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Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin replication (RHR) mechanism initiated at the AAV 3' inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus. Author summary: Efficient adeno-associated virus (AAV) replication requires the presence of helper factors, which can be provided by co-infecting helper viruses such as adenoviruses or herpesviruses. AAV replication has been described to occur as a rolling hairpin replication mechanism. However, we show that during a herpes simplex virus type 1 (HSV-1) supported replication, AAV rolling circle-like replication intermediates are formed. Thus, this study stands in contrast to the textbook model of AAV genome replication. Additionally, we introduce nanopore sequencing as a novel, high-throughput approach to study viral genome replication in unprecedented detail. [ABSTRACT FROM AUTHOR]

The glycosphingolipid (GSL) globoside (Gb4) is essential for parvovirus B19 (B19V) infection. Historically considered the cellular receptor of B19V, the role of Gb4 and its interaction with B19V are controversial. In this study, we applied artificial viral particles, genetically modified cells, and specific competitors to address the interplay between the virus and the GSL. Our findings demonstrate that Gb4 is not involved in the binding or internalization process of the virus into permissive erythroid cells, a function that corresponds to the VP1u cognate receptor. However, Gb4 is essential at a post-internalization step before the delivery of the single-stranded viral DNA into the nucleus. In susceptible erythroid Gb4 knockout cells, incoming viruses were arrested in the endosomal compartment, showing no cytoplasmic spreading of capsids as observed in Gb4-expressing cells. Hemagglutination and binding assays revealed that pH acts as a switch to modulate the affinity between the virus and the GSL. Capsids interact with Gb4 exclusively under acidic conditions and dissociate at neutral pH. Inducing a specific Gb4-mediated attachment to permissive erythroid cells by acidification of the extracellular environment led to a non-infectious uptake of the virus, indicating that low pH-mediated binding to the GSL initiates active membrane processes resulting in vesicle formation. In summary, this study provides mechanistic insight into the interaction of B19V with Gb4. The strict pH-dependent binding to the ubiquitously expressed GSL prevents the redirection of the virus to nonpermissive tissues while promoting the interaction in acidic intracellular compartments as an essential step in infectious endocytic trafficking. Author summary: The neutral glycosphingolipid globoside (Gb4) has been historically considered the cellular receptor of B19V, however, its wide expression profile does not correlate well with the restricted tropism of the virus. Here, we show that Gb4 is essential for the infection at a step following virus uptake and before the delivery of the viral ssDNA into the nucleus. B19V interacts with Gb4 exclusively under acidic conditions, prohibiting the interaction on the plasma membrane and promoting it inside the acidic endosomal compartments, which are engaged by the virus and the GSL after internalization. In the absence of Gb4, incoming viruses are retained in the endocytic compartment and the infection is aborted. This study reveals the mechanism of the interaction between the virus and the glycosphingolipid and redefines the role of Gb4 as an essential intracellular partner required for infectious entry. [ABSTRACT FROM AUTHOR]

Anion‐exchange chromatography (AEX) is used in the downstream purification of monoclonal antibodies to remove impurities and potential viral contamination based on electrostatic interactions. Although the isoelectric point (pI) of viruses is considered a key factor predicting the virus adsorption to the resin, the precise molecular mechanisms involved remain unclear. To address this question, we compared structurally homologous parvoviruses that only differ in their surface charge distribution. A single charged amino acid substitution on the capsid surface of minute virus of mice (MVM) provoked an increased apparent pI (pIapp) 6.2 compared to wild‐type MVM (pIapp = 4.5), as determined by chromatofocusing. Despite their radically different pIapp, both viruses displayed the same interaction profile in Mono Q AEX at different pH conditions. In contrast, the closely related canine parvovirus (pIapp = 5.3) displayed a significantly different interaction at pH 5. The detailed structural analysis of the intricate three‐dimensional structure of the capsids suggests that the charge distribution is critical, and more relevant than the pI, in controlling the interaction of a virus with the chromatographic resin. This study contributes to a better understanding of the molecular mechanisms governing virus clearance by AEX, which is crucial to enable robust process design and maximize safety. [ABSTRACT FROM AUTHOR]

Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells. [ABSTRACT FROM AUTHOR]

Cutthroat trout virus (CTV) is a non-pathogenic fish virus belonging to the Hepeviridae family, and it is distantly related to hepatitis E virus (HEV). Here, we report the development of an efficient cell culture system where CTV can consistently replicate to titers never observed before with a hepevirus. By using the rainbow trout gill (RTGill-W1) cell line, CTV reaches 1010 geq/mL intracellularly and 109 geq/mL extracellularly within 5-6 days in culture. We additionally established a qPCR system to investigate CTV infectivity, and developed a specific antibody directed against the viral capsid protein encoded by ORF2. With these methods, we were able to follow the progressive accumulation of viral RNA and the capsid protein, and their intracellular distribution during virus replication. Virus progeny purified through iodixanol density gradients indicated--that similar to HEV--CTV produced in cell culture is also lipid-associated. The lack of an efficient cell culture system has greatly impeded studies with HEV, a major human pathogen that causes hepatitis worldwide. Although several cell culture systems have recently been established, the replication efficiency of HEV is not robust enough to allow studies on different aspects of the virus replication cycle. Therefore, a surrogate virus that can replicate easily and efficiently in cultured cells would be helpful to boost research studies with hepeviruses. Due to its similarities, but also its key differences to HEV, CTV represents a promising tool to elucidate aspects of the replication cycle of Hepeviridae in general, and HEV in particular. [ABSTRACT FROM AUTHOR]

Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5-80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three -helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system. [ABSTRACT FROM AUTHOR]

The VP1 unique region (VP1u) of human parvovirus B19 (B19V) is the immunodominant part of the viral capsid. Originally inaccessible, the VP1u becomes exposed upon primary attachment to the globoside receptor. To study the function of the exposed VP1u in B19V uptake, we expressed this region as a recombinant protein. Here, we report that purified recombinant VP1u binds and is internalized in UT7/Epo cells. By means of truncations and specific antibodies, we identified the most N-terminal amino acid residues of VP1u as the essential region for binding and internalization. Furthermore, the recombinant VP1u was able to block B19V uptake, suggesting that the protein and the virus undertake the same internalization pathway. Assays with different erythroid and nonerythroid cell lines showed that the N-terminal VP1u binding was restricted to a few cell lines of the erythroid lineage, which were also the only cells that allowed B19V internalization and infection. These results together indicate that the N-terminal region of VP1u is responsible for the internalization of the virus and that the interacting receptor is restricted to B19V-susceptible cells. The highly selective uptake mechanism represents a novel determinant of the tropism and pathogenesis of B19V. [ABSTRACT FROM AUTHOR]

Parvovirus B19 (B19V) is a human pathogen with a marked tropism for erythroid progenitor cells (EPCs). The N-terminal of the VP1 unique region (VP1u) contains a receptor-binding domain (RBD), which mediates virus uptake through interaction with an as-yet-unknown receptor (VP1uR). Considering the central role of VP1uR in the virus tropism, we sought to investigate its expression profile in multiple cell types. To this end, we established a PP7 bacteriophage-VP1u bioconjugate, sharing the size and VP1u composition of native B19V capsids. The suitability of the PP7-VP1u construct as a specific and sensitive VP1uR expression marker was validated in competition assays with B19V and recombinant VP1u. VP1uR expression was exclusively detected in erythroid cells and cells reprogrammed towards the erythroid lineage. Sequence alignment and in silico protein structure prediction of the N-terminal of VP1u (N-VP1u) from B19V and other primate erythroparvoviruses (simian, rhesus, and pig-tailed) revealed a similar structure characterized by a fold of three or four α-helices. Functional studies with simian parvovirus confirmed the presence of a conserved RBD in the N-VP1u, mediating virus internalization into human erythroid cells. In summary, this study confirms the exclusive association of VP1uR expression with cells of the erythroid lineage. The presence of an analogous RBD in the VP1u from non-human primate erythroparvoviruses emphasizes their parallel evolutionary trait and zoonotic potential. [ABSTRACT FROM AUTHOR]

