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1.
Viruses ; 7(12): 6108-26, 2015 Nov 26.
Article in English | MEDLINE | ID: mdl-26703711

ABSTRACT

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. The application of pifithrin µ as an inhibitor of p53 shuttling to mitochondria reduced RV-induced cell death to an extent similar to that of the broad spectrum caspase inhibitor z-VAD-fmk (benzyloxycarbonyl-V-A-D-(OMe)-fmk). However, RV progeny generation was not altered. This indicates that, despite an increased survival rate of its cellular host, induction of apoptosis neither supports nor restricts RV replication. Moreover, some of the examined apoptotic markers were affected in a strain-specific manner and differed between the cell culture-adapted strains: Therien and the HPV77 vaccine on the one hand, and a clinical isolate on the other. In summary, the results presented indicate that the transcription-independent mitochondrial p53 program contributes to RV-induced apoptosis.


Subject(s)
Apoptosis , Host-Pathogen Interactions , Rubella virus/physiology , Signal Transduction , Virus Replication , Animals , Chlorocebus aethiops , Mitochondria/physiology , Mitochondrial Membranes/physiology , Permeability , Tumor Suppressor Protein p53/metabolism , Vero Cells
2.
Am J Obstet Gynecol ; 212(1): 71.e1-71.e8, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25108145

ABSTRACT

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. Cells were infected with several viral and nonviral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student t test or analysis of variance with Bonferroni correction. RESULTS: Infection by rubella and other togaviruses, human immunodeficiency virus-1, and varicella zoster was attenuated in cells preexposed to trophoblast-conditioned medium (P < .05), and a partial effect by the chromosome 19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes. CONCLUSION: Our findings indicate that medium conditioned by primary human trophoblasts attenuates viral infection in nontrophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.


Subject(s)
Disease Resistance , Fetal Diseases/virology , Trophoblasts/physiology , Virus Diseases/immunology , Cells, Cultured , Culture Media, Conditioned , Humans , Infant, Newborn
3.
Ann Transl Med ; 2(10): 97, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25405151

ABSTRACT

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. The addition of animal products introduces batch and lot variations that lead to experimental variability, confounds studies with therapeutic outcomes for cultured cells, and represents a major cost associated with cell culture. Here we report a commercially available serum-free, albumin-free, and xeno free (XF) media (Neuro-Pure(TM)) that is more cost-effective than other commercial medias. Neuro-Pure was used to maintain and differentiate various cells of neuronal lineages, fibroblasts, as well as specific cancer cell lines; without the use of contaminants such serum, albumin, and animal products. Neuro-Pure allows for a controlled and reproducible cell culture environment that is applicable to translational medicine and general tissue culture.

4.
BMC Res Notes ; 7: 757, 2014 Oct 24.
Article in English | MEDLINE | ID: mdl-25343994

ABSTRACT

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. FINDINGS: We found that hNPCs are highly susceptible to SINV infection. Upon infection, the viruses induced apoptosis to hNPCs while not affecting the expression of cell proliferation markers. Lastly, SINV infections result in significant changes in the expression of key regulators of hNPCs' plasticity and homeostasis. CONCLUSION: The robust and versatile signaling, proliferation, and other cell responses of hESCs-derived hNPCs to virus infection demonstrated that it is a good model to study the pathogenesis of viral-induced neurodevelopmental and degenerative diseases. On the other hand, the toxicity of SINV to hNPCs cells cannot be ignored, and therefore extra care should be taken when using SINV as a vector to deliver genes into human stem cell lines.


Subject(s)
Alphavirus Infections/pathology , Alphavirus Infections/virology , Embryonic Stem Cells/pathology , Neural Stem Cells/pathology , Neural Stem Cells/virology , Sindbis Virus/physiology , Animals , Apoptosis , Biomarkers/metabolism , Blotting, Western , Cell Cycle Checkpoints , Cell Differentiation , Cell Proliferation , Humans , Inflammation/pathology , Mice , Multipotent Stem Cells/cytology , Nestin/metabolism , Phenotype , Signal Transduction
5.
Virol J ; 10: 121, 2013 Apr 18.
Article in English | MEDLINE | ID: mdl-23597412

