal region of influenza virus hemagglutinin (HA) has revitalized efforts to develop a universal influenza virus vaccine. This effort will likely require novel immunogens that contain these epitopes but lack the variable and immunodominant epitopes located in the globular head of HA. As a first step toward developing such an immunogen, we investigated whether the 20-residue A-helix of the HA2 chain that forms the major component of the epitope of broadly neutralizing antibodies CR6261, F10, and others is sufficient by itself to elicit antibodies with similarly broad antiviral activity. Here, we report the multivalent display of the A-helix on icosahedral virus-like particles (VLPs) derived from the capsid of Flock House virus. Mice immunized with VLPs displaying 180 copies/particle of the A-helix produced antibodies that recognized trimeric HA and the elicited antibodies had binding characteristics similar to those of CR6261 and F10: they recognized multiple HA subtypes from group 1 but not from group 2. However, the anti-A-helix antibodies did not neutralize influenza virus. These results indicate that further engineering of the transplanted peptide is required and that display of additional regions of the epitope may be necessary to achieve protection.
Assuntos
Anticorpos Antivirais/sangue , Reações Cruzadas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Animais , Anticorpos Neutralizantes/sangue , Portadores de Fármacos/administração & dosagem , Feminino , Vetores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Nodaviridae/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Virossomais/administração & dosagem , Vacinas Virossomais/imunologiaRESUMO
Mitochondria are dynamic organelles that continually respond to cellular stress. Recent studies have demonstrated that mitochondrial stress is relayed from mitochondria to the cytosol by the release of a proteolytic fragment of DELE1 that binds to the eIF2α kinase HRI to initiate integrated stress response (ISR) signaling. We report the cryo-electron microscopy structure of the C-terminal cleavage product of human DELE1, which assembles into a high-order oligomer. The oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. We further show that assembly-impaired DELE1 mutants are compromised in their ability to induce HRI-dependent ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.
Assuntos
Estresse Fisiológico , eIF-2 Quinase , Humanos , Fosforilação , Microscopia Crioeletrônica , eIF-2 Quinase/metabolismo , Mitocôndrias/metabolismoRESUMO
Viral infection has been associated with a starvation-like state in Drosophila melanogaster. Because starvation and inhibiting TOR kinase activity in vivo result in blocked oocyte production, we hypothesized that viral infection would also result in compromised oogenesis. Wild-type flies were injected with flock house virus (FHV) and survival and embryo production were monitored. Infected flies had a dose-responsive loss of fecundity that corresponded to a global reduction in Akt/TOR signaling. Highly penetrant egg chamber destruction mid-way through oogenesis was noted and FHV coat protein was detected within developing egg chambers. As seen with in vivo TOR inhibition, oogenesis was partially rescued in loss of function discs large and merlin mutants. As expected, mutants in genes known to be involved in virus internalization and trafficking [Clathrin heavy chain (chc) and synaptotagmin] survive longer during infection. However, oogenesis was rescued only in chc mutants. This suggests that viral response mechanisms that control fly survival and egg chamber survival are separable. The genetic and signaling requirements for oocyte destruction delineated here represent a novel host-virus interaction with implications for the control of both fly and virus populations.
Assuntos
Drosophila melanogaster/fisiologia , Drosophila melanogaster/virologia , Nodaviridae/patogenicidade , Oócitos/fisiologia , Oogênese , Animais , Células Cultivadas , Cadeias Pesadas de Clatrina/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Feminino , Fertilidade , Camundongos , Mutação , Oócitos/virologia , Ovário/virologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Inanição , Sinaptotagminas/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Porcine circovirus 2 (PCV2) is a T=1 nonenveloped icosahedral virus that has had severe impact on the swine industry. Here we report the crystal structure of an N-terminally truncated PCV2 virus-like particle at 2.3-Å resolution, and the cryo-electron microscopy (cryo-EM) image reconstruction of a full-length PCV2 virus-like particle at 9.6-Å resolution. This is the first atomic structure of a circovirus. The crystal structure revealed that the capsid protein fold is a canonical viral jelly roll. The loops connecting the strands of the jelly roll define the limited features of the surface. Sulfate ions interacting with the surface and electrostatic potential calculations strongly suggest a heparan sulfate binding site that allows PCV2 to gain entry into the cell. The crystal structure also allowed previously determined epitopes of the capsid to be visualized. The cryo-EM image reconstruction showed that the location of the N terminus, absent in the crystal structure, is inside the capsid. As the N terminus was previously shown to be antigenic, it may externalize through viral "breathing."
