ABSTRACT
Human parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2 (PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape.
Subject(s)
Endosomes , Globosides , Golgi Apparatus , Parvovirus B19, Human , Virus Internalization , Endosomes/metabolism , Endosomes/virology , Humans , Golgi Apparatus/metabolism , Golgi Apparatus/virology , Parvovirus B19, Human/metabolism , Parvovirus B19, Human/physiology , Parvovirus B19, Human/genetics , Globosides/metabolism , Phospholipases A2/metabolism , Calcium/metabolismABSTRACT
B19 virus (B19V) is a pathogenic human parvovirus that infects erythroid progenitor cells. Because there are limited in vitro culture systems to propagate this virus, little is known about the molecular mechanisms by which it propagates in cells. In this study, we introduced a HiBiT peptide tag into various loops of VP2 located on the surface of B19V particles and evaluated their ability to form virus-like particles (VLPs). Three independent sites were identified as permissive sites for peptide tag insertion without affecting VLP formation. When the HiBiT tag was introduced into B19V clones (pB19-M20) and transfected into a semipermissive erythroleukemia cell line (UT7/Epo-S1), HiBiT-dependent luciferase activities (HiBiT activities) increased depending on helicase activity of viral NS1. Furthermore, we used a GFP11 tag-split system to visualize VLPs in the GFP1-10-expressing live cells. Time-lapse imaging of green fluorescent protein (GFP)-labeled VLPs revealed that nuclear VLPs were translocated into the cytoplasm only after cell division, suggesting that the breakdown of the nuclear envelope during mitosis contributes to VLP nuclear export. Moreover, HiBiT activities of culture supernatants were dependent on the presence of a detergent, and the released VLPs were associated with extracellular vesicles, as observed under electron microscopy. Treatment with an antimitotic agent (nocodazole) enhanced the release of VLPs. These results suggest that the virions accumulated in the cytoplasm are constitutively released from the cell as membrane-coated vesicles. These properties are likely responsible for viral escape from host immune responses and enhance membrane fusion-mediated transmission. IMPORTANCE Parvovirus particles are expected to be applied as nanoparticles in drug delivery systems. However, little is known about how nuclear-assembled B19 virus (B19V) virions are released from host cells. This study provides evidence of mitosis-dependent nuclear export of B19V and extracellular vesicle-mediated virion release. Moreover, this study provides methods for modifying particle surfaces with various exogenous factors and contributes to the development of fine nanoparticles with novel valuable functions. The pB19-M20 plasmid expressing HiBiT-tagged VP2 is a novel tool to easily quantify VP2 expression. Furthermore, this system can be applied in high-throughput screening of reagents that affect VP2 expression, which might be associated with viral propagation.
Subject(s)
Parvoviridae Infections , Parvovirus B19, Human , Humans , Cell Line , Green Fluorescent Proteins/metabolism , Parvovirus B19, Human/metabolism , Peptides/metabolism , Artificial Virus-Like ParticlesABSTRACT
Parvovirus B19 (B19V) infects human erythroid progenitor cells (EPCs) and causes several hematological disorders and fetal hydrops. Amino acid (aa) 5-68 of minor capsid protein VP1 (VP1u5-68aa) is the minimal receptor binding domain for B19V to enter EPCs. Here, we carried out a genome-wide CRISPR-Cas9 guide RNA screen and identified tyrosine protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. AXL gene silencing in ex vivo expanded EPCs remarkably decreased B19V internalization and replication. Additions of the recombinant AXL extracellular domain or a polyclonal antibody against it upon infection efficiently inhibited B19V infection of ex vivo expanded EPCs. Moreover, B19V VP1u interacted with the recombinant AXL extracellular domain in vitro at a relatively high affinity (KD = 103 nM). Collectively, we provide evidence that AXL is a co-receptor for B19V infection of EPCs.
Subject(s)
Axl Receptor Tyrosine Kinase , Erythema Infectiosum , Parvovirus B19, Human , Humans , Capsid Proteins/genetics , Capsid Proteins/metabolism , Erythema Infectiosum/metabolism , Parvovirus B19, Human/genetics , Parvovirus B19, Human/metabolism , Protein Binding , Axl Receptor Tyrosine Kinase/metabolismABSTRACT
BACKGROUND: Human parvovirus B19 (B19) infection can affect the hematopoietic arrest in fetus by hindering the differentiation and maturation of erythroid progenitor cells. B19 nonstructural protein 1 (NS1) has been shown to inhibit the differentiation of erythroid progenitor cells. The goal of this study is to explore the role of B19 NS1 in the regulation of GATA1 and Notch signaling pathway in hematopoietic cells. METHODS: The B19 NS1 expression plasmid was reconstituted, and the possibility of NS1 regulating GATA1 and GATA2 expression modulated by Notch-Hes pathway was tested by qRT-PCR and western blot. Immunofluorescence assays were conducted to visualize pNS1 in K562 cells. RESULTS: We demonstrate that B19 NS1 inhibited GATA1 and induced Hes1/Hes5, which is involved in the activation of Notch signaling pathway. Meanwhile, NS1 exhibited promoting effects on GATA2 expression. Activation of the Notch signaling pathway up-regulated its downstream transcriptional repressor family Hes, thereby inhibiting the expression of GATA gene in K562 cells. CONCLUSIONS: The results show that B19 NS1 protein negatively regulates GATA1 related nuclear transcription and may interfere with hematopoietic cell differentiation.
