Rubella Virus Structural Proteins Research Articles

Design of a chimeric antigen containing multiple immunodominant epitopes and its use in detection of IgM antibodies against Rubella virus

A chimeric antigen designated B103 containing six immunodominant regions derived from three structural proteins of Rubella virus (RV) was designed and its utility in serological diagnosis was assessed. Protein B103 is comprised of aa 1-30 & aa 96-123 of C protein, aa 31-105 of E2 protein, as well as aa 11-39, aa 154-277 & aa 389-412 of E1 protein. In addition, it contains thioredoxin (TRX) at the N-terminal and His tag at the C-terminal. B103 was expressed in Escherichia coli BL21(DE3) and purified by Streamline Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B103 as verified by Western blotting analysis, we constructed and evaluated a novel capture ELISA for RV-IgM detection. B103 was expressed in a soluble form, accounting for 18.57% of the total bacterial proteins. After purification, the concentration and purity of protein B103 were 3.026 mg/mL and 95.35%, respectively. Western blotting analysis demonstrated that protein B103 could react with acute-phase serum of RV. By ELISA, 40 negative sera and 40 RV-acute phase sera were detected. The sensitivity, specificity, positive predictive value, negative predictive value and coincidence rate of the ELISA were 92.50%, 95.00%, 94.87%, 92.68% and 93.75%, respectively. The McNemer analysis suggested that there was no statistical difference between the 'Gold standard' and the novel ELISA with a kappa coefficient of 0.900, indicating excellent consistency. B103 chimeric protein with excellent antigenicity obtained from prokaryotic expression followed by chromatography purification could prove useful for early diagnosis of RV infection.

Characterization of rubella-specific humoral immunity following two doses of MMR vaccine using proteome microarray technology.

Introduction//BackgroundThe lack of standardization of the currently used commercial anti-rubella IgG antibody assays leads to frequent misinterpretation of results for samples with low/equivocal antibody concentration. The use of alternative approaches in rubella serology could add new information leading to a fuller understanding of rubella protective immunity and neutralizing antibody response after vaccination.MethodsWe applied microarray technology to measure antibodies to all rubella virus proteins in 75 high and 75 low rubella virus-specific antibody responders after two MMR vaccine doses. These data were used in multivariate penalized logistic regression modeling of rubella-specific neutralizing antibody response after vaccination.ResultsWe measured antibodies to all rubella virus structural proteins (i.e., the glycoproteins E1 and E2 and the capsid C protein) and to the non-structural protein P150. Antibody levels to each of these proteins were: correlated with the neutralizing antibody titer (p<0.006); demonstrated differences between the high and the low antibody responder groups (p<0.008); and were components of the model associated with/predictive of vaccine-induced rubella virus-specific neutralizing antibody titers (misclassification error = 0.2).ConclusionOur study supports the use of this new technology, as well as the use of antibody profiles/patterns (rather than single antibody measures) as biomarkers of neutralizing antibody response and correlates of protective immunity in rubella virus serology.

Cryo-Electron Tomography of Rubella Virus

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.

Comparison of Four Methods Using Throat Swabs To Confirm Rubella Virus Infection

Laboratory tests are essential for confirming sporadic cases and outbreaks of rubella. Detection of rubella virus is often necessary to confirm rubella cases and to identify specimens to be used to characterize wild-type rubella viruses. The sensitivities of four methods for detecting rubella virus infection using throat swabs, which had been collected in Henan and Anhui provinces in China, were evaluated. The methods used were reverse transcription (RT)-PCR followed by Southern hybridization using RNA extracted directly from clinical specimens, virus growth in tissue culture followed by virus detection by RT-PCR, low-background immunofluorescence in infected tissue culture cells using monoclonal antibodies to the structural proteins of rubella virus, and a replicon-based method of detecting infectious virus. Among these four methods, direct RT-PCR followed by hybridization was the most sensitive method; the replicon-based method was the least difficult to perform.