Assuntos
Circovirus/química , Proteínas Virais/química , Sequência de Aminoácidos , Microscopia Crioeletrônica , Cristalografia por Raios X , Dados de Sequência Molecular , Proteínas Virais/ultraestrutura , Vírion/química , Vírion/ultraestruturaRESUMO
Multivalent display of heterologous proteins on viral nanoparticles forms a basis for numerous applications in nanotechnology, including vaccine development, targeted therapeutic delivery, and tissue-specific bioimaging. In many instances, precise placement of proteins is required for optimal functioning of the supramolecular assemblies, but orientation- and site-specific coupling of proteins to viral scaffolds remains a significant technical challenge. We have developed two strategies that allow for controlled attachment of a variety of proteins on viral particles using covalent and noncovalent principles. In one strategy, an interaction between domain 4 of anthrax protective antigen and its receptor was used to display multiple copies of a target protein on virus-like particles. In the other, expressed protein ligation and aniline-catalyzed oximation was used to display covalently a model protein. The latter strategy, in particular, yielded nanoparticles that induced potent immune responses to the coupled protein, suggesting potential applications in vaccine development.
Assuntos
Antígenos de Bactérias/química , Toxinas Bacterianas/química , Proteínas Ligantes de Maltose/química , Nanoestruturas/química , Nanotecnologia/métodos , Proteínas Recombinantes/química , Vírion/metabolismo , Compostos de Anilina/química , Animais , Baculoviridae/química , Baculoviridae/genética , Baculoviridae/metabolismo , Western Blotting , Clonagem Molecular , Escherichia coli , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Nanoestruturas/virologia , Oximas/química , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Estereoisomerismo , Ressonância de Plasmônio de Superfície , Transfecção , Vírion/genéticaRESUMO
Assembly of many RNA viruses entails the encapsidation of multiple genome segments into a single virion, and underlying mechanisms for this process are still poorly understood. In the case of the nodavirus Flock House virus (FHV), a bipartite positive-strand RNA genome consisting of RNA1 and RNA2 is copackaged into progeny virions. In this study, we investigated whether the specific packaging of FHV RNA is dependent on an arginine-rich motif (ARM) located in the N terminus of the coat protein. Our results demonstrate that the replacement of all arginine residues within this motif with alanines rendered the resultant coat protein unable to package RNA1, suggesting that the ARM represents an important determinant for the encapsidation of this genome segment. In contrast, replacement of all arginines with lysines had no effect on RNA1 packaging. Interestingly, confocal microscopic analysis demonstrated that the RNA1 packaging-deficient mutant did not localize to mitochondrial sites of FHV RNA replication as efficiently as wild-type coat protein. In addition, gain-of-function analyses showed that the ARM by itself was sufficient to target green fluorescent protein to RNA replication sites. These data suggest that the packaging of RNA1 is dependent on trafficking of coat protein to mitochondria, the presumed site of FHV assembly, and that this trafficking requires a high density of positive charge in the N terminus. Our results are compatible with a model in which recognition of RNA1 and RNA2 for encapsidation occurs sequentially and in distinct cellular microenvironments.
