Rubella virus tropism and single-cell responses in human primary tissue and microglia-containing organoids.

Rubella virus is an important human pathogen that can cause neurological deficits in a developing fetus when contracted during pregnancy. Despite successful vaccination programs in the Americas and many developed countries, rubella remains endemic in many regions worldwide and outbreaks occur wherever population immunity is insufficient. Intense interest since rubella virus was first isolated in 1962 has advanced our understanding of clinical outcomes after infection disrupts key processes of fetal neurodevelopment. Yet it is still largely unknown which cell types in the developing brain are targeted. We show that in human brain slices, rubella virus predominantly infects microglia. This infection occurs in a heterogeneous population but not in a highly microglia-enriched monoculture in the absence of other cell types. By using an organoid-microglia model, we further demonstrate that rubella virus infection leads to a profound interferon response in non-microglial cells, including neurons and neural progenitor cells, and this response is attenuated by the presence of microglia.

Diverse diterpenoids from Callicarpa rubella Lindl. as natural inhibitors of macrophage foam cell formation.

Ten undescribed diterpenoids namely rubellawus E-N of structural types pimarane (1, 3-4), nor-abietane (2), nor-pimarane (5-6), isopimarane (7-9), and nor-isopimarane (10), along with eleven known compounds, were isolated and identified from the aerial parts of Callicarpa rubella Lindl. The structures of the isolated compounds were confirmed by comprehensive spectroscopic analyses and quantum chemical computations. Pharmacologically, almost all the compounds exhibited a potential inhibitory effect on oxidized low-density lipoprotein-induced macrophage foam cell formation, which suggests that these compounds may be promising candidates in the treatment of atherosclerosis.

Dissecting Rubella Placental Infection in an In Vitro Trophoblast Model.

Vertical transmission of rubella virus (RuV) occurs at a high rate during the first trimester of pregnancy. The modes of vertical transmission including the response of trophoblasts to RuV are not well understood. Here, RuV-trophoblast interaction was studied in the BeWo trophoblast cell line. Analysis included early and late time-point kinetics of virus infection rate and the antiviral innate immune response at mRNA and protein level. BeWo characteristics were addressed through metabolic activity by extracellular flux analysis and syncytiotrophoblast formation through incubation with forskolin. We found that RuV infection of BeWo led to profuse type III interferon (IFN) production. Transfecting trophoblast cells with dsRNA analog induced an increase in the production of type I IFN-ß and type III IFNs; however, this did not occur in RuV-infected BeWo trophoblasts. IFN-ß and to a lesser extent type III IFN-λ1 were inhibitory to RuV. While no significant metabolic alteration was detected, RuV infection reduced the cell number in the monolayer culture in comparison to the mock control and resulted in detached and floating cells. Syncytia formation restricted RuV infection. The use of BeWo as a relevant cell culture model for infection of trophoblasts highlights cytopathogenicity in the absence of a type I IFN response as a pathogenic alteration by RuV.

Low Susceptibility of Rubella Virus in First-Trimester Trophoblast Cell Lines.

We recently published an article about myelin oligodendrocyte glycoprotein-independent rubella infection of keratinocytes in vitro, in which first-trimester trophoblast cells were shown as rubella virus (RuV)-resistant. Given an incident rate as high as 90% of congenital rubella syndrome in the first eight weeks of pregnancy, the RuV infection of first-trimester trophoblasts is considered key to opening the gate to transplacental transmission mechanisms. Therefore, with this study, we aimed to verify the susceptibility/resistance of first-trimester trophoblast cell lines, HTR-8/SVneo and Swan.71, against RuV. Cells cultured on multi-well plates were challenged with a RuV clinical strain at a multiplicity of infection from 5 to 10 for 3 h. The infectivity was investigated by immunofluorescence (IF) assay and flow cytometry (FCM) analysis. Supernatants collected during the post-infection period were used to determine virus-progeny production. The scattered signaling of RuV infection of these cells was noted by IF assay, and the FCM analysis showed an average of 4-5% of gated cells infected with RuV. In addition, a small but significant production of virus progeny was also observed. In conclusion, by employing appropriate approaches, we determined the low infectivity of RuV in first-trimester trophoblast cell lines but not resistance as in our previous report.