Preparation of immunoblot test stripes from a Rubella virus-like particles dye crystal complex as antigen

Stably transfected Chinese hamster ovary (CHO24S) cells were the source for Rubella virus-like particles (RVLP) containing all structural proteins (E1, E2, C and their dimers). RVLP are secreted from the CHO24S cells into the medium and the time-point for collecting the medium with the highest yield of >100 kDa proteins (with 17 mg protein from 10 ml cell culture supernatant) was after 2 days of incubation. Different methods for RVLP isolation from the cell culture supernatants were assessed by SDS-PAGE and Western blotting (using sera positive or negative for Rubella virus (RV)-specific antibodies or an anti-E1 monoclonal antibody). A combination of membrane filtration with a rapid, novel gradient ultracentrifugation step (using Coomassie brilliant blue G crystals as adsorbens for RVLP that facilitated virus isolation) was the most suitable technique. 132 RV-positive human sera (RV IgG > 20 IU/ml by commercial ELISA) were tested by our "self made" immunoblot test stripes (using RVLP adsorbed to dye crystals as antigen) for the presence or absence of antibodies specific for RV structural proteins. 57.6% of these sera had antibodies against E1, E2 and C, 31% against E1 and C, and 1.5% against E1 only, whereas 3.8% had no RV specific antibodies and only 6.0% were equivocal which demonstrated that these "self made" test stripes can reliably differentiate RV antibody specificities.

Rubella virus capsid protein modulates viral genome replication and virus infectivity.

The structural proteins (SP) of the Togaviridae can be deleted in defective interfering RNAs. The dispensability of viral SP has allowed construction of noninfectious viral expression vectors and replicons from viruses of the Alphavirus and Rubivirus genera. Nevertheless, in this study, we found that the SP of rubella virus (RUB) could enhance expression of reporter genes from RUB replicons in trans. SP enhancement required capsid protein (CP) expression and was not due to RNA-RNA recombination. Accumulation of minus- and plus-strand RNAs from replicons was observed in the presence of SP, suggesting that SP specifically affects RNA synthesis. By using replicons containing an antibiotic resistance gene, we found 2- to 50-fold increases in the number of cells surviving selection in the presence of SP. The increases depended significantly on the amount of transfected RNA. Small amounts of RNA or templates that replicated inefficiently showed more enhancement. The infectivity of infectious RNA was increased by at least 10-fold in cells expressing CP. Moreover, virus infectivity was greatly enhanced in such cells. In other cells that expressed higher levels of CP, RNA replication of replicons was inhibited. Thus, depending on conditions, CP can markedly enhance or inhibit RUB RNA replication.

Maturation of IgG avidity to individual rubella virus structural proteins

the structural proteins of rubella virus, the capsid protein C and the envelope glycoproteins E1 and E2 were produced in lepidopteran insect cells using baculovirus expression vectors. The C-terminal ends of the corresponding proteins were fused to a polyhistidine tag for easy and gentle purification by metal ion affinity chromatography. to investigate the maturation of natural and vaccinal IgG avidity against individual authentic and recombinant rubella virus (RV) structural proteins. the analysis was carried out using a modified immunoblotting technique where the purified baculovirus-expressed proteins were compared with authentic rubella virus proteins. Altogether, 47 well-characterised serum samples from both naturally infected patients and vaccines were studied. after natural RV infection, IgG antibodies specific for the E1 protein were predominant not only in terms of levels, but also in terms of rate and magnitude of avidity maturation. The avidity development of the IgG antibodies was much slower in vaccines than in patients after a natural RV infection. together, our results indicate that IgG avidity determination in conjunction with immunoblot analysis is useful in the diagnosis of a RV infection. The recombinant proteins showed similar reactivity patterns in the immunoblot analyses as compared with the authentic viral structural proteins, suggesting suitability for serodiagnostics.

Rubella virus E2 signal peptide is required for perinuclear localization of capsid protein and virus assembly.

The rubella virus (RV) structural proteins capsid, E2, and E1 are synthesized as a polyprotein precursor. The signal peptide that initiates translocation of E2 into the lumen of the endoplasmic reticulum remains attached to the carboxy terminus of the capsid protein after cleavage by signal peptidase. Among togaviruses, this feature is unique to RV. The E2 signal peptide has previously been shown to function as a membrane anchor for the capsid protein. In the present study, we demonstrate that this domain is required for RV glycoprotein-dependent localization of the capsid protein to the juxtanuclear region and subsequent virus assembly at the Golgi complex.

Rubella virus capsid associates with host cell protein p32 and localizes to mitochondria.

Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA. The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes. The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein. Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system. One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins. The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria. Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region. The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid. Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.

Immunoblot analysis of natural and vaccine-induced IgG responses to rubella virus proteins expressed in insect cells.

