Papers by Maija Vihinen-ranta
The prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus, an insect pathog... more 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 of RhoA, and of Arf6, a regulator of clathrin-independent entry. Furthermore, the entry of baculovirus induced ruffle formation and triggered the uptake of fluorescent E. coli bioparticles. To conclude, baculovirus enters human cells via a clathrin-independent pathway, which is able to trigger bact...
This article cites 49 articles, 21 of which can be accessed free
The contribution of human DNA polymerase ε to nuclear DNA replication was studied. Antibody K18 t... more The contribution of human DNA polymerase ε to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase ε 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 α. Contrary to the antibody against DNA polymerase α, antibody K18 against DNA polymerase ε did not inhibit SV40 DNA replication in vitro . These results indicate that DNA polymerase ε plays a role in replicative DNA synthesis in proliferating human cells like DNA polymerase α, and that this role for DNA polymerase ε cannot be modeled by SV40 DNA replication.
Biophysical Journal, 2012
Asymmetric cell division in C.Crescentus relies on differentially localizing certain proteins to ... more Asymmetric cell division in C.Crescentus relies on differentially localizing certain proteins to the poles where they bind to the scaffolding protein PopZ that displays a bipolar pattern. On the other hand, expressing PopZ in E. coli favours unipolar patterning. Additionally, the aggregation of misfolded proteins as well as plasmids in bacteria also display unipolar and biopolar patterns, similar to that of PopZ. These different systems have led to the hypothesis that chromosome free regions þ biomolecular aggregation can be sufficient to drive localization in bacterial cells. We have performed Monte-Carlo simulations to show that the entropic force provided by the selfavoiding chromosome confined in the cylindrical geometry of the cell and the energy gained from aggregation results in phase separation between proteins and the polymer. We fully explore the phase space showing how patterning depends on protein concentration, chromosome density, cell shape and aggregation strength. The exploration results in a rich phase diagram of patterns which the observed systems can be fit into. Additionally, the dynamics of pattern formation depends somewhat on the rate at which proteins are added to the system. When proteins are added very slowly to the cell, the unipolar phase dominates, whereas at faster rates the bipolar phase dominates and at yet faster still rate, aggregation at nonpolar locations occurs. Such a rate dependency may explain differences in the observed patterns reported for PopZ induced expression in E. coli cells. Lastly, we show how such a localization process may aid the segregation of other cellular components such as the replication origin which anchors to a pole. We find that adding extra interactions does not destabilize the polar patterning, nor is it sufficient to drive the localization of the origin and indeed other localization and stabilization mechanisms are required.
BMC Biology
Background: Voltage-gated sodium (Na v) channels have traditionally been considered a trademark o... more Background: Voltage-gated sodium (Na v) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Na v 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 Na v 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 Na v channels in RPE both ex vivo and in vitro. Results: Our work demonstrates that several subtypes of Na v channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Na v 1.4, Na v 1.6, and Na v 1.8. Whole cell patch clamp recordings from the hESC-derived RPE monolayers showed that the current was inhibited by TTX and QX-314 and was sensitive to the selective blockers of the main Na v subtypes. Importantly, we show that the Na v channels are involved in photoreceptor outer segment phagocytosis since blocking their activity significantly reduces the efficiency of particle internalization. Consistent with this role, our electron microscopy results and immunocytochemical analysis show that Na v 1.4 and Na v 1.8 accumulate on phagosomes and that pharmacological inhibition of Na v channels as well as silencing the expression of Na v 1.4 with shRNA impairs the phagocytosis process. Conclusions: Taken together, our study shows that Na v channels are present in RPE, giving this tissue the capacity of fast electrical signaling. The channels are critical for the physiology of RPE with an important role in photoreceptor outer segment phagocytosis.
Various types of DNA viruses are known to elicit the formation of a large nuclear viral replicati... more 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 simplex virus 1-sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.
Journal of Physics: Photonics
Kenneth Fahy1,∗, Venera Weinhardt, Maija Vihinen-Ranta, Nicola Fletcher, Dunja Skoko, Eva Pereir... more Kenneth Fahy1,∗, Venera Weinhardt, Maija Vihinen-Ranta, Nicola Fletcher, Dunja Skoko, Eva Pereiro, Pablo Gastaminza, Ralf Bartenschlager, Dimitri Scholz, Axel Ekman and Tony McEnroe 1 SiriusXT Limited, Dublin, Ireland 2 Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany 3 Department of Biological and Environmental Science and Nanoscience Centre, University of Jyväskylä, Jyväskylä, Finland 4 School of Veterinary Medicine, University College Dublin, Dublin, Ireland 5 Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland 6 MISTRAL Beamline, ALBA Synchrotron, Barcelona, Spain 7 Departamento de BiologÃa Celular y Molecular, Centro Nacional de BiotecnologÃa-C.S.I.C., Madrid, Spain 8 Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany ∗ Author to whom any correspondence should be addressed.
