RESUMO
Viral infections during pregnancy are a significant cause of infant morbidity and mortality. Of these, rubella virus infection is a well-substantiated example that leads to miscarriages or severe fetal defects. However, structural information about the rubella virus has been lacking due to the pleomorphic nature of the virions. Here we report a helical structure of rubella virions using cryo-electron tomography. Sub-tomogram averaging of the surface spikes established the relative positions of the viral glycoproteins, which differed from the earlier icosahedral models of the virus. Tomographic analyses of in vitro assembled nucleocapsids and virions provide a template for viral assembly. Comparisons of immature and mature virions show large rearrangements in the glycoproteins that may be essential for forming the infectious virions. These results present the first known example of a helical membrane-enveloped virus, while also providing a structural basis for its assembly and maturation pathway.
Assuntos
Vírus da Rubéola/fisiologia , Rubéola (Sarampo Alemão)/virologia , Montagem de Vírus , Animais , Linhagem Celular , Tomografia com Microscopia Eletrônica , Humanos , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Rubéola (Sarampo Alemão)/embriologia , Rubéola (Sarampo Alemão)/patologia , Vírus da Rubéola/química , Vírus da Rubéola/genética , Vírus da Rubéola/ultraestrutura , TeratogêneseRESUMO
Rubella virus (RV) is a leading cause of birth defects due to infectious agents. When contracted during pregnancy, RV infection leads to severe damage in fetuses. Despite its medical importance, compared with the related alphaviruses, very little is known about the structure of RV. The RV capsid protein is an essential structural component of virions as well as a key factor in virus-host interactions. Here we describe three crystal structures of the structural domain of the RV capsid protein. The polypeptide fold of the RV capsid protomer has not been observed previously. Combining the atomic structure of the RV capsid protein with the cryoelectron tomograms of RV particles established a low-resolution structure of the virion. Mutational studies based on this structure confirmed the role of amino acid residues in the capsid that function in the assembly of infectious virions.
Assuntos
Proteínas do Capsídeo/química , Modelos Moleculares , Conformação Proteica , Vírus da Rubéola/genética , Montagem de Vírus/fisiologia , Animais , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Microscopia Crioeletrônica , Cristalografia por Raios X , Análise Mutacional de DNA , Oligonucleotídeos/genética , Vírus da Rubéola/ultraestrutura , Montagem de Vírus/genéticaRESUMO
Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus. The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface. We also show that the rubella virus nucleocapsid structure often forms a roughly spherical shell which lacks high density at its center. While many rubella virions are approximately spherical and have dimensions similar to that of the icosahedral Ross River virus, the present results indicate that rubella exhibits a large degree of pleomorphy. In addition, we used rotation function calculations and other analyses to show that approximately spherical rubella virions lack the icosahedral organization which characterizes Ross River and other alphaviruses. The present results indicate that the assembly mechanism of rubella virus, which has previously been shown to differ from that of the alphavirus assembly pathway, leads to an organization of the rubella virus structural proteins that is different from that of alphaviruses.
Assuntos
Ross River virus/ultraestrutura , Vírus da Rubéola/ultraestrutura , Animais , Proteínas do Capsídeo/análise , Proteínas do Capsídeo/química , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Congelamento , Glicoproteínas , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/química , Nucleocapsídeo/ultraestrutura , Rubéola (Sarampo Alemão)/virologia , Vírus da Rubéola/química , Células Vero , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/química , Montagem de VírusRESUMO
A radioimmunoassay (RIA) was recently developed for the detection of antiviral IgG and IgM class-specific antibodies using antigen-coated polystyrene balls as the RIA solid-phase. In this communication the attachment and distribution of herpes simplex virus (HSV) capsid and envelope antigens and rubella viruses on the surface of the balls was examined by scanning electron microscopy (SEM). In SEM the surface of the untreated 'clear frosted' polystyrene balls appeared very uneven with innumerable pits and grooves. The viral particles were haphazardly distributed both in the grooves and on the exposed surface of the balls. The strength of adsorption of the viral antigens onto the balls seemed to be remarkably resistant to outside mechanical forces. HSV antigens frequently appeared in clusters, whereas rubella viruses were mostly found as single particles.