The three structural proteins of rubella virus (RV), the capsid protein C and the envelope glycoproteins E1 and E2, were produced individually in soluble form in Sf9 insect cells using the baculovirus system. All proteins were equipped with a polyhistidine tag at their C-terminal ends to enable gentle purification by metal ion affinity chromatography. In addition, the E1 and E2 proteins were engineered to display the FLAG epitope tag at their N-terminal ends. The diagnostic potential of the recombinant purified proteins was evaluated by immunoblot and enzyme immuno assays (EIA) using a total of 57 well-characterised serum samples obtained at various time points after natural RV infection, congenital rubella syndrome (CRS), MMR vaccination or from controls with past RV immunity. In addition, acute and convalescent phase serum pools from a total of 20 patients were evaluated. Authentic RV proteins were used as a reference. The recombinant E1 and C proteins were predominant in eliciting the immune response in both postnatal and vaccinal RV infections, being much weaker in the vaccinal ones. The IgG response to the recombinant C protein was very strong after the first month post infection and decreased with time. The immune response against the recombinant E2 protein, however, was generally poor, but notably stronger after congenital infection. Together, the results showed that the individual recombinant protein antigens could be suitable for diagnosis of RV infection and for study of the immune response to rubella vaccination.

Secretion of Rubella Virions and Virus-like Particles in Cultured Epithelial Cells

Rubella virus (RV) is an enveloped RNA virus that causes systemic infections in humans. More importantly, first trimester in utero infection leads to a collection of devastating birth defects known as congenital rubella syndrome. Epithelial cells are the first line of defense against viruses and consequently, the polarity of virus secretion is an important factor affecting viral spread. As a first step toward understanding how RV interacts with epithelial cells, we have examined the release of RV-like particles and virions from polarized cells in culture. RV structural proteins were targeted to the Golgi complex and virus particle formation occurred on intracellular membranes in three different polarized epithelial cells. Polarized cells could be infected from the apical and basal membranes, indicating that receptors are not confined to one surface. The secretion of virus-like particles and infectious virions varied according to cell type. In two of the three polarized cell lines examined, virus was released primarily from the apical surface, but significant quantities were also secreted from the basolateral membrane. Release of virus from the apical surface may facilitate virus spread from person to person, whereas basolateral secretion could be important for establishing a systemic infection and/or crossing the placenta prior to fetal infection.

Rubella virus-induced cytopathic effect in vitro is caused by apoptosis.

Rubella virus (RV) generally causes a mild disease but it is highly teratogenic when infection occurs during the first trimester of gestation. Under in vitro conditions, RV induces characteristic cytopathic changes on several cell lines, e.g. cell detachment from the monolayer and condensation of chromatin. The purpose of this study was to characterize RV-induced cell death and to determine the factors that might be involved in this process. Both acutely and persistently infected cells exhibited alterations characteristic of apoptosis, including DNA fragmentation, annexin V staining and reduced DNA content. UV-inactivated RV did not induce apoptotic cell death and expression of RV structural proteins in a transfected cell line was not sufficient to induce apoptosis, supporting the interpretation that replicating virus is necessary to provoke apoptosis. Both persistently infected and 24S-transfected cells retained their susceptibility to undergo apoptosis in response to either staurosporine or camptothecin. This indicates that RV does not block chemically induced apoptosis. The signals involved in RV-associated apoptosis appear to be independent of p53 and of the Bcl-2 gene family.

Detection of rubella virus-specific immunoglobulin M antibodies with a baculovirus-expressed E1 protein.

The structural proteins of rubella virus (RV) were expressed in insect cells by using the baculovirus expression vector system. The recombinant E1 envelope glycoprotein was purified by immunoaffinity chromatography and used to detect RV-specific immunoglobulin M antibodies in a time-resolved fluoroimmunoassay. Correlation analysis between the reactivities of antibodies against this recombinant E1 and the reactivities against authentic RV antigen shows that purified E1 can detect RV antibodies of the immunoglobulin M type.

Dimerization of Rubella Virus Capsid Protein Is Not Required for Virus Particle Formation

Rubella virus (RV) virions contain, in addition to the RNA genome, a capsid protein (C) and two envelope glycoproteins (E1 and E2). The C protein in isolated virions has been reported to be a disulfide-linked dimer (C2). There are two cysteine residues (Cys-152 and Cys-196) within the C protein. To define the role of disulfide-linked C2 dimer in virion formation, site-directed mutagenesis was used to alter the Cys-152 residue to serine. The association of mutant C protein with envelope glycoproteins was examined by transient expression of the cDNAs in COS cells. Mutation at the Cys-152 residue completely abolished the formation of C2 dimer. However, C2 dimerization does not appear to be important for the assembly of RV structural proteins into virus-like particles.