Subject(s)
Parvovirus B19, Human , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Humans , K562 Cells , Parvovirus B19, Human/genetics , Parvovirus B19, Human/metabolism , Signal Transduction , Transcription Factors/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
Parvovirus B19 (B19V) is a human pathogen that is the causative agent of fifth disease in children. It is also known to cause hydrops in fetuses, anemia in AIDS patients, and transient aplastic crisis in patients with sickle cell disease. The unique N-terminus of Viral Protein 1 (VP1u) of parvoviruses, including B19V, exhibits phospholipase A2 (PLA2) activity, which is required for endosomal escape. Presented is the structural dynamics of B19V VP1u under conditions that mimic the pHs of cell entry and endosomal trafficking to the nucleus. Using circular dichroism spectroscopy, the receptor-binding domain of B19V VP1u is shown to exhibit an α-helical fold, whereas the PLA2 domain exhibits a probable molten globule state, both of which are pH invariant. Differential scanning calorimetry performed at endosomal pHs shows that the melting temperature (Tm) of VP1u PLA2 domain is tuned to body temperature (37 °C) at pH 7.4. In addition, PLA2 assays performed at temperatures ranging from 25-45 °C show both a temperature and pH-dependent change in activity. We hypothesize that VP1u PLA2 domain differences in Tm at differing pHs have enabled the virus to "switch on/off" the phospholipase activity during capsid trafficking. Furthermore, we propose the environment of the early endosome as the optimal condition for endosomal escape leading to B19V infection.
Subject(s)
Parvovirus B19, Human , Virus Internalization , Capsid Proteins/metabolism , Child , Endosomes/metabolism , Humans , Parvovirus B19, Human/metabolism , Phospholipases A2/chemistry , Viral Proteins/metabolismABSTRACT
Two new structures of the N-terminal domain of the main replication protein, NS1, of human parvovirus B19 (B19V) are presented here. This domain (NS1-nuc) plays an important role in the "rolling hairpin" replication of the single-stranded B19V DNA genome, recognizing origin of replication sequences in double-stranded DNA, and cleaving (i.e., nicking) single-stranded DNA at a nearby site known as the terminal resolution site (trs). The three-dimensional structure of NS1-nuc is well conserved between the two forms, as well as with a previously solved structure of a sequence variant of the same domain; however, it is shown here at a significantly higher resolution (2.4 Å). Using structures of NS1-nuc homologues bound to single- and double-stranded DNA, models for DNA recognition and nicking by B19V NS1-nuc are presented that predict residues important for DNA cleavage and for sequence-specific recognition at the viral origin of replication. IMPORTANCE The high-resolution structure of the DNA binding and cleavage domain of the main replicative protein, NS1, from the human-pathogenic virus human parvovirus B19 is presented here. Included also are predictions of how the protein recognizes important sequences in the viral DNA which are required for viral replication. These predictions can be used to further investigate the function of this protein, as well as to predict the effects on viral viability due to mutations in the viral protein and viral DNA sequences. Finally, the high-resolution structure facilitates structure-guided drug design efforts to develop antiviral compounds against this important human pathogen.