ABSTRACT

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. FINDINGS: The replication of three diverse viruses, Sindbis virus (SINV; family Togaviridae family), vesicular stomatitis virus (VSV; family Rhabdoviridae), and Herpes simplex virus (family Herpesviridae), was quantified by the titer (plaque forming units/ml; pfu/ml) produced in cells treated with one of three anti-microtubule drugs (colchicine, noscapine, or paclitaxel) or the anti-actin filament drug, cytochalasin D. None of these drugs affected the replication these viruses. Specific steps in the SINV infection cycle were examined during drug treatment to determine if alterations in specific steps in the virus replication cycle in the absence of a functional cytoskeletal system could be detected, i.e. redistribution of viral proteins and replication complexes or increases/decreases in their abundance. These investigations revealed that the observable impacts were a colchicine-mediated fragmentation of the Golgi apparatus and concomitant intracellular redistribution of the virion structural proteins, along with a reduction in viral genome and sub-genome RNA levels, but not double-stranded RNA or protein levels. CONCLUSIONS: The failure of poisons affecting the cytoskeleton to inhibit the replication of a diverse set of viruses strongly suggests that viruses do not require a functional cytoskeletal system for replication, either because they do not utilize it or are able to utilize alternate pathways when it is not available.


Subject(s)
Cytoskeleton/metabolism , Herpesvirus 1, Human/physiology , Sindbis Virus/physiology , Vesiculovirus/physiology , Virus Replication , Animals , Cell Line , Colchicine/toxicity , Cytoskeleton/drug effects , Humans , Noscapine/toxicity , Paclitaxel/toxicity , Viral Load , Viral Plaque Assay
6.
J Virol ; 86(20): 11078-85, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22855483

ABSTRACT

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.


Subject(s)
Ross River virus/ultrastructure , Rubella virus/ultrastructure , Animals , Capsid Proteins/analysis , Capsid Proteins/chemistry , Cell Line , Chlorocebus aethiops , Cryoelectron Microscopy , Electron Microscope Tomography , Freezing , Glycoproteins , Membrane Glycoproteins/analysis , Membrane Glycoproteins/chemistry , Nucleocapsid/ultrastructure , Rubella/virology , Rubella virus/chemistry , Vero Cells , Viral Envelope Proteins/analysis , Viral Envelope Proteins/chemistry , Virus Assembly
7.
Structure ; 20(5): 759-66, 2012 May 09.
Article in English | MEDLINE | ID: mdl-22579245

ABSTRACT

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. We describe an approach, metal-tagging TEM (METTEM), that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity, and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before.


Subject(s)
Metals/chemistry , Microscopy, Electron, Transmission/methods , Proteins/chemistry , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cells, Cultured , Cellular Structures/ultrastructure , Eukaryotic Cells/metabolism , Gold/chemistry , Image Processing, Computer-Assisted/methods , Metallothionein/chemistry , Metallothionein/metabolism , Metallothionein/ultrastructure , Metals/metabolism , Microscopy, Fluorescence , Protein Conformation , Proteins/ultrastructure , Rubella virus/enzymology , Rubella virus/metabolism , Sensitivity and Specificity , Viral Proteins/chemistry
8.
Virology ; 429(1): 29-36, 2012 Jul 20.
Article in English | MEDLINE | ID: mdl-22542003

ABSTRACT

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. However, survival was ~9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.


Subject(s)
Cell Line/virology , Replicon , Rubella virus/genetics , Rubella/virology , Animals , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Line/metabolism , Cell Tracking , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Rubella virus/physiology , Transfection , Virus Replication
9.
J Virol ; 86(12): 6457-69, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22491463

ABSTRACT

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. During maturation, P150 and P90 likely interact within the context of P200 and remain in a complex after cleavage. We found that P150-P90 interactions were blocked by mutational disruption of an alpha helix at the N terminus (amino acids [aa] 36 to 49) of P200 and that these mutations also had an effect on NSP targeting, processing, and membrane association. While the P150-P90 interaction also required residues 1700 to 1900 within P90, focus formation required the entire RNA-dependent RNA polymerase (aa 1700 to 2116). Surprisingly, the RUBV capsid protein (CP) rescued RNA synthesis by several alanine-scanning mutations in the N-terminal alpha helix, and packaged replicon assays showed that rescue could be mediated by CP in the virus particle. We hypothesize that CP rescues these mutations as well as internal deletions of the Q domain within P150 and mutations in the 5' and 3' cis-acting elements in the genomic RNA by chaperoning the maturation of P200. CP's ability to properly target the otherwise aggregated plasmid-expressed P200 provides support for this hypothesis.