Assuntos
Antígenos Virais , Poliestirenos , Técnicas Imunológicas , Microscopia Eletrônica de Varredura , Ligação Proteica , Vírus da Rubéola/imunologia , Vírus da Rubéola/ultraestrutura , Simplexvirus/imunologia , Simplexvirus/ultraestrutura , Proteínas Virais/imunologiaRESUMO
Airfuge direct ultracentrifugation of viral samples on electron microscope grids offers a rapid way for concentrating viral particles or subunits to facilitate their detection and study. Using the A-100 fixed angle rotor (30 degrees) with a K factor of 19 at maximum speed (95,000 rpm), samples up to 240 microliters can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10- to 40-fold increase in sensitivity depending on the size of particles. Application of this technique to rubella virus permitted better visualization of viral membrane subunits on the particles. Rubella hemagglutinin immuno-stimulating complexes preparations were also better visualized and their morphology conserved after direct ultracentrifugation on the specimen grids. Similar observations are reported for respiratory syncytial virus associated subunits.
Assuntos
Vírus Sinciciais Respiratórios/ultraestrutura , Vírus da Rubéola/ultraestrutura , Animais , Linhagem Celular , Microscopia Eletrônica , Microesferas , Ultracentrifugação/métodos , Células VeroRESUMO
A new procedure for the positive staining of viruses in suspension, the Tokuyasu staining procedure (TSP), was evaluated using a non-enveloped virus, rotavirus; an enveloped virus, rubella virus and two glutaraldehyde-treated enveloped viruses, Human T Cell Lymphotropic Virus Type I (HTLV-I) and Human Immunodeficiency Virus Type 1 (HIV-1) as models. The TSP involves an initial staining of the virus with uranyl acetate (UA) followed by thin embedding in a mixture of UA and polyvinyl alcohol (PVA). Using aqueous UA for the TSP, a combination of positively and negatively stained particles was seen for both rotavirus and rubella virus. With glutaraldehyde-fixed HTLV-I and HIV-1, stain penetration did not occur and only negative staining was observed. The substitution of methanolic UA for aqueous UA in the TSP resulted in only positive staining of rotavirus and rubella virus. The change in procedure also resulted in stain penetration of the glutaraldehyde-fixed HTLV-I and HIV-1 to give positively stained particles. Some novel morphological features of rotavirus and rubella virus structure were observed by the TSP.
Assuntos
HIV-1/ultraestrutura , Vírus Linfotrópico T Tipo 1 Humano/ultraestrutura , Rotavirus/ultraestrutura , Vírus da Rubéola/ultraestrutura , Coloração e Rotulagem/métodos , Glutaral/farmacologia , HIV-1/efeitos dos fármacos , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Humanos , Microscopia Eletrônica , Compostos Organometálicos , Ácido Fosfotúngstico , SuspensõesRESUMO
A murine monoclonal antibody directed against the E1 membrane glycoprotein of rubella virus was immobilized on an N-hydroxysuccinimide-activated chromatographic support. The antibody was used to purify rubella virus E1-E2 protein complexes from Tween-80/diethyl ether extracts of cell culture supernatants containing virus particles. The adsorption behaviour of immunosorbents with ligand densities of 2.9, 5.4 and 11.1 mg monoclonal antibody per millilitre of gel was investigated using batchwise conditions. Then the immunoaffinity purification process was optimized with regard to adsorption efficiency by adjusting the flow rate, the bed height and the amount of sample loaded onto the column. The optimized immunoaffinity purification process which is reproducible and relatively simple (one-step) had a yield of 73%, a concentration factor of 5-8 and a purification factor of about 2600. No mouse IgG due to ligand leakage could be detected in the immunopurified product using an enzyme immunoassay. High-performance size exclusion chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, immunoblotting and electron microscopy showed that the immunopurified product contained rosette-like structures formed by complexes of E1 and E2 proteins. The product retained its hemagglutinating activity and proved to be suitable for application in a fluorescent enzyme immunoassay for determination of anti-rubella IgG in human serum.
Assuntos
Antígenos Virais/isolamento & purificação , Cromatografia de Afinidade/métodos , Vírus da Rubéola/química , Proteínas do Envelope Viral/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/imunologia , Western Blotting , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Testes de Hemaglutinação , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Ligantes , Vírus da Rubéola/ultraestrutura , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
The morphology of bovine viral diarrhea virus (BVDV) was studied by electron microscopy. The NADL strain of BVDV was plaque purified 3 times, concentrated by polyethylene glycol precipitation, and purified by centrifugation to equilibrium in continuous potassium tartrate density-gradients. The virus was examined by negative-stain electron microscopy in the presence or absence of specific antiserum. The density of BVDV was between 1.101 g/cm3 and 1.174 g/cm3, with the peak at maximum infectivity at 1.122 g/cm3. Oval to pleomorphic viral particles, 120 ( +/- 30) nm in diameter, were enriched in the peak of maximum infectivity. The detailed structure of virions was revealed: a 5- to 7-microns thick unit membrane-like envelope layer with numerous projecting knobs, 4 to 5 nm in diameter, surrounding an interior core-like structure. Viral particles measuring 120 ( +/- 30) nm were found in large aggregates in the presence of specific antiserum.