Subject(s)
Models, Molecular , Parvovirus B19, Human , Viral Nonstructural Proteins , DNA, Viral/genetics , Endonucleases/chemistry , Endonucleases/genetics , Humans , Parvovirus B19, Human/genetics , Parvovirus B19, Human/metabolism , Protein Domains , Protein Structure, Tertiary , Viral Nonstructural Proteins/chemistry , Virus Replication/geneticsABSTRACT
A total of 244 patients with hereditary haemolytic anaemias (HHA) were screened for acute symptomatic human parvovirus B19 infection (HPV-B19) in a prospective study. To assess the risks associated with HPV-B19 infection, patients were classified into Group I and Group II according to presence or absence (symptoms, signs and specific serology) of acute HPV-B19 infection respectively. In all, 131 (53·7%) patients had ß-thalassaemia, 75 (30·7%) hereditary spherocytosis (HS), 27 (11·1%) sickle cell anaemia (SCA) and 11 (4·5%) glucose-6-phosphate dehydrogenase (G6PD) deficiency. Of 33 (13·5%) patients who presented with symptomatic HPV-B19 infection, 19 (57·5%) had HS, nine (27·3%) had ß-thalassaemia and five (15·2%) had SCA. In Group I, there were significant differences in the mean white blood cell, red blood cell and platelet counts, haemoglobin concentration, total bilirubin (TB), alanine aminotransferase, aspartate aminotransferase and serum creatinine (all P < 0·001) compared to Group II. In all, 27 (81·8%) patients had arthropathy and bone marrow failure (BMF); 13 (39·4%) had acute kidney injury (AKI), more in SCA (80%); and 12 (36·4%) patients had hepatitis, more in HS (66·8%). Five (15·2%) patients with HS had BMF, AKI, nervous system involvement and extreme hyperbilirubinaemia (TB range 26·3-84·7 mg/dl). Five (15·2%) patients had haemophagocytic syndrome. Two patients with HS combined with Type-I autoimmune hepatitis presented with transient BMF. Complete recovery or stabilisation was noted at 12 months in every patient except for one patient with SCA who died during the infection. HPV-B19 must be suspected and screened in patients with HHA with typical and atypical presentations with careful follow-up.
Subject(s)
Anemia, Hemolytic, Congenital , Bone Marrow Failure Disorders , Erythema Infectiosum , Hepatitis , Hyperbilirubinemia , Parvovirus B19, Human/metabolism , Acute Disease , Adolescent , Adult , Anemia, Hemolytic, Congenital/blood , Anemia, Hemolytic, Congenital/mortality , Anemia, Hemolytic, Congenital/virology , Bone Marrow Failure Disorders/blood , Bone Marrow Failure Disorders/mortality , Bone Marrow Failure Disorders/virology , Child , Erythema Infectiosum/blood , Erythema Infectiosum/mortality , Female , Follow-Up Studies , Hepatitis/blood , Hepatitis/mortality , Hepatitis/virology , Humans , Hyperbilirubinemia/blood , Hyperbilirubinemia/mortality , Hyperbilirubinemia/virology , Male , Middle Aged , Prospective StudiesABSTRACT
During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5'-GUA AAG CUA CGG GAC GGU-3'), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [KD ] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.
Subject(s)
Gene Expression Regulation, Viral , Introns , Nerve Tissue Proteins/metabolism , Parvovirus B19, Human/genetics , RNA Splicing , RNA-Binding Proteins/metabolism , Viral Nonstructural Proteins/genetics , Cells, Cultured , Erythroid Precursor Cells/virology , Genome, Viral , Hematopoietic Stem Cells , Humans , Nerve Tissue Proteins/genetics , Parvovirus B19, Human/metabolism , Protein Binding , RNA Precursors/genetics , RNA Precursors/metabolism , RNA-Binding Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
OBJECTIVE: In the multicentre Haemoglobinopathy Blood Surveillance Project, to evaluate the seroprevalence of parvovirus B19 and DNA viral load in sickle cell disease (SCD). BACKGROUND: Although the epidemiology of parvovirus B19 seropositivity in SCD has been well documented, there are few studies that have assessed possible persistent parvovirus DNAemia and associated risk factors including blood transfusion. METHODS: A qualitative analysis of parvovirus B19 serology using ELISA and quantitative parvovirus B19 DNA by RT-PCR was performed in patients with SCD. RESULTS: Of 322 patients, 113 (35%) were parvovirus IgG positive and 119 (37%) were IgM positive at enrolment. The prevalence of IgG positivity increased with age. 71/322 (22%) were parvovirus DNA positive at enrolment with a mean viral load of 15 227 ± 55 227 SD. (range 72-329 238 IU/mL). Patients who were positive for parvovirus B19 DNA received a significantly higher red blood cell transfusion volume in the prior year compared to patients who were negative (mean RBC volume = 8310 mL vs 5435 mL, respectively; P = .0073). Seventy-seven patients had follow-up testing approximately 1 year after enrolment and 11/28 (39%) patients had persistently positive IgM. CONCLUSION: Further studies are needed to better understand the natural history of parvovirus B19 infection in SCD especially in relation to RBC transfusion as a risk factor, as well as disease outcome and severity.
