Activation of the Mitochondrial Apoptotic Signaling Platform during Rubella Virus Infection.

Mitochondria- as well as p53-based signaling pathways are central for the execution of the intrinsic apoptotic cascade. Their contribution to rubella virus (RV)-induced apoptosis was addressed through time-specific evaluation of characteristic parameters such as permeabilization of the mitochondrial membrane and subsequent release of the pro-apoptotic proteins apoptosis-inducing factor (AIF) and cytochrome c from mitochondria. Additionally, expression and localization pattern of p53 and selected members of the multifunctional and stress-inducible cyclophilin family were examined.

Chemically defined serum-free and xeno-free media for multiple cell lineages.

Cell culture is one of the most common methods used to recapitulate a human disease environment in a laboratory setting. Cell culture techniques are used to grow and maintain cells of various types including those derived from primary tissues, such as stem cells and cancer tumors. However, a major confounding factor with cell culture is the use of serum and animal (xeno) products in the media.

Cellular responses to Sindbis virus infection of neural progenitors derived from human embryonic stem cells.

Background: Sindbis virus (SINV) causes age-dependent encephalitis in mice, and therefore serves as a model to study viral encephalitis. SINV is used as a vector for the delivery of genes into selected neural stem cell lines; however, the toxicity and side effects of this vector have rarely been discussed. In this context, we investigated the cellular responses of human embryonic stem cell (hESCs) derived neural progenitors (hNPCs) to SINV infection by assessing susceptibility of the cells to SINV infection, analyzing the effect of infection on cell proliferation and cell death, and examining the impact of SINV infection on hNPCs markers of stemness.

Human trophoblasts confer resistance to viruses implicated in perinatal infection.

Objective: Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to nontrophoblast cells. Can trophoblasts protect nontrophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection?

Study Design: Isolated primary term human trophoblasts were cultured for 48-72 hours. Diverse nonplacental human cell lines (U2OS, human foreskin fibroblast, TZM-bl, MeWo, and Caco-2) were preexposed to either trophoblast conditioned medium, nonconditioned medium, or miR-517-3p for 24 hours.

Do viruses require the cytoskeleton?

Background: It is generally thought that viruses require the cytoskeleton during their replication cycle. However, recent experiments in our laboratory with rubella virus, a member of the family Togaviridae (genus rubivirus), revealed that replication proceeded in the presence of drugs that inhibit microtubules. This study was done to expand on this observation.

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.

Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy.

More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell's interior are lagging behind.

Characterization of cell lines stably transfected with rubella virus replicons.

Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer.

Determinants in the maturation of rubella virus p200 replicase polyprotein precursor.

Rubella virus (RUBV), a positive-strand RNA virus, replicates its RNA within membrane-associated replication complexes (RCs) in the cytoplasm of infected cells. RNA synthesis is mediated by the nonstructural proteins (NSPs) P200 and its cleavage products, P150 and P90 (N and C terminal within P200, respectively), which are processed by a protease residing at the C terminus of P150. In this study of NSP maturation, we found that early NSP localization into foci appeared to target the membranes of the endoplasmic reticulum.

Analysis of base and codon usage by rubella virus.

Rubella virus (RUBV), a small, plus-strand RNA virus that is an important human pathogen, has the unique feature that the GC content of its genome (70%) is the highest (by 20%) among RNA viruses. To determine the effect of this GC content on genomic evolution, base and codon usage were analyzed across viruses from eight diverse genotypes of RUBV. Despite differences in frequency of codon use, the favored codons in the RUBV genome matched those in the human genome for 18 of the 20 amino acids, indicating adaptation to the host.

Binding of cellular p32 protein to the rubella virus P150 replicase protein via PxxPxR motifs.

A proline-rich region (PRR) within the rubella virus (RUBV) P150 replicase protein that contains three SH3 domain-binding motifs (PxxPxR) was investigated for its ability to bind cell proteins. Pull-down experiments using a glutathione S-transferase-PRR fusion revealed PxxPxR motif-specific binding with human p32 protein (gC1qR), which could be mediated by either of the first two motifs. This finding was of interest because p32 protein also binds to the RUBV capsid protein.

Rubella virus-like replicon particles: analysis of encapsidation determinants and non-structural roles of capsid protein in early post-entry replication.

Rubella virus (RUBV) contains a plus-strand RNA genome with two ORFs, one encoding the non-structural replicase proteins (NS-ORF) and the second encoding the virion structural proteins (SP-ORF). This study describes development and use of a trans-encapsidation system for the assembly of infectious RUBV-like replicon particles (VRPs) containing RUBV replicons (self replicating genomes with the SP-ORF replaced with a reporter gene). First, this system was used to map signals within the RUBV genome that mediate packaging of viral RNA.

Analysis of subcellular G3BP redistribution during rubella virus infection.