Assuntos
Vírus da Diarreia Viral Bovina/ultraestrutura , Pestivirus/ultraestrutura , Vírion/ultraestrutura , Animais , Complexo Antígeno-Anticorpo/imunologia , Bovinos , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Microscopia Eletrônica , Vírus da Rubéola/ultraestrutura , Vírion/imunologia , Vírion/isolamento & purificaçãoRESUMO
Striking changes in membrane systems occur in the vicinity of replication complexes and mitochondria in rubella virus (RV) infected Vero cells. An electron-dense zone about 22-25 nm in thickness fuses membranes in 3 configurations: between the outer membrane of a mitochondrion and one membrane of the rough endoplasmic reticulum (RER), between the outer membranes of two adjacent mitochondria, and between two apposing membranes of the RER. These junctions were called confronting membranes type 1 (CM-1), confronting membranes type 2 (CM-2) and confronting cisternae (CC), respectively. CM-1, CM-2 and CC were not observed in mock infected or Semliki Forest virus (SFV) infected cells. However, mitochondria were found to cluster around replication complexes in both SFV and RV infected cells. This suggests that replication complexes are sites of high energy requirement because of their key role in virus replication. The electron-dense zones appear to create almost continuous links between RV replication complexes and mitochondria.
Assuntos
Membrana Celular/ultraestrutura , Junções Intercelulares/ultraestrutura , Vírus da Rubéola/fisiologia , Células Vero/virologia , Replicação Viral , Animais , Chlorocebus aethiops , Retículo Endoplasmático Rugoso/ultraestrutura , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Vírus da Rubéola/ultraestrutura , Vírus da Floresta de Semliki/fisiologia , Células Vero/ultraestruturaRESUMO
OBJECTIVE: The rapid growth of the rubella virus in RC-IAL2 with development of cytopathic effect, in response to rubella virus infection, is described. For purposes of comparison, the rubella virus RA-27/3 strain was titered simultaneously in the RC-IAL, Vero, SIRC and RK13 cell lines. METHODS: Rubella virus RA-27/3 strain are inoculated in the RC-IAL cell line (rabbit Kidney, Institute Adolfo Lutz). Plates containing 1.5x10(5) cells/ml of RC-IAL line were inoculated with 0.1ml s RA-27/3 strain virus containing 1x 10(4)TCID50/0.1ml. A 25% cytopathic effect was observed after 48 hours and 100% after 96 hours. The results obtained were compared to those observed with the SIRC, Vero and RK13 cell lines. Rubella virus was detected by immunohistochemistry. RESULTS: With the results, it was possible to conclude that the RC-IAL cell line is a very good substrate for culturing rubella virus. The cells inoculated with rubella virus were examined by phase contrast microscopy and showed the characteristic rounded, bipolar and multipolar cells. The CPE in RC-IAL was observed in the first 48 hours and the curve of the increased infectivity was practically the same as observed in other cell lines. CONCLUSIONS: These findings are important since this is one the few cell lines described in the literature with a cytopathic effect. So it can be used for antigen preparation and serological testing for the diagnosis of specific rubella antibodies.
Assuntos
Vírus da Rubéola/crescimento & desenvolvimento , Animais , Antígenos Virais , Linhagem Celular/patologia , Linhagem Celular/virologia , Chlorocebus aethiops , Efeito Citopatogênico Viral/fisiologia , Técnicas Imunoenzimáticas , Coelhos , Rubéola (Sarampo Alemão)/virologia , Vírus da Rubéola/ultraestrutura , Sensibilidade e Especificidade , Células Vero/patologia , Células Vero/virologia , Cultura de Vírus/métodos , Replicação ViralRESUMO
Rubella virus (RUB) assembles its replication complexes (RCs) in modified organelles of endo-lysosomal origin, known as cytopathic vacuoles (CPVs). These peculiar structures are key elements of RUB factories, where rough endoplasmic reticulum, mitochondria, and Golgi are recruited. Bicistronic RUB replicons expressing an antibiotic resistance gene either in the presence or the absence of the RUB capsid (C) gene were used to study the structure of RCs in transfected cells. Confocal microscopy showed that the RUB replicase components P90 and P150 localized to CPVs, as did double-stranded RNA (dsRNA), a marker for RNA synthesis. Electron microscopy (EM) showed that replicons generated CPVs containing small vesicles and large vacuoles, similar to CPVs from RUB-infected cells and that the replicase proteins were sufficient for organelle recruitment. Some of these CPVs contained straight membranes. When cross-sectioned, these rigid membranes appeared to be sheets of closely packed proteins. Immuno-EM revealed that these sheets, apparently in contact with the cytosol, contained both P150 and P90, as well as dsRNA, and thus could be two-dimensional arrays of functional viral replicases. Labelling of dsRNA after streptolysin-O permeabilization showed that replication of viral genome takes place on the cytoplasmic side of CPVs. When present, C accumulated around CPVs. Mitochondrial protein P32 was detected within modified CPVs, the first demonstration of involvement of this protein, which interacts with C, with RCs.
