Exploring nucleic acid condensation and release from individual parvovirus particles with different physicochemical cues.

In the infection cycle, viruses release their genome in the host cell during uncoating. Here we use a variety of physicochemical procedures to induce and monitor the in vitro uncoating of ssDNA from individual Minute Virus of Mice (MVM) particles. Our experiments revealed two pathways of genome release: i) filamentous ssDNA appearing around intact virus particles when using gradual mechanical fatigue and heating at moderate temperature (50 °C). ii) thick structures of condensed ssDNA appearing when the virus particle is disrupted by mechanical nanoindentations, denaturing agent guanidinium chloride and high temperature (70 °C). We propose that in the case of filamentous ssDNA, when the capsid integrity is conserved, the genome is externalized through one channel of the capsid pores. However, the disruption of virus particles revealed a native structure of condensed genome. The mechanical analysis of intact particles after DNA strands ejection confirm the stabilization role of ssDNA in MVM.

Nuclear entry and egress of parvoviruses.

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 by currently undefined events of viral genome uncoating. After genome release, viral replication compartments are initiated and infection proceeds. Parvoviral genomes replicate during cellular S phase followed by nuclear capsid assembly during virus-induced S/G2 cell cycle arrest. Nuclear egress of capsids occurs upon nuclear envelope degradation during apoptosis and cell lysis. An alternative pathway for nuclear export has been described using active transport through the NPC mediated by the chromosome region maintenance 1 protein, CRM1, which is enhanced by phosphorylation of the N-terminal domain of VP2. However, other alternative but not yet uncharacterized nuclear export pathways cannot be excluded.

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 ß-mediated access to the nuclear pore complex and nucleoporin 153-mediated interactions on the nuclear side. The importin ß-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 envelope, also allowing the passive entry of importin ß-capsid complexes into the nucleus.IMPORTANCE Parvoviruses are small DNA viruses that deliver their DNA into the postmitotic nuclei, which is an important step for parvoviral gene and cancer therapies. Limitations in virus-receptor interactions or endocytic entry do not fully explain the low transduction/infection efficiency, indicating a bottleneck after virus entry into the cytoplasm. We thus investigated the transfer of parvovirus capsids from the cytoplasm to the nucleus, showing that the nuclear import of the parvovirus capsid follows a unique strategy, which differs from classical nuclear import and those of other viruses.

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 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.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

[Advances in Parvovirus Non-structural Protein NS1 Induced Apoptosis].

Until now, more than seventeen parvovirus have been reported which can infect mammals and poultries. The infected cells appeared different properties of apoptosis and death, present a typical cytopathic effect. NS1 is a major nonstructural protein of parvovirus, with a conservative structure and function, which plays an important role in the viral life cycle. In addition to the influence on viral replication, the NS1 also participates in apoptosis induced by viruses. Parvovirus induced apoptosis which is mainly mediated by mitochondrial pathway, this review summarized the latest research progresses of parvovirus induced apoptosis.

Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

Parvovirus glycan interactions.

Members of the Parvoviridae utilize glycan receptors for cellular attachment and subsequent interactions determine transduction efficiency or pathogenic outcome. This review focuses on the identity of the glycan receptors utilized, their capsid binding footprints, and a discussion of the overlap of these sites with tropism, transduction, and pathogenicity determinants. Despite high sequence diversity between the different genera, most parvoviruses bind to negatively charged glycans, such as sialic acid and heparan sulfate, abundant on cell surface membranes. The capsid structure of these viruses exhibit high structural homology enabling common regions to be utilized for glycan binding. At the same time the sequence diversity at the common footprints allows for binding of different glycans or differential binding of the same glycan.

Interaction of parvoviruses with the nuclear envelope.

Parvoviruses are serious pathogens but also serve as platforms for gene therapy or for using their lytic activity in experimental cancer treatment. Despite of their growing importance during the last decade little is known on how the viral genome is transported into the nucleus of the infected cell, which is crucial for replication. As nucleic acids are not karyophilic per se nuclear import must be driven by proteins attached to the viral genome. In turn, presence and conformation of these proteins depend upon the entry pathway of the virus into the cell. This review focuses on the trafficking of the parvoviral genome from the cellular periphery to nucleus. Despite of the uncertainties in knowledge about the entry pathway we show that parvoviruses developed a unique strategy to pass the nuclear envelope by hijacking enzymes involved in mitosis.

Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against cervical and pancreatic carcinomas.

The rat parvovirus H-1PV has oncolytic and tumour-suppressive properties potentially exploitable in cancer therapy. This possibility is being explored and results are encouraging, but it is necessary to improve the oncotoxicity of the virus. Here we show that this can be achieved by co-treating cancer cells with H-1PV and histone deacetylase inhibitors (HDACIs) such as valproic acid (VPA). We demonstrate that these agents act synergistically to kill a range of human cervical carcinoma and pancreatic carcinoma cell lines by inducing oxidative stress, DNA damage and apoptosis. Strikingly, in rat and mouse xenograft models, H-1PV/VPA co-treatment strongly inhibits tumour growth promoting complete tumour remission in all co-treated animals. At the molecular level, we found acetylation of the parvovirus nonstructural protein NS1 at residues K85 and K257 to modulate NS1-mediated transcription and cytotoxicity, both of which are enhanced by VPA treatment. These results warrant clinical evaluation of H-1PV/VPA co-treatment against cervical and pancreatic ductal carcinomas.

Multiplex polymerase chain reaction assay for the detection of minute virus of mice and mouse parvovirus infections in laboratory mice.

Mouse parvoviruses are among the most prevalent infectious pathogens in contemporary mouse colonies. To improve the efficiency of routine screening for mouse parvovirus infections, a multiplex polymerase chain reaction (PCR) assay targeting the VP gene was developed. The assay detected minute virus of mice (MVM), mouse parvovirus (MPV) and a mouse housekeeping gene (α-actin) and was able to specifically detect MVM and MPV at levels as low as 50 copies. Co-infection with the two viruses with up to 200-fold differences in viral concentrations can easily be detected. The multiplex PCR assay developed here could be a useful tool for monitoring mouse health and the viral contamination of biological materials.

Generation of an adenovirus-parvovirus chimera with enhanced oncolytic potential.

In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.

Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine.

The parvoviral capsid controls an intracellular phase of infection essential for efficient killing of stepwise-transformed human fibroblasts.

Members of the rodent subgroup of the genus Parvovirus exhibit lytic replication and spread in many human tumor cells and are therefore attractive candidates for oncolytic virotherapy. However, the significant variation in tumor tropism observed for these viruses remains largely unexplained. We report here that LuIII kills BJ-ELR 'stepwise-transformed' human fibroblasts efficiently, while MVM does not. Using viral chimeras, we mapped this property to the LuIII capsid gene, VP2, which is necessary and sufficient to confer the killer phenotype on MVM. LuIII VP2 facilitates a post-entry, pre-DNA-amplification step early in the life cycle, suggesting the existence of an intracellular moiety whose efficient interaction with the incoming capsid shell is critical to infection. Thus targeting of human cancers of different tissue-type origins will require use of parvoviruses with capsids that effectively make this critical interaction.

The non-structural protein NS-2 of Bombyx mori parvo-like virus is localized to the nuclear membrane.

Bombyx mori parvo-like virus (BmPLV) has two complementary single-stranded DNA genome (VD1 and VD2) and owns a self-encoding DNA polymerase motif, but its replication mechanism is unclear. In our previous research, a protein encoded by VD1-ORF1 was indentified in the midgut of BmPLV China Zhenjiang isolate-(BmPLV-Z) infected silkworm larvae via two-dimensional gel electrophoresis (2-DE). This protein was named as non-structural protein 2 (NS2), which showed no similarity to that of parvoviruses. To date, little is known about it. In this study, sequence alignment results showed that NS2 shared homology with some chromosomal replication initiator protein dnaA and DNA-binding response regulators. The ns2 was cloned and expressed in E. coli, and then a polyclonal antibody of the NS2 protein was prepared successfully. The data from real-time quantitative PCR displayed that the transcription of VD1-ORF1 from BmPLV-Z-infected midguts started from 28-h post inoculation (h p.i.) in low amounts, but in high amounts at late stages of infection. Immunofluorescence showed that NS2 ultimately concentrated on the nuclear membrane in BmN cells at late stages, indicating that NS2 might be associated with integral membrane protein.