Immunoglobulin G subclass antibodies to rubella virus in chronic liver disease, acute rubella and healthy controls

Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.

Immunoglobulin G subclass antibodies to rubella virus in chronic liver disease, acute rubella and healthy controls.

Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.

Expression of the rubella virus structural proteins by an infectious Sindbis virus vector.

To assess the potential of an infectious Sindbis virus vector (dsSIN) as a eukaryotic expression vector, dsSIN recombinants which contain each of the rubella virus (RUB) structural proteins (C, E2, and E1) individually were constructed. Expression of the RUB proteins by each dsSIN recombinant was robust and processing was similar to that observed when these proteins were expressed using other vectors. The C and E2 recombinants, each of which contains a cassette of less than 1,000 nts, were stable through low multiplicity amplification; however, the E1 recombinant, which contains a 1700 nt cassette, was not. Therefore, use of the E1 recombinant is restricted to stock derived from the original transfection. The replication rate of dsSIN and the C and E2 recombinants was similar, however, the replication rate of the E1 recombinant was slower. No phenotypic mixing of any of the RUB proteins in recombinant dsSIN virions could be detected.

Brefeldin A and monensin arrest cell surface expression of membrane glycoproteins and release of rubella virus.

The maturation of rubella virus (RV) glycoproteins E2 and E1 was examined by using brefeldin A (BFA) and monensin. BFA, which induces the rapid redistribution of Golgi enzymes residing in the Golgi complex into the endoplasmic reticulum (ER), was used to locate the intracellular site for the modification of carbohydrate side-chains on RV E1 and E2 proteins. The monovalent ionophore monensin, which inhibits intracellular transport of proteins through the ER-Golgi complex, was used to block the transport of E1 and E2 glycoproteins through the Golgi complex. BFA and monensin effectively blocked the cell surface expression of RV E2 and E1 proteins, secretion of an anchor-free form of E2 and budding of RV from the plasma membrane. For O-linked glycosylation, addition of N-acetylgalactosamine and galactose to E2 protein was found to take place in the medial to the trans Golgi. A dramatic change in the intracellular distribution of RV structural proteins was observed when transfected COS cells were treated with BFA or monensin, although the proteolytic processing of RV structural protein precursor was not affected. In the presence of BFA or monensin, virus release from infected Vero cells was only 0.1% of the intracellular virus, and the intracellular virus titre decreased as well. Our results suggest that O-linked glycosylation on the E2 protein occurred in the post-ER region and the transport of RV structural proteins to the Golgi complex and post-Golgi compartment may be a rate-limiting step in RV assembly and budding.

Synthesis and processing of the rubella virus p110 polyprotein precursor in baculovirus-infected Spodoptera frugiperda cells

In order to study the processing of rubella virus (RV) structural proteins (capsid, protein, of 33 kDa; E2 of 42–47 kDa; and E1 of 58 kDa) in Spodoptera frugiperda (fall armyworm) cells, a 24S cDNA encoding the polyprotein precursor, p110, was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during viral infection. By immunoblot analysis using antibodies directed against whole RV and the individual structural proteins, evidence is presented that polypeptides similar to those synthesized in RV-infected B-Vero cells are expressed in this lepidopteran insect cell line infected with the recombinant baculovirus, VL1392-RV24S. The identity of the recombinant proteins was further confirmed using human convalescent sera. By expressing the recombinant proteins in the presence and absence of tunicamycin, we have further demonstrated that the 24S transcription-translation unit of RV, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared to B-Vero cells.

Expression and characterization of a soluble rubella virus E1 envelope protein

Individual specific antigenic rubella virus (RV) structural proteins are required for accurate serological diagnosis of acute and congenital rubella infections as well as rubella immune status. The RV envelope glycoprotein E1 is the major target antigen and plays an important role in viral-specific immune responses. The native virion is difficult to produce in large quantities and the protein subunits are also difficult to isolate without loss of antigenicity. The production of a soluble RV E1 (designated E1 delta Tm) using the baculovirus-insect cell expression system is described. In contrast to wild-type RV E1, the genetically engineered E1 delta Tm protein lacks a transmembrane anchor. It behaved as a secretory protein and was secreted abundantly from insect cells. Pulse-chase studies were used to examine the synthesis, glycosylation, and secretion of E1 delta Tm by the insect cells. The secreted E1 delta Tm protein was purified from serum-free medium by one-step immunochromatography. The purified E1 delta Tm protein retained full antigenicity and may be a convenient source of E1 protein for use in diagnostic assay and rubella vaccine development.