Subject(s)
Polyproteins/chemistry , Polyproteins/metabolism , RNA-Dependent RNA Polymerase/metabolism , Rubella virus/enzymology , Rubella/virology , Viral Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Humans , Molecular Sequence Data , Polyproteins/genetics , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Processing, Post-Translational , Protein Transport , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , Rubella virus/chemistry , Rubella virus/genetics , Sequence Alignment , Viral Proteins/chemistry , Viral Proteins/genetics
10.
Arch Virol ; 157(5): 889-99, 2012 May.
Article in English | MEDLINE | ID: mdl-22322905

ABSTRACT

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. Although usage patterns were conserved in corresponding genes in the diverse genotypes, within-genome comparison revealed that both base and codon usages varied regionally, particularly in the hypervariable region (HVR) of the P150 replicase gene. While directional mutation pressure was predominant in determining base and codon usage within most of the genome (with the strongest tendency being towards C's at third codon positions), natural selection was predominant in the HVR region. The GC content of this region was the highest in the genome (>80%), and it was not clear if selection at the nucleotide level accompanied selection at the amino acid level. Dinucleotide frequency analysis of the RUBV genome revealed that TpA usage was lower than expected, similar to mammalian genes; however, CpG usage was not suppressed, and TpG usage was not enhanced, as is the case in mammalian genes.


Subject(s)
Codon , Genome, Viral , Rubella virus/genetics , Base Composition , Base Sequence , Evolution, Molecular , Humans , Rubella/virology , Rubella virus/chemistry , Rubella virus/isolation & purification
11.
J Gen Virol ; 93(Pt 4): 807-816, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22238231

ABSTRACT

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. Binding of p32 to P150 was confirmed and was abolished by mutation of the first two motifs. When mutations in the first two motifs were introduced into a RUBV cDNA infectious clone, virus replication was significantly impaired. However, virus RNA synthesis was found to be unaffected, and subsequent immunofluorescence analysis of RUBV-infected cells revealed co-localization of p32 and P150 but little overlap of p32 with RNA replication complexes, indicating that p32 does not participate directly in virus RNA synthesis. Thus, the role of p32 in RUBV replication remains unresolved.


Subject(s)
Carrier Proteins/metabolism , Mitochondrial Proteins/metabolism , Proline-Rich Protein Domains/physiology , RNA-Dependent RNA Polymerase/metabolism , Rubella virus/physiology , Animals , Capsid Proteins/metabolism , Capsid Proteins/physiology , Chlorocebus aethiops , Humans , Proline-Rich Protein Domains/genetics , Protein Binding , RNA, Viral/metabolism , RNA, Viral/physiology , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/physiology , Rubella virus/genetics , Rubella virus/metabolism , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Nonstructural Proteins/physiology , Virus Replication/genetics , Virus Replication/physiology , src Homology Domains/physiology
12.
J Gen Virol ; 93(Pt 2): 267-274, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21994324

ABSTRACT

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. Active virus RNA replication was required for induction of these granules, but they were found to differ from SGs induced by arsenite treatment both in composition (they did not uniformly contain other SG proteins, such as PABP and TIA-1) and in resistance to cycloheximide treatment. Thus, bona fide SGs do not appear to be induced during RUBV infection. The distribution of G3BP, either on its own or in granules, did not overlap with that of dsRNA-containing replication complexes, indicating that it played no role in virus RNA synthesis. However, G3BP did co-localize with viral ssRNAs in perinuclear clusters, suggesting an interaction that could possibly be important in a post-replicative role in virus replication, such as encapsidation.