Assuntos
Proteínas do Capsídeo/metabolismo , Nodaviridae/fisiologia , RNA Viral/metabolismo , Montagem de Vírus , Replicação Viral , Motivos de Aminoácidos , Substituição de Aminoácidos/genética , Arginina/genética , Proteínas do Capsídeo/genética , Mitocôndrias/química , Mutagênese Sítio-Dirigida , Ligação Proteica , Transporte ProteicoRESUMO
Recent studies have established that several nonenveloped viruses utilize virus-encoded lytic peptides for host membrane disruption. We investigated this mechanism with the "gamma" peptide of the insect virus Flock House virus (FHV). We demonstrate that the C terminus of gamma is essential for membrane disruption in vitro and the rescue of immature virus infectivity in vivo, and the amphipathic N terminus of gamma alone is not sufficient. We also show that deletion of the C-terminal domain disrupts icosahedral ordering of the amphipathic helices of gamma in the virus. Our results have broad implications for understanding membrane lysis during nonenveloped virus entry.
Assuntos
Proteínas do Capsídeo/genética , Membrana Celular/virologia , Nodaviridae/fisiologia , Internalização do Vírus , Mutação , Nodaviridae/genética , Estrutura Terciária de ProteínaRESUMO
The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic gamma peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the gamma peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of gamma peptides from the virus particle, and (iii) the exposed/released gamma peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.
Assuntos
Proteínas do Capsídeo/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Nodaviridae/fisiologia , Internalização do Vírus , Endocitose , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Modelos BiológicosRESUMO
As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.
Assuntos
Modelos Moleculares , Nodaviridae/química , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/química , Proteínas Virais/química , Cristalografia , Dimerização , Ligação Proteica , Conformação Proteica , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismoRESUMO
The infectivity of flock house virus (FHV) requires autocatalytic maturation cleavage of the capsid protein at residue 363, liberating the C-terminal 44-residue gamma peptides, which remain associated with the particle. In vitro studies previously demonstrated that the amphipathic, helical portion (amino acids 364 to 385) of gamma is membrane active, suggesting a role for gamma in RNA membrane translocation during infection. Here we show that the infectivity of a maturation-defective mutant of FHV can be restored by viruslike particles that lack the genome but undergo maturation cleavage. We propose that the colocalization of the two defective particle types in an entry compartment allows the restoration of infectivity by gamma.
Assuntos
Proteínas do Capsídeo/fisiologia , Nodaviridae/fisiologia , Fragmentos de Peptídeos/fisiologia , Vírion/fisiologia , Internalização do Vírus , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Drosophila/virologia , Nodaviridae/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genéticaRESUMO
The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.
Assuntos
Vacinas contra Antraz , Antraz/prevenção & controle , Antitoxinas/química , Antitoxinas/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Animais , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Vetores Genéticos , Iridoviridae/química , Iridoviridae/imunologia , Masculino , Proteínas de Membrana/imunologia , Microscopia Eletrônica , Nanopartículas , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptores de PeptídeosRESUMO
Virus assembly occurs in a complex environment and is dependent upon viral and cellular components being properly correlated in time and space. The simplicity of the flock house virus (FHV) capsid and the extensive structural, biochemical and genetic characterization of the virus make it an excellent system for studying in vivo virus assembly. The tetracysteine motif (CCPGCC), that induces fluorescence in bound biarsenical compounds (FlAsH and ReAsH), was genetically inserted in the coat protein, to visualize this gene product during virus infection. The small size of this modification when compared to those made by traditional fluorescent proteins minimizes disruption of the coat proteins numerous functions. ReAsH not only fluoresces when bound to the tetracysteine motif but also allows correlated electron microscopy (EM) of the same cell following photoconversion and osmium staining. These studies demonstrated that the coat protein was concentrated in discrete patches in the cell. High pressure freezing (HPF) followed by freeze substitution (FS) of infected cells showed that these patches were formed by virus particles in crystalline arrays. EM tomography (EMT) of the HPF/FS prepared samples showed that these arrays were proximal to highly modified mitochondria previously established to be the site of RNA replication. Two features of the mitochondrial modification are approximately 60 nm spherules that line the outer membrane and the large chamber created by the convolution induced in the entire organelle.