An Intraocular Inflammatory Profile of Rubella Associated Uveitis.

Purpose: To analyze intraocular cytokine levels and cell profiles in patients with rubella virus-associated uveitis (RVU). Methods: We collected intraocular fluid samples from patients with RVU (n = 10), uveitis of other causes (n = 27), and cataract (n = 22). Levels of 15 cytokines (IL-1ß, IL-1ra, IL-2, IL-6, IL-6rα, IL-7, IL-8, IL-10, IL-17A, IL-23, TARC, MCP-1, TNF-α, PlGF, and VEGF) were measured using multiplex assay, and intraocular cell populations were determined by multiparameter flowcytometry. Clinical characteristics of RVU patients were collected and compared to laboratory outcomes. Results: RVU patients exhibited high intraocular levels of MCP-1, IL-6rα, and TARC, whilst patients with noninfectious uveitis were characterized by high levels of PlGF. Cataract patients showed high levels of IL-2 and IL-23. Intraocular cell population of RVU patients disclosed mainly T-cells and monocytes/macrophages and B-cells were scarcely detected. Conclusion: RVU patients exhibit a cytokine profile distinct from noninfectious uveitis and cataract.

Rubella Virus Strain-Associated Differences in the Induction of Oxidative Stress Are Independent of Their Interferon Activation.

Rubella virus (RV) infection impacts cellular metabolic activity in a complex manner with strain-specific nutritional requirements. Here we addressed whether this differential metabolic influence was associated with differences in oxidative stress induction and subsequently with innate immune response activation. The low passaged clinical isolates of RV examined in this study induced oxidative stress as validated through generation of the reactive oxygen species (ROS) cytoplasmic hydrogen peroxide and mitochondrial superoxide. The addition of the cytoplasmic and mitochondrial ROS scavengers N-acetyl-l-cysteine and MitoTEMPO, respectively, reduced RV-associated cytopathogenicity and caspase activation. While the degree of oxidative stress induction varied among RV clinical isolates, the level of innate immune response and interferon-stimulated gene activation was comparable. The type III IFNs were highly upregulated in all cell culture systems tested. However, only pre-stimulation with IFN ß slightly reduced RV replication indicating that RV appears to have evolved the ability to counteract innate immune response mechanisms. Through the data presented, we showed that the ability of RV to induce oxidative stress was independent of its capacity to stimulate and counteract the intrinsic innate immune response.

Myelin Oligodendrocyte Glycoprotein-Independent Rubella Infection of Keratinocytes and Resistance of First-Trimester Trophoblast Cells to Rubella Virus In Vitro.

Rubella virus (RuV), which belongs to the family Togaviridae and genus Rubivirus, causes systemic infection in children and young adults and congenital rubella syndrome in developing fetuses if the infection occurs during pregnancy. The mechanisms of fetal infection by RuV are not completely understood. Myelin oligodendrocyte glycoprotein (MOG) is reported to be a cellular receptor for RuV; however, it is mainly expressed in the central nervous system. Therefore, it is thought that other receptors are also responsible for virus entry into susceptible cells. In this study, we found that first-trimester trophoblast cells were resistant to RuV. In addition, we showed that HaCaT cells (an immortalized keratinocyte cell line) that did not express MOG on their surface were infected with RuV. This finding is one of the first demonstrations of MOG-independent RuV infection of susceptible host cells and suggests that it is important to continue searching for alternative RuV receptors. In addition, this study reports the resistance of first-trimester trophoblast cells to RuV and suggests that utilizing an epithelial-mesenchymal transition approach to study the mechanisms of transplacental vertical RuV infection.

Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry.

