showing 56 related works from this author
Cytoplasmic Parvovirus Capsids Recruit Importin Beta for Nuclear Delivery
2019
Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps, including nuclear import, are inefficient, limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: the classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family or the direct attachment of the capsid to the nuclear pore complex leading to the local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analyses combined with coimmunoprecipitation, we show that infection requires importin β-mediated access to the nuclear pore complex and …
Computational Modeling of Protein Dynamics in Eukaryotic Cells
2012
Proteins have important functions inside the cell, traveling diffusively or being actively transported to various cellular sites where their activity is needed. Protein motion in the cellular environment is therefore an important topic to understand. However, the cell provides a very complex environment for that motion, which poses problems especially for any modeling effort designed to interpret experimentally observed features. So as to gain a realistic picture of protein dynamics inside the cell, we have recently introduced advanced numerical methods for describing that dynamics [1]. The starting point is an accurate numerical duplicate of the cell determined by LSCM, which can be used a…
Compact Cell Imaging Device (CoCID) provides insights into the cellular origins of viral infections
2021
The overall CoCID concept is centred on providing virologists with a next-generation imaging device, which, through increased penetration and depth of focus, as well as through high natural contrast and sensitivity to organelle density (including virus-related organelles), will produce higher-fidelity ultrastructural images of whole intact cells. These insights will, in turn, help increase our understanding of the links between the structural reorganisation of cells and the mechanisms of viral entry, replication, assembly, and egress in cells. CoCID will provide this valuable imaging capability in the form of a compact lab-scale device that will greatly improve the accessibility of soft X-r…
Protein diffusion in mammalian cell cytoplasm.
2011
We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribut…
Epigenetic upregulation of endogenous VEGF-A reduces myocardial infarct size in mice.
2014
“Epigenetherapy” alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral in vivo delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed in vivo that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upr…
Culture medium induced vimentin reorganization associates with enhanced baculovirus-mediated gene delivery.
2009
Baculoviruses can express transgenes under mammalian promoters in a wide range of vertebrate cells. However, the success of transgene expression is dependent on both the appropriate cell type and culture conditions. We studied the mechanism behind the substantial effect of the cell culture medium on efficiency of the baculovirus transduction in different cell lines. We tested six cell culture mediums; the highest transduction efficiency was detected in the presence of RPMI 1640 medium. Vimentin, a major component of type III intermediate filaments, was reorganized in the optimized medium, which associated with enhanced nuclear entry of baculoviruses. Accordingly, the phosphorylation pattern…
Intracellular route of canine parvovirus entry.
1998
ABSTRACT The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18°C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membr…
Parvovirus nonstructural protein 2 interacts with chromatin-regulating cellular proteins
2022
Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodel…
Tumor targeting of baculovirus displaying a lymphatic homing peptide.
2008
Background Tumor-associated cells and vasculature express attractive molecular markers for site-specific vector targeting. To attain tumor-selective tropism, we recently developed a baculovirus vector displaying the lymphatic homing peptide LyP-1, originally identified by ex vivo/in vivo screening of phage display libraries, on the viral envelope by fusion to the transmembrane anchor of vesicular stomatitis virus G-protein. Methods In the present study, we explored the specificity and kinetics of viral binding and internalization as well as in vivo tumor homing of the LyP-1 displaying virus to elucidate the applicability of baculovirus for targeted therapies. Results We demonstrated that th…
Extinct type of human parvovirus B19 persists in tonsillar B cells
2017
Parvovirus B19 (B19V) DNA persists lifelong in human tissues, but the cell type harbouring it remains unclear. We here explore B19V DNA distribution in B, T and monocyte cell lineages of recently excised tonsillar tissues from 77 individuals with an age range of 2–69 years. We show that B19V DNA is most frequent and abundant among B cells, and within them we find a B19V genotype that vanished from circulation >40 years ago. Since re-infection or re-activation are unlikely with this virus type, this finding supports the maintenance of pathogen-specific humoral immune responses as a consequence of B-cell long-term survival rather than continuous replenishment of the memory pool. Moreover, we …
A neutralizing antibody against human DNA polymerase epsilon inhibits cellular but not SV40 DNA replication.