Subject(s)
Anemia, Sickle Cell , Antibodies, Viral/blood , DNA, Viral/blood , Erythema Infectiosum , Immunoglobulin G/blood , Immunoglobulin M/blood , Parvovirus B19, Human/metabolism , Adolescent , Adult , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/epidemiology , Anemia, Sickle Cell/therapy , Anemia, Sickle Cell/virology , Centers for Disease Control and Prevention, U.S. , Child , Child, Preschool , Erythema Infectiosum/blood , Erythema Infectiosum/epidemiology , Female , Humans , Infant , Infant, Newborn , Male , Risk Factors , United StatesABSTRACT
Human parvovirus B19 (B19V) infection has symptoms similar to those of antiphospholipid syndrome (APS). Antibodies against B19VVP1 unique region (VP1u) exhibit activity similar to that of antiphospholipid antibodies (aPLs) by inducing vascular endothelial cell adhesion factors and APSlike syndrome. Previous studies have identified an effect of aPLs on angiogenesis. However, little is understood regarding the effect of antiB19VVP1u antibodies on angiogenesis. The present study investigated the effects of antiB19VVP1u antibodies on the expression of adhesion molecules and angiogenic signaling using an aPLinduced human umbilical vein endothelial cell (HUVEC) model, and trypan blue staining and western blotting. The effect of B19VVP1u antibodies on vascular endothelial growth factor (VEGF) expression in A549 cells, another wellknown model used to study angiogenesis, was also examined. Significantly higher intracellular adhesion molecule 1 expression was observed following treatments with 10% fetal calf serum (FCS), aPL immunoglobulin G (IgG), B19VVP1u IgG or B19VNS1 IgG, compared with in the normal human (NH) IgGtreated cells. Conversely, significantly higher vascular cellular adhesion molecule 1 was only detected in HUVECs treated with B19VVP1u IgG. Significantly increased integrin ß1 was detected in HUVECs treated with aPL IgG or B19VVP1u IgG, whereas no difference in integrin ß1 was observed in those treated with 10% FCS, NH IgG or B19VNS1 IgG. No difference in AKTmTORS6 ribosomal protein (S6RP) signaling was observed in HUVECs treated with B19VP1u IgG or B19VNS1 IgG, compared with NH IgGtreated cells. Significantly higher human inducible factor1α was detected in HUVECs treated with 10% FCS, aPL IgG, B19VVP1u IgG or B19VNS1 IgG, compared with in NH IgGtreated cells. However, there was no difference in the level of VEGF observed among HUVECs treated with NH IgG, B19VVP1u IgG or B19VNS1 IgG. Notably, no difference in VEGF level was observed in A549 cells treated with NH IgG, aPL IgG, B19VVP1u IgG or B19VNS1 IgG. These findings suggest that antiB19VVP1u antibodies may serve a role in activating adhesion molecules, but not in AKTmTORS6RP signaling.
Subject(s)
Antibodies, Viral/pharmacology , Antiphospholipid Syndrome , Erythema Infectiosum , Immunoglobulin G/pharmacology , Neovascularization, Physiologic/drug effects , Parvovirus B19, Human/metabolism , Signal Transduction/drug effects , A549 Cells , Antibodies, Antiphospholipid/pharmacology , Antiphospholipid Syndrome/metabolism , Antiphospholipid Syndrome/pathology , Antiphospholipid Syndrome/virology , Erythema Infectiosum/metabolism , Erythema Infectiosum/pathology , Human Umbilical Vein Endothelial Cells , HumansABSTRACT
OBJECTIVE: Blood-derived products from patient with hemophilia treated by factor VIII concentrates are potential sources of transfusion-transmitted infections, including human immunodeficiency virus, hepatitis, human pegivirus-1 (HPgV-1), B19 virus, and also human hepegivirus-1 (HHpgV-1). In the current study, we investigated the impact of blood transfusion on the prevalence of HHpgV-1, HPgV-1, and B19 virus in plasma of Iranian patient with hemophilia after direct-acting antiviral treatment of hepatitis C virus (HCV) infections for the first time. MATERIALS AND METHODS: A total of 170 patients with hemophilia who received direct-acting antivirals were enrolled in this study. Among them, 92 patients had a history of blood transfusion. The presence of HHpgV-1, HPgV-1, and B19 virus was detected by nested polymerase chain reaction analysis using the conserved primers. The plasmids harboring 5'-UTR and NS3 were used as positive controls for HPgV-1 and HHpgV-1, respectively. RESULTS: Our data identified 3 individuals with HHpgV-1 viremia (1.76%), 11 individuals with HPgV-1 viremia (6.47%), and 33 individuals with B19 viremia (19.4%). All patients were negative for hepatitis B virus, human immunodeficiency virus, and HCV infections. These findings indicated lower transmissibility or higher rates of virus clearance for HHpgV-1, HPgV-1, and B19 virus as compared with other bloodborne human flaviviruses such as HCV. However, the prevalence of B19 virus was significantly higher than the other 2 viruses. CONCLUSION: In general, these findings showed that the history of blood transfusion could increase the risk of viral transmission of bloodborne viruses among patient with hemophilia.