Rubella virus (RuV) causes a systemic infection, and transplacental fetal infection causes congenital rubella syndrome. In this study, we showed that treatment of cells with sphingomyelinase inhibited RuV infection. Assays using inhibitors of serine palmitoyl transferase and ceramide transport protein demonstrated the contribution of sphingomyelin (SM) to RuV infection. Compelling evidence for direct binding of RuV to lipid membranes at neutral pH was obtained using liposome coflotation assays. The absence of either SM or cholesterol (Chol) abrogated the RuV-liposome interaction. SM and Chol (SM/Chol) were also critical for RuV binding to erythrocytes and lymphoid cells. Removal of Ca2+ from the assay buffer or mutation of RuV envelope E1 protein Ca2+-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various nonlymphoid adherent cell lines independently of extracellular Ca2+ or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca2+-binding sites and cellular SM/Chol were essential for the early stage of RuV infection, possibly affecting envelope-membrane fusion in acidic compartments. Myelin oligodendrocyte glycoprotein (MOG) has recently been identified as a cellular receptor for RuV. However, RuV bound to MOG-negative cells in a Ca2+-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca2+ dependent and the other is Ca2+ independent. Ca2+-dependent binding observed in lymphoid cells occurs by the direct interaction between E1 protein fusion loops and SM/Chol-enriched membranes. Clarification of the mechanism of Ca2+-independent RuV binding is an important next step in understanding the pathology of RuV infection.IMPORTANCE Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Even though effective rubella vaccines are available, rubella outbreaks still occur in many countries. We studied the entry mechanism of rubella virus (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca2+ at neutral pH. This Ca2+-dependent binding is specifically directed to membranes enriched in sphingomyelin and cholesterol and is critical for RuV infection. Importantly, RuV also binds to many cell lines in a Ca2+-independent manner. An unidentified RuV receptor(s) is involved in this Ca2+-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes.

UNLABELLED: The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. IMPORTANCE: Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment.

Rubella virus: first calcium-requiring viral fusion protein.

Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world. RuV is a small enveloped RNA virus that infects target cells by receptor-mediated endocytosis and low pH-dependent membrane fusion. The structure of the RuV E1 fusion protein was recently solved in its postfusion conformation. RuV E1 is a member of the class II fusion proteins and is structurally related to the alphavirus and flavivirus fusion proteins. Unlike the other known class II fusion proteins, however, RuV E1 contains two fusion loops, with a metal ion complexed between them by the polar residues N88 and D136. Here we demonstrated that RuV infection specifically requires Ca(2+) during virus entry. Other tested cations did not substitute. Ca(2+) was not required for virus binding to cell surface receptors, endocytic uptake, or formation of the low pH-dependent E1 homotrimer. However, Ca(2+) was required for low pH-triggered E1 liposome insertion, virus fusion and infection. Alanine substitution of N88 or D136 was lethal. While the mutant viruses were efficiently assembled and endocytosed by host cells, E1-membrane insertion and fusion were specifically blocked. Together our data indicate that RuV E1 is the first example of a Ca(2+)-dependent viral fusion protein and has a unique membrane interaction mechanism.

[Progress on mechanism of cell apoptosis induced by rubella virus].

Rubella virus (RV), a member of the family Togaviridae, can induce apoptosis of host cells in vitro. Protein kinases of the Ras-Raf-MEK-ERK pathway and PI3K-Akt pathway play essential roles in virus multiplication, cell survival and apoptosis. Proteins p53 and TAp63 that bind to specific DNA sequences stimulate Bax in a manner to produce functional pores that facilitate release of mitochondrial cytochrome c and downstream caspase activation. In this review, the molecular mechanisms of RV-induced cell apoptosis, including RV-infected cell lines, pathological changes in cell components and apoptosis signaling pathways are summarized.

The Rubella virus capsid is an anti-apoptotic protein that attenuates the pore-forming ability of Bax.

Apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. Accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. Interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. Whereas large DNA viruses have the luxury of encoding accessory proteins whose primary function is to undermine programmed cell death pathways, it is generally thought that most RNA viruses do not encode these types of proteins. Here we report that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis. Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur. Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation. Importantly, data from reverse genetic studies are consistent with a scenario in which the anti-apoptotic activity of capsid protein is important for virus replication. If so, this would be among the first demonstrations showing that blocking apoptosis is important for replication of an RNA virus. Finally, it is tempting to speculate that other slowly replicating RNA viruses employ similar mechanisms to avoid killing infected cells.