1999
The contribution of human DNA polymerase epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA polymerase alpha. Contrary to the antibody against DNA polymerase alpha, antibody K18 against DNA polymerase epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA polymerase e…
Clathrin-independent entry of baculovirus triggers uptake of E. coli in non-phagocytic human cells
2008
The prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus, an insect pathogen, holds great potential as a gene therapy vector. To develop transductional targeting and gene delivery by baculovirus, we focused on characterizing the nature and regulation of its uptake in human cancer cells. Baculovirus entered the cells along fluid-phase markers from the raft areas into smooth-surfaced vesicles devoid of clathrin. Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake. The internalization and nuclear uptake was, however, affected by mutants o…
Nuclear entry and egress of parvoviruses.
2022
Parvoviruses are small non-enveloped single-stranded DNA viruses, which depend on host cell nuclear transcriptional and replication machinery. After endosomal exposure of nuclear localization sequence and a phospholipase A2 domain on the capsid surface, and escape into the cytosol, parvovirus capsids enter the nucleus. Due to the small capsid diameter of 18–26 nm, intact capsids can potentially pass into the nucleus through nuclear pore complexes (NPCs). This might be facilitated by active nuclear import, but capsids may also follow an alternative entry pathway that includes activation of mitotic factors and local transient disruption of the nuclear envelope. The nuclear entry is followed b…
Herpes simplex virus 1 induces egress channels through marginalized host chromatin
2016
AbstractLytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. We used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, co…
Exploitation of Microtubule Cytoskeleton and Dynein during Parvoviral Traffic toward the Nucleus
2003
ABSTRACT Canine parvovirus (CPV), a model virus for the study of parvoviral entry, enters host cells by receptor-mediated endocytosis, escapes from endosomal vesicles to the cytosol, and then replicates in the nucleus. We examined the role of the microtubule (MT)-mediated cytoplasmic trafficking of viral particles toward the nucleus. Immunofluorescence and immunoelectron microscopy showed that capsids were transported through the cytoplasm into the nucleus after cytoplasmic microinjection but that in the presence of MT-depolymerizing agents, viral capsids were unable to reach the nucleus. The nuclear accumulation of capsids was also reduced by microinjection of an anti-dynein antibody. More…
Subcellular localization of bacteriophage PRD1 proteins in Escherichia coli
2014
Bacteria possess an intricate internal organization resembling that of the eukaryotes. The complexity is especially prominent at the bacterial cell poles, which are also known to be the preferable sites for some bacteriophages to infect. Bacteriophage PRD1 is a well-known model serving as an ideal system to study structures and functions of icosahedral internal membrane-containing viruses. Our aim was to analyze the localization and interactions of individual PRD1 proteins in its native host Escherichia coli. This was accomplished by constructing a vector library for production of fluorescent fusion proteins. Analysis of solubility and multimericity of the fusion proteins, as well as their …
Sodium channels enable fast electrical signaling and regulate phagocytosis in the retinal pigment epithelium
2019
Background Voltage-gated sodium (Nav) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Nav channels in several non-excitable cells, such as astrocytes and macrophages, demonstrating that the roles of these channels are more diverse than was previously thought. Despite the earlier discoveries, the presence of Nav channel-mediated currents in the cells of retinal pigment epithelium (RPE) has been dismissed as a cell culture artifact. We challenge this notion by investigating the presence and possible role of Nav channels in RPE both ex vivo and in vitro. Results Our work demonstrates that several subtypes of Nav cha…
Magnetic resonance imaging of viral particle biodistribution in vivo
2006
We describe here a technique for the visualization of viral vector delivery by magnetic resonance imaging (MRI) in vivo. By conjugating avidin-coated baculoviral vectors (Baavi) with biotinylated ultra-small superparamagnetic iron oxide particles (USPIO), we are able to produce vector-related MRI contrast in the choroid plexus cells of rat brain in vivo over a period of 14 days. Ten microlitres of 2.5 x 10(10) PFU/ml nuclear-targeted LacZ-encoding Baavi with bUSPIO coating was injected into rat brain ventricles and visualized by MRI at 4.7 T. As baculoviruses exhibit restricted cell-type specificity in the rat brain, altered MRI contrast was detected in the choroid plexus of the injected ve…
Reactive Self-Assembly and Specific Cellular Delivery of NCO-sP(EO-stat-PO)-Derived Nanogels
2018
This study presents the reactive self-assembly of isocyanate functional and amphiphilic six-arm, star-shaped polyether prepolymers in water into nanogels. Intrinsic molecular amphiphilicity, mainly driven by the isophorone moiety at the distal endings of the star-shaped molecules, allows for the preparation of spherical particles with an adjustable size of 100-200 nm by self-assembly and subsequent covalent cross-linking without the need for organic solvents or surfactants. Covalent attachment of a fluorescence dye and either the cell-penetrating TAT peptide or a random control peptide sequence shows that only TAT-labeled nanogels are internalized by HeLa cells. The nanogels thus specifical…
Chromatin organization regulates viral egress dynamics.