Subject(s)
Blood Transfusion , DNA, Viral/blood , Erythema Infectiosum/blood , Hemophilia A/blood , Hepacivirus/metabolism , Hepatitis C/blood , Parvovirus B19, Human/metabolism , Adolescent , Adult , Child , Child, Preschool , Cross-Sectional Studies , Erythema Infectiosum/epidemiology , Erythema Infectiosum/etiology , Female , Hemophilia A/epidemiology , Hemophilia A/therapy , Hemophilia A/virology , Hepatitis C/epidemiology , Hepatitis C/therapy , Hepatitis C/virology , Humans , Iran/epidemiology , Male , Middle Aged , PrevalenceABSTRACT
Adenoviral viral vectors have been widely used for gene-based therapeutics, but commonly used serotype 5 shows poor transduction efficiency into hematopoietic cells. In this study, we aimed to generate a recombinant adenovirus serotype 5 (rAd5) vector that has a high efficiency in gene transfer to megakaryocytic leukemic cells with anticancer potential. We first modified the rAd5 backbone vector with a chimeric fiber gene of Ad5 and Ad11p (rAd5F11p) to increase the gene delivery efficiency. Then, the nonstructural protein NS1 of human parvovirus B19 (B19V), which induces cell cycle arrest at the G2/M phase and apoptosis, was cloned into the adenoviral shuttle vector. As the expression of parvoviral NS1 protein inhibited Ad replication and production, we engineered the cytomegalovirus (CMV) promoter, which governs NS1 expression, with two tetracycline operator elements (TetO2). Transfection of the rAd5F11p proviral vectors in Tet repressor-expressing T-REx-293 cells produced rAd in a large quantity. We further evaluated this chimeric rAd5F11p vector in gene delivery in human leukemic cells, UT7/Epo-S1. Strikingly, the novel rAd5F11p-B19NS1-GFP vector, exhibited a transduction efficiency much higher than the original vector, rAd5-B19NS1-GFP, in UT7/Epo-S1 cells, in particular, when they were transduced at a relatively low multiplicity of infection (100 viral genome copies/cell). After the transduction of rAd5F11p-B19NS1-GFP, over 90% of the UT7/Epo-S1 cells were arrested at the G2/M phase, and approximately 40%-50% of the cells were undergoing apoptosis, suggesting the novel rAd5F11P-B19NS1-GFP vector holds a promise in therapeutic potentials of megakaryocytic leukemia.
Subject(s)
Adenoviridae/genetics , Gene Transfer Techniques , Genetic Vectors/genetics , Leukemia, Megakaryoblastic, Acute/therapy , Parvovirus B19, Human/genetics , Viral Nonstructural Proteins/genetics , Adenoviridae/metabolism , Apoptosis , Cell Cycle Checkpoints , Gene Expression , Gene Transfer Techniques/instrumentation , Genetic Vectors/metabolism , Humans , Leukemia, Megakaryoblastic, Acute/physiopathology , Parvovirus B19, Human/metabolism , Viral Nonstructural Proteins/metabolismABSTRACT
Breast cancer is a worldwide health problem, and the complexity of the disease, as well as the lack of treatment specificity, generates an urgent need for developing prophylactic and therapeutic measures. Searching for novel epitope-based approaches able to induce tumour immunity, we designed virus-like particles (VLPs) derived from Human parvovirus B19 assembled of chimeric VP2 proteins displaying two epitopes from the insulin-like growth factor-1 receptor (IGF-1R). Here, we present the generation of two chimeric VP2s that retain the stability, solubility and conditions of purification and assembly of the native VP2. We generated versatile chimeric multiepitope anti-cancer vaccine candidates, which prevented and delayed tumour growth when used in a prophylactic scheme of 4 weekly immunizations prior to 4T1 cell inoculation in female BALB/c mice. The presence of specific antibodies against the displayed epitopes suggests their participation in the protective effect; in contrast, no significant proliferative T-cell responses were recorded following stimulation by specific epitopes. The results comprise an approach whereby fusing desired epitopes from cancer to the N-terminus of B19 VP2 protein can generate a library of chimeric VP2-desired epitopes for further assembly in a designed and personalized epitope delivery system.