Analysis of the function of cytoplasmic fibers formed by the rubella virus nonstructural replicase proteins.

The P150 and P90 replicase proteins of rubella virus (RUBV), a plus-strand RNA Togavirus, produce a unique cytoplasmic fiber network resembling microtubules. Pharmacological and mutagenic approaches were used to determine if these fibers functioned in virus replication. The pharmacological approach revealed that microtubules were required for fiber formation, but neither was necessary for virus replication. Through the mutagenic approach it was found that α-helices near both termini of P150 were necessary for fiber assembly and infectivity, but fiber formation and viability could not be correlated because most of these mutations were lethal. The N-terminal α-helix of P150 affected both proteolytic processing of P150 and P90 from the P200 precursor and targeting of P200, possibly through directing conformational folding of P200. Finally, we made the unexpected discovery that RUBV genomes can spread from cell-to-cell without virus particles, a process that we hypothesize utilizes RUBV-induced cytoplasmic projections containing fibers and replication complexes.

Rubella virus and birth defects: molecular insights into the viral teratogenesis at the cellular level.

BACKGROUND: In utero rubella virus (RV) infection of a fetus can result in birth defects that are often collectively referred to as congenital rubella syndrome (CRS). In extreme cases, fetal death can occur. In spite of the availability of a safe and effective vaccine against rubella, recent worldwide estimates are that more than 100,000 infants are born with CRS annually. RECENT PROGRESS: Recently, several significant findings in the field of cell biology, as well as in the RV replication and virus-cell interactions, have originated from the authors' laboratory, and other researchers have provided insights into RV teratogenesis. It has been shown that 1) an RV protein induces cell-cycle arrest by generating a subpopulation of tetraploid nuclei (i.e., 4N DNA) cells, perhaps representative of the tetraploid state following S phase in the cell cycle, due to its interaction with citron-K kinase (CK); 2) RV infection induces apoptosis in cell culture, and 3) CK functional perturbations lead to tetraploidy, followed by apoptosis, in specific cell types. CONCLUSIONS: Based on several similarities between known RV-associated fetal and cellular manifestations and CK deficiency-associated phenotypes, it is reasonable to postulate that P90-CK interaction in RV-infected cells interferes with CK function and induces cell-cycle arrest following S phase in a subpopulation, perhaps representative of tetraploid stage, which could lead to subsequent apoptosis in RV infection. Taking all these observations to the fetal organogenesis level, it is plausible that P90-CK interaction could perhaps be one of the initial steps in RV infection-induced apoptosis-associated fetal birth defects in utero.

Distinct serum cytokine levels in drug- and measles-induced exanthema.

BACKGROUND: Macular or maculopapular skin reactions are frequent events in drug allergy as well as in viral infections. Clinically, the differentiation may be difficult in the absence of a clear relationship to drug intake or failure to detect virus-specific antibodies of the IgM class. Studies on drug-specific T cell lines and T cell clones isolated from drug-allergic patients have suggested that these cells may represent a significant source of IL-5. On the other hand, viral infections are frequently associated with elevated IFN-gamma levels. OBJECTIVE: Determination of serum-cytokine levels to differentiate between drug- and virally induced skin eruptions. PATIENTS: 18 patients suffering from acute drug allergy and 19 patients with acute measles, rubella or parvovirus infection. MEASUREMENTS: Cytokine-ELISA (IL-5, IL-4 and IFN-gamma) of sera collected during acute drug allergy or during acute measles, rubella or parvovirus infection. RESULTS: In 12/18 patients with drug allergy, IL-5 and/or IL-4 were elevated. A significant correlation (r(Spearman) = 0.84) between IL-5 serum levels and eosinophil counts in the blood was found. No correlation was detected between IL-4 and blood eosinophilia or between IL-4 and IL-5 levels. After remission, IL-5 and IL-4 decreased to undetectable levels. IFN-gamma on the other hand was not measurable in patients with drug allergy while elevated IFN-gamma serum levels were detected in 17/19 patients with measles, rubella or parvovirus infection; 2 patients with acute virus infection had elevated IL-5, and/or IL-4 and IFN-gamma levels. CONCLUSION: These data underline the distinct pathogenesis of these morphologically similar exanthemas and suggest that the combined analysis of eosinophilia in the blood, IL-4 and IFN-gamma might help in differentiating skin eruptions.