2017
Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walk modelling of herpes s…
Distribution and dynamics of transcription-associated proteins during parvovirus infection.
2012
Canine parvovirus (CPV) infection leads to reorganization of nuclear proteinaceous subcompartments. Our studies showed that virus infection causes a time-dependent increase in the amount of viral nonstructural protein NS1 mRNA. Fluorescence recovery after photobleaching showed that the recovery kinetics of nuclear transcription-associated proteins, TATA binding protein (TBP), transcription factor IIB (TFIIB), and poly(A) binding protein nuclear 1 (PABPN1) were different in infected and noninfected cells, pointing to virus-induced alterations in binding dynamics of these proteins. peerReviewed
Distribution and Dynamics of Transcription-Associated Proteins during Parvovirus Infection
2012
ABSTRACT Canine parvovirus (CPV) infection leads to reorganization of nuclear proteinaceous subcompartments. Our studies showed that virus infection causes a time-dependent increase in the amount of viral nonstructural protein NS1 mRNA. Fluorescence recovery after photobleaching showed that the recovery kinetics of nuclear transcription-associated proteins, TATA binding protein (TBP), transcription factor IIB (TFIIB), and poly(A) binding protein nuclear 1 (PABPN1) were different in infected and noninfected cells, pointing to virus-induced alterations in binding dynamics of these proteins.
Release of canine parvovirus from endocytic vesicles
2003
Canine parvovirus (CPV) is a small nonenveloped virus with a single-stranded DNA genome. CPV enters cells by clathrin-mediated endocytosis and requires an acidic endosomal step for productive infection. Virion contains a potential nuclear localization signal as well as a phospholipase A(2) like domain in N-terminus of VP1. In this study we characterized the role of PLA(2) activity on CPV entry process. PLA(2) activity of CPV capsids was triggered in vitro by heat or acidic pH. PLA(2) inhibitors inhibited the viral proliferation suggesting that PLA(2) activity is needed for productive infection. The N-terminus of VP1 was exposed during the entry, suggesting that PLA(2) activity might have a …
Concepts to Reveal Parvovirus–Nucleus Interactions
2021
Parvoviruses are small single-stranded (ss) DNA viruses, which replicate in the nucleoplasm and affect both the structure and function of the nucleus. The nuclear stage of the parvovirus life cycle starts at the nuclear entry of incoming capsids and culminates in the successful passage of progeny capsids out of the nucleus. In this review, we will present past, current, and future microscopy and biochemical techniques and demonstrate their potential in revealing the dynamics and molecular interactions in the intranuclear processes of parvovirus infection. In particular, a number of advanced techniques will be presented for the detection of infection-induced changes, such as DNA modification…
Coxsackievirus B3-Induced Cellular Protrusions: Structural Characteristics and Functional Competence▿†
2011
ABSTRACT Virus-induced alterations in cell morphology play important roles in the viral life cycle. To examine the intracellular events of coxsackievirus B3 (CVB3) infection, green monkey kidney (GMK) cells were either inoculated with the virus or transfected with the viral RNA. Various microscopic and flow cytometric approaches demonstrated the emergence of CVB3 capsid proteins at 8 h posttransfection, followed by morphological transformation of the cells. The morphological changes included formation of membranous protrusions containing viral capsids, together with microtubules and actin. Translocation of viral capsids into these protrusions was sensitive to cytochalasin D, suggesting the …
Internalization of novel non-viral vector TAT-streptavidin into human cells
2007
BMC Biotechnology, 7 (1)
Soft X-ray Tomography Reveals HSV-1-Induced Remodeling of Human B Cells.
