Rubella Virions Research Articles

Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus.

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.

Enhanced expression of HIV and SIV vaccine antigens in the structural gene region of live attenuated rubella viral vectors and their incorporation into virions

Despite the urgent need for an HIV vaccine, its development has been hindered by virus variability, weak immunogenicity of conserved epitopes, and limited durability of the immune response. For other viruses, difficulties with immunogenicity were overcome by developing live attenuated vaccine strains. However, there is no reliable method of attenuation for HIV, and an attenuated strain would risk reversion to wild type. We have developed rubella viral vectors, based on the live attenuated vaccine strain RA27/3, which are capable of expressing important HIV and SIV vaccine antigens. The rubella vaccine strain has demonstrated safety, immunogenicity, and long lasting protection in millions of children. Rubella vectors combine the growth and immunogenicity of live rubella vaccine with the antigenicity of HIV or SIV inserts. This is the first report showing that live attenuated rubella vectors can stably express HIV and SIV vaccine antigens at an insertion site located within the structural gene region. Unlike the Not I site described previously, the new site accommodates a broader range of vaccine antigens without interfering with essential viral functions. In addition, antigens expressed at the structural site were controlled by the strong subgenomic promoter, resulting in higher levels and longer duration of antigen expression. The inserts were expressed as part of the structural polyprotein, processed to free antigen, and incorporated into rubella virions. The rubella vaccine strain readily infects rhesus macaques, and these animals will be the model of choice for testing vector growth in vivo and immunogenicity.

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.

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.

Structural gene products and morphogenesis of a hybrid between rubella virus and a hamster latent retrovirus

We studied the structural gene products and morphogenesis of the hybrid between rubella virus (RV) and a latent retrovirus (R-type virus or R virus) of a baby hamster kidney cell line, BHK21/WI-2. In electron microscopic analysis, the rubella virion measures 80 nm in diameter and has a round nucleoid form with an electron-lucid centre, while R-type virus is 110 nm in diameter and has a round nucleoid with radial spokes. The type 2 hybrid (H2) virion is pleomorphic, ranging from 85 to 110 nm in diameter, and has a nucleoid structure similar to R virus, with short spokes. Indirect immunofluorescence staining of H2-infected cells with polyclonal and monoclonal antibodies (mAb) against the RV structural proteins demonstrated expression of the homologous RV gene products. SDS-PAGE of 35S-methionine-labelled proteins in RV- and H2-infected cells revealed that they both produce four major immunoprecipitable proteins, three of which (E2a, E2b and C) are products of the two genetic homologous loci (e2 and c). In nonhomologous regions, RV-infected cells exhibited E1 protein, while H2-infected cells produced the Env protein of R virus. Western immunoblotting analysis of RV and H2 viral proteins with mAb confirmed that H2 virions carry rubella viral E2 and C proteins and that E1 protein is carried by RV but not H2 virion. These results explained the absence of serological cross-neutralization of haemagglutination and plaque-forming ability of RV and H2 viruses.

Structure of rubella E1 glycoprotein epitopes established by multiple peptide synthesis

Essential parts of epitopes have been identified on rubella virion envelope glycoprotein E1, by scanning with overlapping octapeptides situated between amino acids 243-286 in a previously determined antigenic domain.

Rubella virus antigens: localization of epitopes involved in hemagglutination and neutralization by using monoclonal antibodies

Monoclonal antibodies (MAbs) against the rubella virion were used to locate epitopes involved in hemagglutination and neutralization. The MAbs exhibiting high-level hemagglutination-inhibiting activity were shown by Western blot analysis to be specific for the E1 polypeptide; this is consistent with the presence of the hemagglutinin on the E1 polypeptide. Some of the E1-specific MAbs also neutralized viral infectivity. However, hemagglutination-inhibiting activity and neutralizing activity did not always correlate. Three distinct functional epitopes were identified on the E1 polypeptide by competition analyses: one which reacted with MAbs with high-level hemagglutination-inhibiting activity and with neutralizing activity, one which reacted with MAbs with low-level hemagglutination-inhibiting activity and with neutralizing activity, and one which reacted with MAbs with only hemagglutination-inhibiting activity. A MAb specific for the E2 polypeptide exhibited neutralizing activity. This E2-specific MAb and two E1-specific MAbs with neutralizing activity were capable of precipitating intact virus which indicates that at least three epitopes involved in neutralization are accessible on the surface of the virion.

Immunological characterisation of the rubella E 1 glycoprotein

Three epitopes have been identified on rubella virion envelope polypeptide E 1 using monoclonal antibodies. Antibodies to two of the epitopes, E 1EP1 and E 1EP2, show both haemagglutination inhibition and neutralization activities whereas antibodies to the remaining epitope, E 1EP3, show neutralizing activity only.

A model of the structural organization of rubella virions.

Rubella virus contains three major structural polypeptides designated E1, E2, and C with molecular weights of 62,000, 47,000-54,000 (a complex), and 38,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced conditions. Limited-digest peptide maps confirm that each of these polypeptides is distinct and the E2 is a series of three closely related glycopolypeptides. Both E1 and E2 are glycosylated and covalently incorporate [3H]palmitic acid. Enzymatic digestion of intact virus with trypsin completely degrades both E1 and E2, while the C polypeptide remains intact. E1 has an isoelectric point of pH 6.5. E2 exhibits at least 15 different isoelectric species, which focus over the pH range of 5.0-8.6, and C has two distinct isoelectric species of pH 8.8 and pH 9.5. Under unreduced conditions, E1 exists as a disulfide-bonded dimer (E1-E1) with a molecular weight of 105,000; a disulfide-bounded heterodimer (E1-E2) with a molecular weight of 95,000; and in monomeric form (E1). E2 is found predominantly in heterodimeric form (E1-E2), and C is found only in dimeric form when unreduced. Functional-inhibition studies with selected monoclonal antibodies show at least three distinct antigenic domains on E1 that include sites involved in hemagglutination and lysis of red blood cells.