Novel adenovirus-based helper system to support production of recombinant parvovirus.

Preclinical studies using various cell culture and animal systems highlight the potential of recombinant rodent parvoviruses (recPVs) for cancer therapy. Production of these viruses is, however, not efficient and this hampers the clinical applications of these agents. In this study, we show that the adenovirus genes E2a, E4(orf6) and VA RNA increase the production of recPVs by more than 10-fold and reduce the time of production from 3 to 2 days in HEK293T cells. The helper effects of these genes can be observed with different recPVs, regardless of the nature and size of the inserted transgene. Furthermore, we generated a recombinant Adenovirus 5 carrying the parvovirus VP transcription unit. This helper, named Ad-VP, allows recPVs to be efficiently produced through a protocol based only on cell infection, making possible to use cell lines, such as NB324K, which are good producers of parvoviruses but are hardly transfectable. Hence, we could further improve viral titers and reduce time and costs of production. This Ad-VP helper-based protocol could be scaled up to a bioreactor format for the generation of the large amounts of recPVs needed for future clinical applications.

Erythroid progenitor cells expanded from peripheral blood without mobilization or preselection: molecular characteristics and functional competence.

BACKGROUND: Continued development of in-vitro procedures for expansion and differentiation of erythroid progenitor cells (EPC) is essential not only in hematology and stem cell research but also virology, in light of the strict erythrotropism of the clinically important human parvovirus B19. METHODOLOGY/PRINCIPAL FINDINGS: We cultured EPC directly from ordinary blood samples, without ex vivo stem cell mobilization or CD34+ cell in vitro preselection. Profound increase in the absolute cell number and clustering activity were observed during culture. The cells obtained expressed the EPC marker combination CD36, CD71 and glycophorin, but none of the lymphocyte, monocyte or NK markers. The functionality of the generated EPC was examined by an in vitro infection assay with human parvovirus B19, tropic for BFU-E and CFU-E cells. Following infection (i) viral DNA replication and mRNA production were confirmed by quantitative PCR, and (ii) structural and nonstructural proteins were expressed in >50% of the cells. As the overall cell number increased 100-200 fold, and the proportion of competent EPC (CD34+ to CD36+) rose from <0.5% to >50%, the in vitro culture procedure generated the EPC at an efficiency of >10,000-fold. Comparative culturing of unselected PBMC and ex vivo-preselected CD34+ cells produced qualitatively and quantitatively similar yields of EPC. CONCLUSIONS/SIGNIFICANCE: This approach yielding EPC directly from unmanipulated peripheral blood is gratifyingly robust and will facilitate the study of myeloid infectious agents such as the B19 virus, as well as the examination of erythropoiesis and its cellular and molecular mechanisms.

Microinjection of Xenopus laevis oocytes as a system for studying nuclear transport of viruses.

Microinjection of Xenopus laevis oocytes is an excellent system for studying nuclear transport because of the large size of the oocyte and its high nuclear pore complex (NPC) density. In addition, the fact that Xenopus oocytes are not permissive for most mammalian viruses makes this system especially useful for studying nuclear transport of viruses in the absence of the confounding factor of virus replication. In this article, we briefly discuss the contribution of microinjection of Xenopus oocytes to the field of nuclear transport. We then describe the protocols we have developed using microinjection of Xenopus oocytes to study nuclear transport of viral capsids, and summarize variations of the technique that can be used to address many different questions about the nuclear transport of viruses.

The choice of translation initiation site of the rep proteins from goose parvovirus P9-generated mRNA is governed by splicing and the nature of the excised intron.

The goose parvovirus (GPV) Rep 1 and Rep 2 proteins are encoded by P9-generated mRNAs that are either unspliced or spliced within the rep gene region, respectively. These mRNAs are present in an approximately equal ratio. The translation of Rep 1 was initiated from the first AUG in unspliced P9-generated mRNA; however, this AUG was bypassed in spliced P9-generated RNA and Rep 2 translation initiated predominately at the next initiating AUG downstream. We show that the choice of the site of initiation of translation of GPV Rep-encoding mRNAs is governed both by the splicing process itself and by the nature of the excised intron.

Parvovirus induced alterations in nuclear architecture and dynamics.

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 photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analysis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.