Subject(s)
Breast Neoplasms/prevention & control , Epitopes/metabolism , Parvovirus B19, Human/metabolism , Receptor, IGF Type 1/immunology , Vaccines, Virus-Like Particle/administration & dosage , Animals , Apoptosis , Breast Neoplasms/immunology , Cancer Vaccines/administration & dosage , Cancer Vaccines/immunology , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Line, Tumor , Cell Survival , Epitopes/genetics , Female , Humans , Immunization , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Parvovirus B19, Human/genetics , Treatment Outcome , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunologyABSTRACT
Lytic infection of human parvovirus B19 (B19V) takes place exclusively in human erythroid progenitor cells of bone marrow and fetal liver, which disrupts erythropoiesis. During infection, B19V expresses three nonstructural proteins (NS1, 11-kDa, and 7.5-kDa) and two structural proteins (VP1 and VP2). While NS1 is essential for B19V DNA replication, 11-kDa enhances viral DNA replication significantly. In this study, we confirmed the enhancement role of 11-kDa in viral DNA replication and elucidated the underlying mechanism. We found that 11-kDa specially interacts with cellular growth factor receptor-bound protein 2 (Grb2) during virus infection and in vitro We determined a high affinity interaction between 11-kDa and Grb2 that has an equilibrium dissociation constant (KD ) value of 18.13 nM. In vitro, one proline-rich motif was sufficient for 11-kDa to sustain a strong interaction with Grb2. In consistence, in vivo during infection, one proline-rich motif was enough for 11-kDa to significantly reduce phosphorylation of extracellular signal-regulated kinase (ERK). Mutations of all three proline-rich motifs of 11-kDa abolished its capability to reduce ERK activity and, accordingly, decreased viral DNA replication. Transduction of a lentiviral vector encoding a short hairpin RNA (shRNA) targeting Grb2 decreased the expression of Grb2 as well as the level of ERK phosphorylation, which resulted in an increase of B19V replication. These results, in concert, indicate that the B19V 11-kDa protein interacts with cellular Grb2 to downregulate ERK activity, which upregulates viral DNA replication.IMPORTANCE Human parvovirus B19 (B19V) infection causes hematological disorders and is the leading cause of nonimmunological fetal hydrops during pregnancy. During infection, B19V expresses two structural proteins, VP1 and VP2, and three nonstructural proteins, NS1, 11-kDa, and 7.5-kDa. While NS1 is essential, 11-kDa plays an enhancing role in viral DNA replication. Here, we elucidated a mechanism underlying 11-kDa protein-regulated B19V DNA replication. 11-kDa is tightly associated with cellular growth factor receptor-bound protein 2 (Grb2) during infection. In vitro, 11-kDa interacts with Grb2 with high affinity through three proline-rich motifs, of which at least one is indispensable for the regulation of viral DNA replication. 11-kDa and Grb2 interaction disrupts extracellular signal-regulated kinase (ERK) signaling, which mediates upregulation of B19V replication. Thus, our study reveals a novel mechanism of how a parvoviral small nonstructural protein regulates viral DNA replication by interacting with a host protein that is predominately expressed in the cytoplasm.
Subject(s)
GRB2 Adaptor Protein/metabolism , Parvoviridae Infections/metabolism , Parvovirus B19, Human/physiology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Amino Acid Motifs , Binding Sites , DNA Replication , Humans , Molecular Weight , Mutation , Parvovirus B19, Human/metabolism , Phosphorylation , Proline/metabolism , Protein Binding , Virus ReplicationABSTRACT
Both human parvovirus B19 (B19V) and human bocavirus (HBoV) are known to be important human pathogens of the Parvoviridae family. Our earlier investigation demonstrated that both B19V-VP1u and HBoV-VP1u have a significantly disruptive effect on tight junctions (TJs) in A549 cells, implying the essential role of parvovirus in airway infection and lung injury. However, no direct evidence that B19V-VP1u and HBoV-VP1u induce lung injury exists. The present study further investigates the induction of lung injury by B19V-VP1u and HBoV-VP1u in naïve Balb/c mice following subcutaneous injection of PBS, recombinant B19V-VP1u or HBoV-VP1u. The experimental results reveal significantly increased activity, protein expression and ratio of matrix metalloproteinase-9 (MMP-9) to MMP-2 in Balb/c mice that received B19V-VP1u or HBoV-VP1u compared to those that received PBS. Significantly higher levels of inflammatory cytokines, including IL-6 and IL-1ß, and greater lymphocyte infiltration in lung tissue sections were detected in mice that received B19V-VP1u or HBoV-VP1u. Additionally, significantly increased levels of phosphorylated p65 (NF-κB) and MAPK signaling proteins were observed in lung tissue of mice that received B19V-VP1u or HBoV-VP1u compared to those of mice that received PBS. These findings demonstrate for the first time that B19V-VP1u and HBoV-VP1u proteins induce lung inflammatory reactions through p65 (NF-κB) and MAPK signaling.