Assuntos
Capsídeo/ultraestrutura , Drosophila/virologia , Nodaviridae/ultraestrutura , Montagem de Vírus/fisiologia , Animais , Proteínas Luminescentes , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , TomografiaRESUMO
Virus-like particles (VLPs) are self-assembling platforms composed of viral structural proteins. They are used for a variety of purposes, ranging from the study of virus assembly to vaccine development. VLPs can be produced in plants, bacteria, yeast, and insect and mammalian cells. The baculovirus expression system is one of the most commonly used systems for production of VLPs in eukaryotic cells. This chapter provides a brief overview of the main strategies used to generate recombinant baculoviruses and the applications of insect virus-derived VLPs in basic and applied research. It then describes detailed protocols for generation of recombinant baculoviruses, screening for their expression of VLPs in insect cells, and VLP purification.
Assuntos
Insetos/virologia , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Baculoviridae/genética , Humanos , Proteínas Estruturais Virais/genética , Montagem de Vírus/genéticaRESUMO
Specific targeting of tumor cells is an important goal for the design of nanotherapeutics for the treatment of cancer. Recently, viruses have been explored as nano-containers for specific targeting applications, however these systems typically require modification of the virus surface using chemical or genetic means to achieve tumor-specific delivery. Interestingly, there exists a subset of viruses with natural affinity for receptors on tumor cells that could be exploited for nanotechnology applications. For example, the canine parvovirus (CPV) utilizes transferrin receptors (TfRs) for binding and cell entry into canine as well as human cells. TfRs are over-expressed by a variety of tumor cells and are widely being investigated for tumor-targeted drug delivery. We explored whether the natural tropism of CPV to TfRs could be harnessed for targeting tumor cells. Towards this goal, CPV virus-like particles (VLPs) produced by expression of the CPV-VP2 capsid protein in a baculovirus expression system were examined for attachment of small molecules and delivery to tumor cells. Structural modeling suggested that six lysines per VP2 subunit are presumably addressable for bioconjugation on the CPV capsid exterior. Between 45 and 100 of the possible 360 lysines/particle could be routinely derivatized with dye molecules depending on the conjugation conditions. Dye conjugation also demonstrated that the CPV-VLPs could withstand conditions for chemical modification on lysines. Attachment of fluorescent dyes neither impaired binding to the TfRs nor affected internalization of the 26 nm-sized VLPs into several human tumor cell lines. CPV-VLPs therefore exhibit highly favorable characteristics for development as a novel nanomaterial for tumor targeting.
RESUMO
Aggregation of protein-based therapeutics is a challenging problem in the biopharmaceutical industry. Of particular concern are implications for product efficacy and clinical safety because of potentially increased immunogenicity of the aggregates. We used transmission electron microscopy (TEM) to characterize biophysical and morphological features of antibody aggregates formed upon controlled environmental stresses. TEM results were contrasted with results obtained in parallel by independent methods, including size-exclusion chromatography, dynamic light scattering, microflow imaging, and nanoparticle tracking. For TEM, stressed samples were imaged by negative staining and in the frozen-hydrated state. In both cases, aggregates appeared amorphous but differed in fine structural detail. Specifically, negatively stained aggregates were compact and consisted of smaller globular structures that had a notable three-dimensional character. Elements of the native IgG structure were retained, suggesting that the aggregates were not assembled from denatured protein. In contrast, aggregates in frozen-hydrated samples appeared as extended, branched protein networks with large surface area. Using multiple scales of magnification, a wide range of particle sizes was observed and semiquantitatively characterized. The detailed information provided by TEM extended observations obtained with the independent methods, demonstrating the suitability of TEM as a complementary approach to submicron particle analysis.