Rubella virus (RUBV) replicates slowly and to low titre in vertebrate cultured cells, with minimal cytopathology. To determine whether a cellular stress response is induced during such an infection, the formation of Ras-GAP-SH3 domain-binding protein (G3BP)-containing stress granules (SGs) in RUBV-infected cells was examined. Late in infection, accumulation of G3BP granules was detected, albeit in fewer than half of infected cells.

Lack of processing of the expressed ORF1 gene product of hepatitis E virus.

Background: Proteolytic processing is a common mechanism among plus strand RNA viruses and the replicases of all plus strand RNA viruses of animals thus far characterized undergo such processing. The replicase proteins of hepatitis E virus (HEV) are encoded by ORF1. A previous report published by our group 1 provided data that processing potentially occurred when ORF1 (Burma strain; genotype 1) was expressed using a vaccinia virus-based expression system.

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.

Three-dimensional structure of Rubella virus factories.

Viral factories are complex structures in the infected cell where viruses compartmentalize their life cycle. Rubella virus (RUBV) assembles factories by recruitment of rough endoplasmic reticulum (RER), mitochondria and Golgi around modified lysosomes known as cytopathic vacuoles or CPVs. These organelles contain active replication complexes that transfer replicated RNA to assembly sites in Golgi membranes.

Quantitative and qualitative assay of rubella IgA antibody in breast milk.

Breast milk contains immunological factors, such as IgA antibody, which help to prevent infectious diseases. A total of 197 paired samples of colostrum and breast milk was collected from postpartum mothers in Gunma City, Japan, and examined for anti-rubella IgA antibody by enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB). The anti-rubella virus IgA ranged from 0.

Calcium-dependent association of calmodulin with the rubella virus nonstructural protease domain.

The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca(2+)- and Zn(2+)-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca(2+)-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca(2+)/CaM.

Determinants of subcellular localization of the rubella virus nonstructural replicase proteins.

The rubella virus (RUBV) nonstructural replicase proteins (NSPs), P150 and P90, are proteolytically processed from a P200 precursor. To understand the NSPs' function in the establishment of virus RNA replication complexes (RCs), the NSPs were analyzed in virus-infected cells or cells transfected with NSP-expressing plasmids. In infected cells, P150 was localized in cytoplasmic foci at 24 hpi and in cytoplasmic fibers, unique to RUBV, by 48 hpi.

Viral calciomics: interplays between Ca2+ and virus.

Ca(2+) is one of the most universal and versatile signaling molecules and is involved in almost every aspect of cellular processes. Viruses are adept at utilizing the universal Ca(2+) signal to create a tailored cellular environment that meets their own demands. This review summarizes most of the known mechanisms by which viruses perturb Ca(2+) homeostasis and utilize Ca(2+) and cellular Ca(2+)-binding proteins to their benefit in their replication cycles.

Functional replacement of a domain in the rubella virus p150 replicase protein by the virus capsid protein.

The rubella virus (RUBV) capsid (C) protein rescues mutants with a lethal deletion between two in-frame NotI sites in the P150 replicase gene, a deletion encompassing nucleotides 1685 to 2192 of the RUBV genome and amino acids (aa) 548 to 717 of P150 (which has a total length of 1,301 aa). The complete domain rescuable by the C protein was mapped to aa 497 to 803 of P150. Introduction of aa 1 to 277 of the C protein (lacking the C-terminal E2 signal sequence) between the NotI sites in the P150 gene in a replicon construct yielded a viable construct that synthesized viral RNA with wild-type kinetics, indicating that C and this region of P150 share a common function.

A cysteine-rich metal-binding domain from rubella virus non-structural protein is essential for viral protease activity and virus replication.

The protease domain within the RUBV (rubella virus) NS (non-structural) replicase proteins functions in the self-cleavage of the polyprotein precursor into the two mature proteins which form the replication complex. This domain has previously been shown to require both zinc and calcium ions for optimal activity. In the present study we carried out metal-binding and conformational experiments on a purified cysteine-rich minidomain of the RUBV NS protease containing the putative Zn(2+)-binding ligands.

Analysis of gene expression in fetal and adult cells infected with rubella virus.

Congenital infection with rubella virus (RUB) leads to persistent infection and congenital defects and we showed previously that primary human fetal fibroblasts did not undergo apoptosis when infected with RUB, which could promote fetal virus persistence [Adamo, P., Asís, L., Silveyra, P.

Rubella virus-induced superinfection exclusion studied in cells with persisting replicons.

For the first time, homologous superinfection exclusion was documented for rubella virus (RUB) by using Vero cells harbouring persisting RUB replicons. Infection with wild-type RUB was reduced by tenfold, whereas Sindbis virus infection was unaffected. Replication following infection with packaged replicons and transfection with replicon transcripts was also restricted in these cells, indicating that restriction occurred after penetration and entry.

Analysis of the selective advantage conferred by a C-E1 fusion protein synthesized by rubella virus DI RNAs.

During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.