Immunological characterisation of rubella virion polypeptides.

Antibodies specific for rubella virion polypeptide, VPI, secreted by clones of hybridoma cells or produced in rabbits in response to specific antigenic stimulation, located determinants for haemagglutination inhibiting (HI) and neutralising (Nt) antibodies on this envelope component.

Further studies on the restriction of rubella virus replication in rat glial cells in culture.

We have shown previously that normal rat glial cells in tissue culture are nonpermissive for rubella virus (RV) replication for up to 2 weeks postinfection and that during that time, only five of the seven intracellular polypeptides associated with a normal productive infection could be observed following autoradiography. In this note we report three changes observed to occur simultaneously upon continuous subculture of these infected glia. Cell morphology and growth characteristics were significantly altered, infectious virus particles could be detected in tissue culture media, and analysis of intracellular viral polypeptides revealed the synthesis of ll seven polypeptides associated with a productive RV infection. These data indicate that under the appropriate conditions normal rubella virions may be reexpressed in a manner characteristic of many persistent viral infections. In addition, immunofluorescence techniques have been employed which show that during a nonproductive infection RV antigens are expressed in some glia which bear a striking morphological resemblance to oligodendrocytes.

Rubella virion polypeptides: characterization by polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping.

Four polypeptides with molecular weights of 55 K, 47 K, 45 K, and 33 K have been resolved by polyacrylamide gel electrophoresis of immune precipitated rubella virus. The 47 K and 45 K components have similar peptide maps but different isoelectric points so that the same polypeptide may exist in more than one charged form. The 55 K and 45 K components have similar isoelectric points but different peptide maps showing that similarity of isoelectric point is not evidence of identity.

Effects of 17beta-estradiol on the replication of rubella virus in an estrogen-responsive, continuous cell line

A 70% reduction in the number of infectious rubella virions normally produced in MCF-7 cells occurred when the cells were stimulated with 17beta-estradiol. Analysis of virus-induced RNA indicated a delay in the conversion of replicative intermediate to virion RNA.

Hemolytic activity of rubella virus

Hemolytic activity of rubella virus was demonstrated by chelating Ca 2+ with EDTA after adsorption of the virus to erythrocytes in the presence of the ion. The hemolysis was inhibited by rabbit serum immunized with the virus. Furthermore, the hemolytic activity co-sedimented in sucrose density gradient centrifugation with the hemagglutinating and plaque-forming activity of the virus, suggesting the association of the hemolytic activity with rubella virions.

Fate of rubella genome ribonucleic acid after immune and nonimmune virolysis in the presence of ribonuclease.

To determine whether rubella virion ribonucleic acid (RNA) becomes accessible to nuclease attack after immune lysis of the viral envelope, virions containing radioactively labeled RNA were examined in three ways with the following results. (i) Incubation of purified virus with heat-inactivated rubella convalescent human serum and guinea pig complement resulted in an increase in acid-soluble RNA. Antibody was required; the reaction was temperature dependent and was blocked by ethylenediaminetetraacetic acid. When exogenous nuclease was added prior to lysis, radioactivity in virions was reduced to 15% of that in unlysed control pellets (ii) Sucrose gradient sedimentation profiles of RNA released from lysed and unlysed virions under controlled conditions showed that the nuclease content of serum-virus mixtures was sufficient to eliminate all RNA of genome size, although degradation was not complete. (iii) Virions were also lysed by unheated human immune sera in the absence of guinea pig complement and by some, but not all, unheated antibody-negative sera.

Antibody Response in Rubella Reinfection

AbstractSera were collected from 292 army recruits before and after a rubella outbreak in winter 1971. Of the 37 initially susceptible men, 34 (92%) had serologic evidence of rubella infection, but only 6 (16%) had a rash. A 4-fold or greater booster response in rubella hemagglutination-inhibiting (HI) antibodies was found in 15 (5.9%) of the initially immune men. Paired sera from the booster cases were also titrated by complement fixation (CF) using purified rubella virions (V) and virus-free rubella soluble (S) antigen preparation as antigens, and also by platelet aggregation (PA) using rubella S antigen. In 8 cases reinfection was evident by all serologic techniques, in the others the antibody responses to rubella S antigen were weak or absent.

Precipitin responses to rubella vaccine RA 27-3.

formation of precipitating antibodies against two or more specific antigens [1-3]. The two major antigens, which appear to be structural components of the rubella virion, but which also occur as soluble proteins in infected cell cultures, have been designated theta and iota [4]. In contrast to the natural infection, vaccination with the attenuated rubella virus strains HPV-77 (DE5) and Cendehill stimulates moderate to high levels of anti-theta antibody, but usually produces no detectable anti-iota at all. At best, only minimal amounts of the latter antibody are formed in a minority of vaccinees, and they are almost exclusively children [5, 6]. The present note describes the precipitin responses in recipients of rubella vaccine RA 27/3 [7, 8], and in particular the appearance of anti-iota precipitin, even in adolescents and young adults. We suggest that this elaboration of antiiota antibody may be associated with a degree and kind of immunity resembling that acquired after natural infection with wild rubella virus.