2022
Upon infection, viruses hijack the cell machinery and remodel host cell structures to utilize them for viral proliferation. Since viruses are about a thousand times smaller than their host cells, imaging virus-host interactions at high spatial resolution is like looking for a needle in a haystack. Scouting gross cellular changes with fluorescent microscopy is only possible for well-established viruses, where fluorescent tagging is developed. Soft X-ray tomography (SXT) offers 3D imaging of entire cells without the need for chemical fixation or labeling. Here, we use full-rotation SXT to visualize entire human B cells infected by the herpes simplex virus 1 (HSV-1). We have mapped the temporo…
Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein
2013
ABSTRACT The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mu…
Gap junctions and connexin hemichannels both contribute to the electrical properties of retinal pigment epithelium.
2022
Gap junctions are intercellular channels that permit the transfer of ions and small molecules between adjacent cells. These cellular junctions are particularly dense in the retinal pigment epithelium (RPE), and their contribution to many retinal diseases has been recognized. While gap junctions have been implicated in several aspects of RPE physiology, their role in shaping the electrical properties of these cells has not been characterized in mammals. The role of gap junctions in the electrical properties of the RPE is particularly important considering the growing appreciation of RPE as excitable cells containing various voltage-gated channels. We used a whole-cell patch clamp to measure …
Persistence of Human Bocavirus 1 in Tonsillar Germinal Centers and Antibody-Dependent Enhancement of Infection
2021
Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown.
Morphological characterization of baculovirus Autographa californica multiple nucleopolyhedrovirus
2009
The budded form of baculovirus Autographa californica multiple nucleopolyhedrovirus is used widely in biotechnological applications. In this study, we observed the morphology of baculovirus in nanometer scale by atomic force microscopy. Additionally, the correlation between transduction efficiency and virus stock storage time was evaluated. By atomic force microscopy, asymmetrical baculovirus particles with enlarged head regions were detected. Observed virus stocks contained variable-length particles, 256 ± 40 nm, along with disintegrated particles and/or cellular components. Long-term storage of stocks led to virus aggregation and decreased cellular entry and transgene expression in mammal…
Kadonneeksi luultu virustyyppi löytyi ikääntyvien B-soluista
2017
Functional roles of the membrane-associated AAV protein MAAP
2021
AbstractWith a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved over-lapping genes to maximise the usage of its genome. An example is the recently found ORF in the cap gene, encoding membrane-associated accessory protein (MAAP), located in the same genomic region as the VP1/2 unique domain, but in a different reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. Our studies confirm that MAAP translation initiates from the first CTG codon found in the VP1 ORF2. We have further observed MAAP localised in the plasma membrane, in the membranous structures in close proximity to the nucleus and to the nuclea…
Dynamics and interactions of parvoviral NS1 protein in the nucleus
2007
Summary Nuclear positioning and dynamic interactions of viral proteins with nuclear substructures play essen- tial roles during infection with DNA viruses. Visual- ization of the intranuclear interactions and motility of the parvovirus replication protein (NS1) in living cells gives insight into specific parvovirus protein- cellular structure interactions. Confocal analysis of highly synchronized infected Norden Laboratory Feline Kidney cells showed accumulation of nuclear NS1 in discrete interchromosomal foci. NS1 fused with enhanced yellow fluorescence protein (NS1- EYFP) provided a marker in live cells for dynamics of NS1 traced by photobleaching techniques. Fluo- rescence Recovery after…
Effect of ATP Binding and Hydrolysis on Dynamics of Canine Parvovirus NS1▿ †
2010
ABSTRACT The replication protein NS1 is essential for genome replication and protein production in parvoviral infection. Many of its functions, including recognition and site-specific nicking of the viral genome, helicase activity, and transactivation of the viral capsid promoter, are dependent on ATP. An ATP-binding pocket resides in the middle of the modular NS1 protein in a superfamily 3 helicase domain. Here we have identified key ATP-binding amino acid residues in canine parvovirus (CPV) NS1 protein and mutated amino acids from the conserved A motif (K406), B motif (E444 and E445), and positively charged region (R508 and R510). All mutations prevented the formation of infectious viruse…
Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection
2015
ABSTRACT The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging and in situ proximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized …
Cytoplasmic parvovirus capsids recruit importin beta for nuclear delivery
2020
Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps including nuclear import are inefficient limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family, or direct attachment of the capsid to the nuclear pore complex leading to local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analysis combined with co-immunoprecipitation we showed that infection requires importin β-mediated access into the nuclear pore complex and nucleopori…
Parvovirus induced alterations in nuclear architecture and dynamics.