Scientific Reports
With a limited coding capacity of 4.7Â kb, adeno-associated virus (AAV) genome has evolved over-la... more With a limited coding capacity of 4.7Â 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 nuclear envelope by co-transfecting with plasmids encoding the wild-type AAV (wt-AAV) genome and adenovirus (Ad) helper genes. While keeping VP1/2 protein sequence identical, both inactivation and truncation of MAAP translation affected the emergence and intracellular distribution of the AAV capsid proteins. We...
Viruses
Parvoviruses are small single-stranded (ss) DNA viruses, which replicate in the nucleoplasm and a... more 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 and damage, as well as protein–chromatin interactions.
mBio
Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-thr... more Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-...
Journal of Virology
Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the foll... more 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 nucleoporin 153-mediated interactions on the nuclear side. The importin β-capsid interaction continued within the nucleoplasm, which suggests a mixed model of nuclear entry in which the classical nuclear import across the nuclear pore complex is accompanied by transient ruptures of the nuclear env...
Macromolecular Bioscience
This study presents the reactive self-assembly of isocyanate functional and amphiphilic sixarm st... more This study presents the reactive self-assembly of isocyanate functional and amphiphilic sixarm star-shaped polyether prepolymers in water into nanogels. Intrinsic molecular amphiphilicity, mainly driven by the isophorone moiety at the distal endings of the starshaped molecules allowed 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 tensides. Covalent attachment of a fluorescence dye and either the cellpenetrating TAT peptide or a random control peptide sequence showed that only TAT labeled nanogels are internalized by HeLa cells. The nanogels thus specifically entered the cells and accumulated in the perinuclear area in a time-and concentration-dependent manner.
Viruses
Protoparvoviruses target the nucleus due to their dependence on the cellular reproduction machine... more 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 changes in the nuclear envelope, and is completed by intranuclear disassembly of capsids and chromatinization of the viral genome. This review discusses the nuclear import strategies of protoparvoviruses and describes its dynamics comprising active and passive movement, and directed and diffusive motion of capsids in the molecularly crowded environment of the cell.
Viruses
During lytic herpes simplex virus 1 (HSV-1) infection, the expansion of the viral replication com... more 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 around the viral replication compartments correlated with their expansion. We also observed a gradual transfer of capsids to the nuclear envelope. Later in the infection, random walk modeling indicated a gradually faster transport of capsids to the nuclear envelope that correlated with an increase in ...
Scientific reports, Jan 18, 2018
Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multis... more Parvoviral 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 image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin β revealing previously unknown dynamics of the entry pathway.
Microscopy and Microanalysis
DNA viruses target the cell nucleus due to their need of the cellular DNA reproduction machinery.... more DNA viruses target the cell nucleus due to their need of the cellular DNA reproduction machinery. Lytic infection with herpes simplex virus 1 (HSV-1) is known to elicit the formation of a large nuclear viral replication compartment and redistribution of the cell chromatin. The final nuclear steps of infection include the assembly of viral capsids in the replication compartment area and and their egress through the nuclear envelope. To reach the nuclear envelope, the viral capsids have to be transported through the host chromatin. The infection-induced quantitative changes on the spatial and molecular organization of chromatin, and their effect on the viral capsid translocation toward the nuclear egress site have remained unknown.
Scientific reports, Jan 16, 2017
Various types of DNA viruses are known to elicit the formation of a large nuclear viral replicati... more 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 simplex virus 1-sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site...
Nature Communications, 2017
Parvovirus B19 (B19V) DNA persists lifelong in human tissues, but the cell type harbouring it rem... more 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 440 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 demonstrate the mechanism of B19V internalization to be antibody dependent in two B-cell lines as well as in ex vivo isolated tonsillar B cells. This study provides direct evidence for a cell type accountable for B19V DNA tissue persistence.
Scientific reports, Jun 28, 2016
Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cel... more Lytic 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, confocal and electron microscopy analysis showed that these gaps frequently contained viral nucleocapsids. These results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear en...
Uploads
Papers by Maija Vihinen-ranta