Cytoplasmic Parvovirus Capsids Recruit Importin Beta for Nuclear Delivery

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 …

Parvovirus nonstructural protein 2 interacts with chromatin-regulating cellular proteins

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…

Extinct type of human parvovirus B19 persists in tonsillar B cells

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 …

Reactive Self-Assembly and Specific Cellular Delivery of NCO-sP(EO-stat-PO)-Derived Nanogels

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.

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…

Canine parvovirus infection : events at the nuclear envelope and its vicinity

Concepts to Reveal Parvovirus–Nucleus Interactions

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…

Ecological conditions alter cooperative behaviour and its costs in a chemically defended sawfly

The evolution of cooperation and social behaviour is often studied in isolation from the ecology of organisms. Yet, the selective environment under which individuals evolve is much more complex in nature, consisting of ecological and abiotic interactions in addition to social ones. Here, we measured the life-history costs of cooperative chemical defence in a gregarious social herbivore, Diprion pini pine sawfly larvae, and how these costs vary under different ecological conditions. We ran a rearing experiment where we manipulated diet (resin content) and attack intensity by repeatedly harassing larvae to produce a chemical defence. We show that forcing individuals to allocate more to coope…

Kadonneeksi luultu virustyyppi löytyi ikääntyvien B-soluista

Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection

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

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…

Quantitative microscopy reveals stepwise alteration of chromatin structure during herpesvirus infection

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…

G2/M checkpoint regulation and apoptosis facilitate the nuclear egress of parvoviral capsids

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…

Viral highway to nucleus exposed by image correlation analyses.

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 …

Electronic Supplementary Material from Ecological conditions alter cooperative behaviour and its costs in a chemically defended sawfly.

The evolution of cooperation and social behaviour is often studied in isolation from the ecology of organisms. Yet, the selective environment under which individuals evolve is much more complex in nature, consisting of ecological and abiotic interactions in addition to social ones. Here, we measured the life-history costs of cooperative chemical defence in a gregarious social herbivore, Diprion pini pine sawfly larvae, and how these costs vary under different ecological conditions. We ran a rearing experiment where we manipulated diet (resin content) and attack intensity by repeatedly harassing larvae to produce a chemical defence. We show that forcing individuals to allocate more to cooper…

Reorganization of Nuclear Pore Complexes and the Lamina in Late-Stage Parvovirus Infection

Article

Import and impact : characterization of parvovirus-nucleus interactions

Protoparvovirus Knocking at the Nuclear Door

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…

Data from: Ecological conditions alter cooperative behaviour and its costs in a chemically defended sawfly

The evolution of cooperation and social behaviour is often studied in isolation from the ecology of organisms. Yet, the selective environment under which individuals evolve is much more complex in nature, consisting of ecological and abiotic interactions in addition to social ones. Here we measured the life-history costs of cooperative chemical defence in a gregarious social herbivore, Diprion pini pine sawfly larvae, and how these costs vary under different ecological conditions. We ran a rearing experiment where we manipulated diet (resin content) and attack intensity by repeatedly harassing larvae to produce a chemical defence. We show that forcing individuals to allocate more to coopera…