2009
The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after ph…
Quantitative microscopy reveals stepwise alteration of chromatin structure during herpesvirus infection
2019
During lytic herpes simplex virus 1 (HSV-1) infection, the expansion of the viral replication compartments leads to an enrichment of the host chromatin in the peripheral nucleoplasm. We have shown previously that HSV-1 infection induces the formation of channels through the compacted peripheral chromatin. Here, we used three-dimensional confocal and expansion microscopy, soft X-ray tomography, electron microscopy, and random walk simulations to analyze the kinetics of host chromatin redistribution and capsid localization relative to their egress site at the nuclear envelope. Our data demonstrated a gradual increase in chromatin marginalization, and the kinetics of chromatin smoothening arou…
Infection-induced chromatin modifications facilitate translocation of herpes simplex virus capsids to the inner nuclear membrane
2021
Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although …
Mapping antigenic epitopes of potato virus Y with antibodies affinity-purified by using overlapping synthetic peptides
1994
Synthetic, overlapping peptides representing the entire amino acid sequence of potato virus Y (PVY) coat protein were used to affinity-purify antibodies from polyclonal antisera to PVY. In testing the binding of the purified antibodies to PVY particles, antigenic epitopes were identified. The N-terminal and C-terminal regions of the PVY coat protein were found to contain most of the antigenic epitopes. The results will facilitate the development of detection methods for PVY based on synthetic peptides.
Diffusion through thin membranes: Modeling across scales
2016
From macroscopic to microscopic scales it is demonstrated that diffusion through membranes can be modeled using specific boundary conditions across them. The membranes are here considered thin in comparison to the overall size of the system. In a macroscopic scale the membrane is introduced as a transmission boundary condition, which enables an effective modeling of systems that involve multiple scales. In a mesoscopic scale, a numerical lattice-Boltzmann scheme with a partial-bounceback condition at the membrane is proposed and analyzed. It is shown that this mesoscopic approach provides a consistent approximation of the transmission boundary condition. Furthermore, analysis of the mesosco…
G2/M checkpoint regulation and apoptosis facilitate the nuclear egress of parvoviral capsids
2022
The nuclear export factor CRM1-mediated pathway is known to be important for the nuclear egress of progeny parvovirus capsids in the host cells with virus-mediated cell cycle arrest at G2/M. However, it is still unclear whether this is the only pathway by which capsids exit the nucleus. Our studies show that the nuclear egress of DNA-containing full canine parvovirus. capsids was reduced but not fully inhibited when CRM1-mediated nuclear export was prevented by leptomycin B. This suggests that canine parvovirus capsids might use additional routes for nuclear escape. This hypothesis was further supported by our findings that nuclear envelope (NE) permeability was increased at the late stages…
Subcellular localization of bacteriophage PRD1 proteins in Escherichia coli
2014
Bacteria possess an intricate internal organization resembling that of the eukaryotes. The complexity is especially prominent at the bacterial cell poles, which are also known to be the preferable sites for some bacteriophages to infect. Bacteriophage PRD1 is a well-known model serving as an ideal system to study structures and functions of icosahedral internal membrane-containing viruses. Our aim was to analyze the localization and interactions of individual PRD1 proteins in its native host Escherichia coli. This was accomplished by constructing a vector library for production of fluorescent fusion proteins. Analysis of solubility and multimericity of the fusion proteins, as well as their …
Viral highway to nucleus exposed by image correlation analyses.
2018
AbstractParvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of …
Characterization of a nuclear localization signal of canine parvovirus capsid proteins.