Subject(s)
Carrier Proteins/analysis , Rubella virus/physiology , Virus Replication , Animals , Chlorocebus aethiops , DNA Helicases , Poly-ADP-Ribose Binding Proteins , RNA Helicases , RNA Recognition Motif Proteins , RNA, Viral/biosynthesis , Vero Cells
13.
J Gen Virol ; 93(Pt 3): 516-525, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22113006

ABSTRACT

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. Mutations within a proposed packaging signal did not significantly affect relative packaging efficiency. The insertion of various fragments derived from the RUBV genome into Sindbis virus replicons revealed that there are several regions within the RUBV genome capable of enhancing encapsidation of heterologous replicon RNAs. Secondly, the trans-encapsidation system was used to analyse the effect of alterations within the capsid protein (CP) on release of VRPs and subsequent initiation of replication in newly infected cells. Deletion of the N-terminal eight amino acids of the CP reduced VRP titre significantly, which could be partially complemented by native CP provided in trans, indicating that this mutation affected an entry or post-entry event in the replication cycle. To test this hypothesis, the trans-encapsidation system was used to demonstrate the rescue of a lethal deletion within P150, one of the virus replicase proteins, by CP contained within the virus particle. This novel finding substantiated the functional role of CP in early post-entry replication.


Subject(s)
Capsid Proteins/metabolism , Rubella virus/physiology , Virus Assembly , Virus Replication , Animals , Capsid Proteins/genetics , Cell Line , Genetic Complementation Test , RNA, Viral/genetics , Replicon , Sequence Deletion , Sindbis Virus
14.
Virol J ; 8: 245, 2011 May 20.
Article in English | MEDLINE | ID: mdl-21595991

ABSTRACT

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. FINDINGS: To further test for processing and to rule out artifacts associated with the expression system, ORF1 was re-expressed using a plasmid-based expression vector with the result that the previous processing profile could not be confirmed. When ORF1 from an HEV infectious cDNA clone (US swine strain; genotype 3) was expressed using the plasmid-based system, the only species detected was the 185 kDa precursor of ORF1. A putative papain-like cysteine protease 2 had been predicted within ORF1 using the original HEV genomic sequence. However, analysis of subsequent ORF1 sequences from a large number of HEV isolates reveals that this protease motif is not conserved. CONCLUSIONS: The expressed HEV ORF1 gene product does not undergo proteolytic processing, indicating that the replicase precursor of HEV is potentially unique in this regard.


Subject(s)
Hepatitis E virus/physiology , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Cell Line , Humans , Molecular Sequence Data , Protein Processing, Post-Translational
15.
Virology ; 406(2): 212-27, 2010 Oct 25.
Article in English | MEDLINE | ID: mdl-20696450

ABSTRACT

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.


Subject(s)
Cytoplasm/metabolism , Rubella virus/physiology , Rubella/metabolism , Viral Nonstructural Proteins/metabolism , Amino Acid Sequence , Animals , Chlorocebus aethiops , Cytoplasm/virology , Humans , Microtubule-Organizing Center/metabolism , Microtubule-Organizing Center/virology , Molecular Sequence Data , Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , Rubella/virology , Rubella virus/genetics , Vero Cells , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Virus Replication
16.
Virology ; 405(2): 579-91, 2010 Sep 30.
Article in English | MEDLINE | ID: mdl-20655079

ABSTRACT

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. We have studied the structure of RUBV factory in three dimensions by electron tomography and freeze-fracture. CPVs contain stacked membranes, rigid sheets, small vesicles and large vacuoles. These membranes are interconnected and in communication with the endocytic pathway since they incorporate endocytosed BSA-gold. RER and CPVs are coupled through protein bridges and closely apposed membranes. Golgi vesicles attach to the CPVs but no tight contacts with mitochondria were detected. Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities.