Subject(s)
Human bocavirus/metabolism , Lung Injury/pathology , Parvovirus B19, Human/metabolism , Viral Proteins/metabolism , Animals , Female , Humans , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Lung/immunology , Lung/metabolism , Lung/pathology , Lung Injury/veterinary , Lung Injury/virology , Lymphocytes/cytology , Lymphocytes/immunology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Tight Junctions/metabolism , Transcription Factor RelA/metabolism , Viral Proteins/geneticsABSTRACT
Human parvovirus B19 (B19) and human bocavirus 1 (HBoV) are the only known pathogenic parvoviruses, and are responsible for a variety of diseases in human beings. Mounting evidence indicates a strong association between B19 infection and cardiac disorders including myocarditis, dilated cardiomyopathy and heart failure. However, very limited information about the role of HBoV in cardiac disorders is known. To elucidate the effects of B19 and HBoV on cardiac disorders, we expressed EGFPconjugate constructs of B19VP1 unique region (VP1u) and HBoVVP1u, along with the mutants EGFPB19VP1uD175A and EGFPHBoVVP1uV12A, in H9c2 cells by stable transfection. The protein expression levels of EGFP, EGFPB19VP1u, EGFPB19VP1uD175A, EGFPHBoVVP1u and EGFPHBoVVP1uV12A in H9c2 cells were observed under a fluorescence microscope and confirmed by western blotting. Secreted phospholipase A2 (sPLA2) activity was detected in B19VP1u and HBoVVP1u but not B19VP1uD175A and HBoVVP1uV12A recombinant proteins. Significantly higher expression levels of MCP2 and IP10 mRNA were detected in H9c2 cells that were transfected with pEGFPB19VP1u, compared with in those cells transfected with pEGFPHBoVVP1u, pEGFPB19VP1uD175A or pEGFPHBoVVP1uV12A. Significantly higher protein levels of IL1ß and IL6 were detected in H9c2 cells transfected with pEGFPB19VP1u or pEGFPHBoVVP1u, compared with in those cells transfected with pEGFPB19VP1uD175A or pEGFPHBoVVP1uV12A. Notably, significantly higher expression of both TNFα and NFκB was observed only in H9c2 cells transfected with pEGFPB19VP1u, but not in those cells transfected with pEGFPHBoVVP1u, pEGFPB19VP1uD175A or pEGFPHBoVVP1uV12A. These findings, to our knowledge for the first time, reveal the difference between B19VP1u and HBoVVP1u in H9c2 cells and provide insight into the roles of B19VP1u and HBoVVP1u in the pathogenesis of cardiac inflammation.
Subject(s)
Capsid Proteins/metabolism , Human bocavirus/metabolism , Inflammation/pathology , Myocytes, Cardiac/metabolism , Parvovirus B19, Human/metabolism , Animals , Chemokine CCL8/genetics , Chemokine CCL8/metabolism , Chemokine CXCL10/genetics , Chemokine CXCL10/metabolism , Cytokines/metabolism , Humans , Inflammation Mediators/metabolism , Mice , NF-kappa B/metabolism , Phospholipases A2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolismABSTRACT
The natural properties of virus-like particles (VLPs), like their nanometric size, polyvalence, monodispersity and biocompatibility, had called the attention of scientists from different fields. VLPs constitute an excellent platform for the development nanomaterials with a broad spectrum of applications, ranging from physics of soft matter to the development of vaccines and biological nanocarriers. To expand the repertoire of functions of VLPs, they can be decorated with different molecules. In this research, the α-glucosidase Ima1p of Saccharomyces cerevisiae was attached to the surface of in vitro assembled VLPs of parvovirus B19, by using the SpyTag/SpyCatcher system. The resulting particles were structurally characterized displaying a noticeable increase in size compared to the non-decorated VLPs. The study of the biochemical properties of the coupled enzyme indicate that it increased its Vmax by three-fold toward p-nitrophenyl-α-D-glucopyranoside (p-NPG) as substrate. In addition, the linked enzyme displayed a notorious 10⯰C shift in its optimal temperature, from 35⯰C for the non-attached enzyme, to 45⯰C for the enzyme attached to VLPs. The decorated VLPs were also able to act on glycogen; therefore, these particles may be further developed as part of the therapy for treatment of lysosomal storage diseases derived from defects in the human acid α-glucosidase.