Phagocytosis of latex beads is defective in cultured human retinal pigment epithelial cells with persistent rubella virus infection.

Phagocytosis, a secondary function of retinal pigment epithelial (RPE) cells essential to sight, was significantly decreased, when measured with latex beads, during persistent rubella virus (RV) infection of human cultured RPE cells. A target for RV in vivo, RPE cells infected with RV (RPE/RV) ingested fewer fluorescent microspheres (26%) than did uninfected RPE cells (68%) (P < 0.001), as measured by flow cytometry. In RPE/RV cells, with characteristic RPE monolayer appearance and normal growth during subculturing over 6 months, persistent RV infection was shown by specific RV antigen immunofluorescence, by the presence of the RV genome in RPE/RV cell messenger RNA, and by recovery of cell-free RV after cocultivation with Vero cells. The adhesion of latex beads to apical cell surfaces of RPE/RV and uninfected RPE cells appeared similar, as imaged by scanning electron microscopy. Cytoskeletal actin, a component of phagocytosis in RPE, appeared altered in 60 to 75% of RPE/RV cells by antiactin immunofluorescence staining, as previously described in other RV-infected cells, but its role in the disturbed phagocytosis of latex beads was not determined. Persistently RV-infected human RPE is an additional example of RV-associated secondary cellular dysfunction in the absence of cytopathic effects.

Infection of cultured human fetal pancreatic islet cells by rubella virus.

A high incidence of insulin-dependent diabetes mellitus (IDDM) has been reported in children and young adults previously afflicted with congenital rubella syndrome (CRS). The authors have studied the effect of rubella virus infection on human pancreatic islet cells in tissue culture. These experiments were performed with the use of both monolayers and free-floating human fetal islets of Langerhans tissue. Levels of production of immunoreactive insulin by islet cells that had been infected by rubella virus were lower than those observed in control cultures, under conditions of high glucose concentration (11.1 mmol/L) in the medium. The presence of rubella viral antigens in human pancreatic beta and non-beta cells was demonstrated by double-label immunofluorescence. These results suggest that rubella virus can infect human pancreatic islet cells and that such infection may lead to significant reductions in levels of secreted insulin.

[Difference in the capacity to repair DNA damage in human cells chronically infected with the rubella virus]. / Razlichie v sposobnosti k reparatsii povrezhdenii DNK kletok cheloveka, khronicheski infitsirovannykh virusom krasnukhi.

Centrifugation of cell lysates in alkaline sucrose gradients and chromatography on hydroxyapatite columns were used to demonstrate inhibition of reparation of mitomycin C-induced DNA damages at the stage of reunification of single-strand breaks of DNA in human HEp-2 cell cultures chronically infected with rubella virus. At the same time, reparation of single-strand breaks of DNA caused by bleomycin occurs with similar intensity both in chronically infected and noninfected HEp-2 cultures. The experimental results suggest that the chronic course of infection in human cells leads to disorders in reparative synthesis of cellular DNA and/or is due to disconnected effect of reparation enzymes in this system.

Characterization of immune complexes in progressive rubella panencephalitis.

In two patients with the slow virus disease progressive rubella panencephalitis, high levels of immune complexes were found in serum and, in one case, spinal fluid. These complexes contained immunoglobulin G. Serum complexes sedimented over a wide density range compared with the more homogeneous spinal fluid complexes. Complexes from both serum and spinal fluid contained antibody directed against rubella virus. Serum complexes were also shown to contain rubella antigen (or antigens) that appeared to differ between the two patients.