Subject(s)
Imaging, Three-Dimensional/methods , Organelles , Rubella virus/metabolism , Vacuoles , Virus Assembly , Virus Replication , Animals , Cell Line , Cricetinae , Electron Microscope Tomography , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Freeze Fracturing , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Golgi Apparatus/virology , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondria/virology , Organelles/metabolism , Organelles/ultrastructure , Organelles/virology , Vacuoles/metabolism , Vacuoles/ultrastructure , Vacuoles/virology
17.
J Med Virol ; 82(8): 1475-9, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20572092

ABSTRACT

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 0.5 to 78.5 U/ml with a mean of 6.05 U/ml and a median of 3.6 U/ml in colostrum, and from 0.5 to 32.7 U/ml with a mean of 2.74 U/ml and a median of 2 U/ml in milk. The differences between the means of titers of total IgA and anti-rubella virus IgA in colostrum and in milk were significant statistically. The levels of anti-rubella virus IgA in both colostrum and breast milk correlated positively with the anti-rubella virus hemagglutination inhibition (HI) titers in the sera of mother, indicating that the levels of these different classes of antibodies correlated. Based on WB, anti-rubella virus IgA in both colostrum and breast milk reacted with the rubella viral protein E1 and C, but not with the E2 protein.


Subject(s)
Antibodies, Viral/analysis , Colostrum/immunology , Immunoglobulin A/analysis , Milk, Human/immunology , Rubella virus/immunology , Adolescent , Adult , Antibodies, Viral/blood , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Female , Hemagglutination Inhibition Tests , Humans , Infant, Newborn , Japan , Pregnancy , Viral Core Proteins/immunology , Viral Envelope Proteins/immunology , Young Adult
18.
J Biol Chem ; 285(12): 8855-68, 2010 Mar 19.
Article in English | MEDLINE | ID: mdl-20086014

ABSTRACT

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. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152-1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca(2+)/CaM with a dissociation constant of 100-300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a "wrapping around" mode of interaction between RUBpep and Ca(2+)/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity.


Subject(s)
Calcium/chemistry , Calmodulin/chemistry , Rubella virus/enzymology , Viral Nonstructural Proteins/chemistry , Animals , Binding Sites , Chlorocebus aethiops , Cysteine Proteases/chemistry , Magnetic Resonance Spectroscopy/methods , Mutagenesis, Site-Directed , Peptides/chemistry , Protein Structure, Tertiary , Spectrometry, Fluorescence/methods , Vero Cells , Zinc/chemistry
19.
Virology ; 390(2): 315-23, 2009 Aug 01.
Article in English | MEDLINE | ID: mdl-19539969

ABSTRACT

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. RCs, marked by dsRNA, colocalized with P150-foci, but only occasionally with the endosome/lysosome marker LAMP-2, indicating that RNA synthesis occurs at other sites rather than exclusively in endosomes/lysosomes as was previously thought. An expressed cleavage-deficient form of P200 also localized to cytoplasmic foci, suggesting that the precursor is required for targeting to sites of RC establishment. P150 was found to be the determinant of fiber formation and the NSP membrane-binding domain was mapped to the N-terminus of P150.


Subject(s)
RNA-Dependent RNA Polymerase/metabolism , Rubella virus/physiology , Viral Nonstructural Proteins/metabolism , Animals , Binding Sites , Cell Membrane/chemistry , Chlorocebus aethiops , Cytoplasm/chemistry , Microscopy, Confocal , Protein Binding , Protein Transport , RNA-Dependent RNA Polymerase/genetics , Vero Cells , Viral Nonstructural Proteins/genetics
20.
Cell Calcium ; 46(1): 1-17, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19535138

ABSTRACT

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. Ca(2+) plays important roles in virion structure formation, virus entry, viral gene expression, posttranslational processing of viral proteins and virion maturation and release. As part of the review, we introduce an algorithm to identify linear "EF-hand" Ca(2+)-binding motifs which resulted in the prediction of a total of 93 previously unrecognized Ca(2+)-binding motifs in virus proteins. Many of these proteins are nonstructural proteins, a class of proteins among which Ca(2+) interactions had not been formerly appreciated. The presence of linear Ca(2+)-binding motifs in viral proteins enlarges the spectrum of Ca(2+)-virus interplay and expands the total scenario of viral calciomics.


Subject(s)
Calcium-Binding Proteins/metabolism , Calcium/metabolism , Viral Proteins/metabolism , Virus Diseases/metabolism , Calcium Channels/metabolism , Calcium Signaling , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , HIV-1/metabolism , Homeostasis , Host-Pathogen Interactions , Humans , Membrane Potential, Mitochondrial , Virus Diseases/virology
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