Subject(s)
Capsid Proteins/chemistry , Drug Delivery Systems/methods , Nanoparticles/chemistry , Nanoparticles/metabolism , Parvovirus B19, Human/chemistry , alpha-Glucosidases/chemistry , Capsid Proteins/genetics , Capsid Proteins/metabolism , Glucosides/metabolism , Glycogen/metabolism , Hydrogen-Ion Concentration , Kinetics , Nanoparticles/ultrastructure , Particle Size , Parvovirus B19, Human/genetics , Parvovirus B19, Human/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Temperature , Virus Assembly , alpha-Glucosidases/genetics , alpha-Glucosidases/metabolismABSTRACT
Human parvovirus B19 (B19V) causes myriads of clinical diseases; however, owing to lack of awareness and undetermined clinical impact, it has failed to become a virus pathogen of global concern. Cryptically, B19V causes significant morbidity and mortality. Half of the world population and 60 per cent of Indians are known to be serologically naive and are at risk of acquiring B19V infections. Cumulatively, our data showed 21.3 per cent B19V-infected patients with juvenile chronic arthropathy, recurrent abortions, multi-transfused thalassaemia and leukaemia. In addition, B19V-infected cases that ended fatally included patients with pure red cell aplasia, fulminant hepatitis and haemophagocytic syndrome. Novel clinical associations of B19V observed were amegakaryocytic thrombocytopaenia, myositis and non-occlusive ischaemic gangrene of bowel. B19V possesses multiple receptors which are distributed widely in human tissues. Vascular endothelial cell infection by B19V causes endothelialitis and vasculitic injuries besides antibody-dependent enhancement which empowered B19V to cause multiorgan diseases. Owing to lack of suitable animal model for B19V, true causal role remains to be determined, but numerous reports on B19V infections substantiate a causal role in multiorgan diseases. Hence, B19V infections need to be recognized, investigated and treated besides making efforts on vaccine developments.
Subject(s)
Parvoviridae Infections/complications , Parvoviridae Infections/epidemiology , Parvovirus B19, Human/pathogenicity , Cardiovascular Diseases/epidemiology , Cardiovascular Diseases/virology , Female , Hematologic Diseases/epidemiology , Hematologic Diseases/virology , Humans , India/epidemiology , Kidney Diseases/epidemiology , Kidney Diseases/virology , Liver Diseases/epidemiology , Liver Diseases/virology , Nervous System Diseases/epidemiology , Nervous System Diseases/virology , Parvoviridae Infections/transmission , Parvovirus B19, Human/metabolism , Pregnancy , Pregnancy Complications/epidemiology , Seroepidemiologic StudiesABSTRACT
Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication.IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication.
Subject(s)
Cell Division , DNA Replication , Host-Pathogen Interactions , Parvoviridae Infections/virology , Parvovirus B19, Human/genetics , Parvovirus B19, Human/metabolism , Virus Replication , Bromodeoxyuridine/metabolism , CD36 Antigens/analysis , CD36 Antigens/metabolism , Cell Cycle , Cell Cycle Checkpoints , Cell Line , DNA Damage , DNA Polymerase III , DNA Polymerase beta , DNA Repair , DNA, Single-Stranded/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/virology , Fetal Death , Gene Expression Regulation, Viral/physiology , Genome, Viral , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/physiology , Humans , Parvovirus B19, Human/pathogenicity , Phosphorylation , Protein Interaction Maps , Red-Cell Aplasia, Pure/virology , Replication Protein A/genetics , S Phase , Transcriptome , Viremia/virologyABSTRACT
Decoration of virus-like particles (VLPs) expands the repertory of functions these particles can display. In the last years, VLPs have successfully been used as scaffolds to present different molecules, frequently through the specific reaction of chemical groups on the surface of the particles, or by protein engineering when the presentation of peptides or proteins is the primary goal. VLPs of parvovirus B19 (B19V), have been previously produced in vitro and its stability and ability to assemble into hybrid particles composed of wild-type and chimeric proteins evidenced their potential as research tools. Herein, we report the presentation of functional proteins on the surface of B19V VLPs, through the fusion of the gene coding for the heterologous protein within the gene coding for the structural protein VP2. Two model proteins were used for the construction of chimeras, a lipase from Bacillus pumilus (BplA) and the enhanced green fluorescent protein (EGFP). Both chimeras were folded and successfully assembled in vitro into VLPs. While the BplA chimera exhibited esterase activity, the chimera of EGFP showed no fluorescence. We replaced the EGFP by its fast-folding derivative "super folder GFP" (sfGFP) flanked by larger linkers to increase its movement freedom, which resulted in fluorescent protein able to assemble fluorescent VLPs. These results expand the toolbox for VLP decoration as well as for the construction of new nanobiomaterials.