Assuntos
Replicon/genética , Vírus da Rubéola/genética , Replicação Viral , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Imunofluorescência , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Organelas/ultraestrutura , Organelas/virologia , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Vírus da Rubéola/crescimento & desenvolvimento , Vírus da Rubéola/ultraestrutura , Transfecção , Vacúolos/ultraestrutura , Vacúolos/virologia , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Thin section electron microscopy was used to investigate cellular changes associated with the replication of rubella virus (RV) in Vero cells and to compare these changes to those of the related alphavirus, Semliki Forest virus (SFV). Conspicuous membrane-bound cytoplasmic vacuoles analogous to the alphavirus replication complexes were observed in RV infected cells but not in mock infected cells. The vacuoles were characterised by membrane-bound vesicles measuring about 60 nm which often displayed an irregular dense core and/or a network of fibres. These vesicles were morphologically distinct from RV particles and were generally located at regular intervals on the inner side of the surrounding membrane of the RV replication complex. Degenerating cellular material was often found in the membrane-bound vacuole of a replication complex. The replication complexes were intimately associated with the rough endoplasmic reticulum (RER), which was localised 45-75 nm from the surrounding membrane of the replication complex. Parallel studies of replication complexes in SFV infected cells did not reveal such an intimate association with the RER. RV replication complexes appeared as early as 8 h post infection (p.i.), before detection of RV particles by electron microscopy, and their peak production at 24 h p.i. coincided with the time of maximum virus titre.
Assuntos
Vírus da Rubéola/fisiologia , Replicação Viral , Animais , Vírus da Rubéola/ultraestrutura , Células VeroRESUMO
Degradation of purified rubella virus by heat treatment (37, 45, or 56 degrees C) revealed the following structures. The viral envelope, a modified cellular membrane, bears spherical subunits, 5-6 nm in diameter, hexamers, or pentamers. Two glycoproteins, VP-2 (50 000 daltons) and VP-3 (63 000 daltons), are associated with the envelope. The nucleocapsid if formed by the condensation of the viral ribonucleic acid on acentral structure 10 nm in diameter. Only one protein, VP-1 (35 000 daltons) is present in the nucleocapsid. Similarity between rubella virus and Togaviruses is discussed.
Assuntos
Vírus da Rubéola/ultraestrutura , Proteínas Virais , Membrana Celular/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Glicoproteínas , Microscopia Eletrônica , Peso Molecular , RNA Viral , Cultura de VírusRESUMO
Fusion of rubella virus-infected cells was induced by their brief treatment at pH below 6.0. Exposure of rubella virus to pH 5 caused an irreversible conformational change of the viral envelope glycoproteins, E1 and E2. The change was manifested in the marked reduction in both infectivity and haemagglutinating activity of the virus, the increased resistance of E1 and decreased resistance of E2 polypeptides to proteolytic digestion with trypsin, and the acquisition of liposome-binding activity of the virus. The above changes are presumed to mimic the events occurring in the acidic environment within endosomes following endocytosis of the virus.