1998
We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4-13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of…
Reorganization of Nuclear Pore Complexes and the Lamina in Late-Stage Parvovirus Infection
2015
Article
Protoparvovirus Knocking at the Nuclear Door
2017
Protoparvoviruses target the nucleus due to their dependence on the cellular reproduction machinery during the replication and expression of their single-stranded DNA genome. In recent years, our understanding of the multistep process of the capsid nuclear import has improved, and led to the discovery of unique viral nuclear entry strategies. Preceded by endosomal transport, endosomal escape and microtubule-mediated movement to the vicinity of the nuclear envelope, the protoparvoviruses interact with the nuclear pore complexes. The capsids are transported actively across the nuclear pore complexes using nuclear import receptors. The nuclear import is sometimes accompanied by structural chan…
CMR 2005: 12.06: Ultra-small iron oxide nanoparticle (USPIO)-labeled baculoviruses as novel MRI agents for imaging viral vector biodistributionin vivo
2006
Pathways of Cell Infection by Parvoviruses and Adeno-Associated Viruses
2004
Animal viruses have developed various strategies for infecting cells, and all begin with adsorption to cell surface receptors, penetration into the cytosol, uncoating or release of the viral genome, and targeting the genome and any required accessory proteins toward the correct cellular organelle or compartment for replication (26, 48, 63). Since genome delivery and release require the rearrangement of the viral structures, infection is normally a multistep process involving various viral and cellular components. Viruses that replicate in the nucleus must have mechanisms for transporting the genome and other components to the vicinity of the nuclear pore and into the nucleus (84). The endos…
Baculovirus-mediated immediate-early gene expression and nuclear reorganization in human cells
2007
Baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), has the ability to transduce mammalian cell lines without replication. The general objective of this study was to detect the transcription and expression of viral immediate-early genes in human cells and to examine the interactions between viral components and subnuclear structures. Viral capsids were seen in large, discrete foci in nuclei of both dividing and non-dividing human cells. Concurrently, the transcription of viral immediate-early transregulator genes (ie-1, ie-2) and translation of IE-2 protein were detected. Quantitative microscopy imaging and analysis showed that virus transduction altered the size of …
Improvement in Nuclear Entry and Transgene Expression of Baculoviruses by Disintegration of Microtubules in Human Hepatocytes
2005
ABSTRACT Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), a potent virus for mammalian cell gene delivery, possesses an ability to transduce mammalian cells without viral replication. We examined the role of the cellular cytoskeleton in the cytoplasmic trafficking of viral particles toward the nucleus in human hepatic cells. Microscopic studies showed that capsids were found in the nucleus after either viral inoculation or cytoplasmic microinjection of nucleocapsids. The presence of microtubule (MT) depolymerizing agents caused the amount of nuclear capsids to increase. Overexpression of p50/dynamitin, an inhibitor of dynein-dependent endocytic trafficking from peripheral e…
Characterization of antigenic epitopes of potato virus Y.
1993
Immunochemical analysis of overlapping synthetic hexapeptides covering the entire length of the coat protein of potato virus Y (PVY) revealed immunodominant regions both at the N-terminal and at the C-terminal end of the coat protein. Immunization of rabbits with synthetic peptides representing N- and C-terminal regions of the coat protein resulted in production of antibodies that reacted with PVY. Antigenicity of PVY peptides was found to correlate with predicted beta turns, with hydrophilicity and with predicted chain flexibility. Characterization of the immunochemical properties of PVY will facilitate the development of detection methods for potyviruses.
Role of Recycling Endosomes and Lysosomes in Dynein-Dependent Entry of Canine Parvovirus
2002
ABSTRACT Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes r…
Creation of ordered 3D tubes out of DNA origami lattices
2023
Funding Information: Funding from the Jane and Aatos Erkko Foundation (J.J.T. and A.K./M.V.-R.) and the Academy of Finland (#330584 and #350797 J.J.T./#308992 A.K. and A.K.N./#330896 M.V.-R.) is gratefully acknowledged. The authors also acknowledge the provision of facilities and technical support by Aalto University at OtaNano - Nanomicroscopy Center (Aalto-NMC). Publisher Copyright: © 2023 The Royal Society of Chemistry. Hierarchical self-assembly of nanostructures with addressable complexity has been a promising route for realizing novel functional materials. Traditionally, the fabrication of such structures on a large scale has been achievable using top-down methods but with the cost of…
Internalization of novel non-viral vector TAT-streptavidin into human cells
2007
Background. The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT47–57-streptavidin (TAT-SA, 60 kD). SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes. Results. By confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accu…