Assuntos
Vírus da Rubéola/ultraestrutura , Proteínas do Envelope Viral/ultraestrutura , Hemaglutininas Virais/fisiologia , Concentração de Íons de Hidrogênio , Lipossomos , Peso Molecular , Testes de Precipitina , Conformação Proteica , Tripsina/metabolismo , Proteínas Virais de Fusão/ultraestruturaRESUMO
Rubella virus is a small enveloped virus that assembles in association with Golgi membranes. Freeze-substitution electron microscopy of rubella virus-infected cells revealed a previously unrecognized virion polymorphism inside the Golgi stacks: homogeneously dense particles without a defined core coexisting with less dense, mature virions that contained assembled cores. The homogeneous particles appear to be a precursor form during the virion morphogenesis process as the forms with mature morphology were the only ones detected inside secretory vesicles and on the exterior of cells. In mature virions potential remnants of C protein membrane insertion were visualized as dense strips connecting the envelope with the internal core. In infected cells Golgi stacks were frequently seen close to cytopathic vacuoles, structures identified as the sites for viral RNA replication, along with the rough endoplasmic reticulum and mitochondria. These associations could facilitate the transfer of viral genomes from the cytopathic vacuoles to the areas of rubella assembly in Golgi membranes.
Assuntos
Complexo de Golgi/virologia , Vírus da Rubéola/crescimento & desenvolvimento , Montagem de Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Complexo de Golgi/ultraestrutura , Humanos , Vírus da Rubéola/química , Vírus da Rubéola/ultraestrutura , Células VeroRESUMO
Rubella virus (RV) infection induces a variety of morphological changes in the host cell including the modification of lysosomes to produce "replication complexes" and the alteration of mitochondrial morphology and distribution. The morphogenesis of RV was further characterized with particular emphasis on the localization of RV core particles. Thin-section electron microscopy (TSEM) studies indicated that RV core-like particles, measuring approximately 33 nm in diameter, were found associated with RV replication complexes. Immunogold-labeling electron microscopy (EM) using monoclonal antibodies to RV capsid proteins confirmed that these particles were viral cores. RV core particles were also detected in association with mitochondria as observed by TSEM and immunogold-labeling EM using monoclonal antibodies to capsid or polyclonal antibodies to RV virions. The results of this study indicate that the localization of RV core particles in relation to replication complexes is similar to that found for the alphaviruses. However, the association of RV core particles with mitochondria appears unique within the family Togaviridae.
Assuntos
Vírus da Rubéola/ultraestrutura , Células Vero/virologia , Vírion/ultraestrutura , Animais , Anticorpos Antivirais , Chlorocebus aethiops , Imuno-Histoquímica , Microscopia Eletrônica , Nucleocapsídeo/imunologia , Células Vero/ultraestrutura , Replicação ViralRESUMO
cDNA clones encoding the envelope glycoprotein E1 of rubella virus (RV) were altered by site-directed mutagenesis at consensus sites for addition of N-linked glycans. The resulting plasmids were introduced into COS cells and the mutant E1 proteins were analyzed by indirect immunofluorescence, radioimmunoprecipitation, and immunoblotting. We found that RV E1 contains three N-linked oligosaccharides, each approximately 2 kDa in size. Although lack of glycosylation did not appear to affect targeting of E1 to the Golgi region, mutants lacking N-linked glycans at Asn 177 and Asn 209 failed to bind anti-E1 antibodies under nonreducing conditions. Our results suggest that glycosylation may be important for expression of important immunologic epitopes on RV E1.
Assuntos
Vírus da Rubéola/genética , Proteínas do Envelope Viral/genética , Animais , Sequência de Bases , Células Cultivadas , DNA Viral/biossíntese , DNA Viral/química , Expressão Gênica , Glicosilação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Vírus da Rubéola/ultraestrutura , Proteínas do Envelope Viral/biossínteseRESUMO
A feature of the rubella virus (RV) replication cycle is the formation of cytoplasmic vesicle-containing structures known as replication complexes. Following detergent treatment of RV-infected cells, pre-embedding immunogold labeling electron microscopy using antiserum to double-stranded (ds) RNA was employed to characterize the replication complexes. Concentrations of gold particles were found associated with amorphous material located within the RV replication complex. Unlabeled long fine strands, 3-5 nm in width, were also frequently seen associated with this gold-labeled material. On some occasions gold-labeled vesicles within the replication complexes were also detected. The gold-labeled amorphous material was first detected in RV replication complexes at 12 hr postinfection, soon after the reported latent period of 8 hr. Concentrations of gold particles were not detected in mock-infected cells. The findings in this study indicate that the amorphous material is released from detergent-disrupted vesicles within the replication complex and that the vesicles contain the dsRNA. When cells were infected with the related Semliki Forest virus (SFV) and examined using the same antibody, similar gold-labeled material associated with unlabeled fine strands was also observed in SFV replication complexes. For both RV and SFV, the vesicles which line the inner membrane of the replication complexes contain the dsRNA which represent the viral replicative forms and replicative intermediates.