The viral protein 1 unique region (VP1u) of human parvovirus B19 (B19V) is a multifunctional capsid protein with essential roles in virus tropism, uptake, and subcellular trafficking. These functions reside on hidden protein domains, which become accessible upon interaction with cell membrane receptors. A receptor-binding domain (RBD) in VP1u is responsible for the specific targeting and uptake of the virus exclusively into cells of the erythroid lineage in the bone marrow. A phospholipase A2 domain promotes the endosomal escape of the incoming virus. The VP1u is also the immunodominant region of the capsid as it is the target of neutralizing antibodies. For all these reasons, the VP1u has raised great interest in antiviral research and vaccinology. Besides the essential functions in B19V infection, the remarkable erythroid specificity of the VP1u makes it a unique erythroid cell surface biomarker. Moreover, the demonstrated capacity of the VP1u to deliver diverse cargo specifically to cells around the proerythroblast differentiation stage, including erythroleukemic cells, offers novel therapeutic opportunities for erythroid-specific drug delivery. In this review, we focus on the multifunctional role of the VP1u in B19V infection and explore its potential in diagnostics and erythroid-specific therapeutics. [ABSTRACT FROM AUTHOR]

Virus filtration is a highly effective method in the downstream processing of biotherapeutic products to provide effective removal of potential infectious agents based on a size exclusion mechanism. The direct visualization of viruses retained inside the filter membrane represents a valuable tool to get a deeper understanding of the filtration process and to explain observations of virus breakthrough under particular operating conditions. Parvoviruses, which are used as worst-case models in validation studies, were purified and labeled with fluorescent dyes to detect their retention pattern inside the filter membrane using laser scanning microscopy. Critical factors influencing the reproducibility and accuracy of the approach were identified and optimized. The retention profiles revealed detectable differences between viruses, suggesting that the use of bacteriophages or nanoparticles as surrogates is limited in their applicability to accurately predict the behavior of parvoviruses in filter membranes. The established method enables a direct and quantitative analysis of the virus retention profile, adding a valuable tool to the conventional measurement of the viral load reduction to better understand the mechanism underlying the removal of viruses during nanofiltration of biotherapeutic products. Image 1 • The established method visualizes parvovirus retention in virus filter membranes. • Virus-specific detection requires careful verification of spike quality. • Small capsid modifications affect the behavior of viruses during filtration. • Parvoviruses and bacteriophage particles show a significantly different retention. [ABSTRACT FROM AUTHOR]

Human parvovirus B19 (B19V) traffics to the cell nucleus where it delivers the genome for replication. The intracellular compartment where uncoating takes place, the required capsid structural rearrangements and the cellular factors involved remain unknown. We explored conditions that trigger uncoating in vitro and found that prolonged exposure of capsids to chelating agents or to buffers with chelating properties induced a structural rearrangement at 4 °C resulting in capsids with lower density. These lighter particles remained intact but were unstable and short exposure to 37 °C or to a freeze-thaw cycle was sufficient to trigger DNA externalization without capsid disassembly. The rearrangement was not observed in the absence of chelating activity or in the presence of MgCl2 or CaCl2, suggesting that depletion of capsid-associated divalent cations facilitates uncoating. The presence of assembled capsids with externalized DNA was also detected during B19V entry in UT7/Epo cells. Following endosomal escape and prior to nuclear entry, a significant proportion of the incoming capsids rearranged and externalized the viral genome without capsid disassembly. The incoming capsids with accessible genomes accumulated in the nuclear fraction, a process that was prevented when endosomal escape or dynein function was disrupted. In their uncoated conformation, capsids immunoprecipitated from cytoplasmic or from nuclear fractions supported in vitro complementary-strand synthesis at 37 °C. This study reveals an uncoating strategy of B19V based on a limited capsid rearrangement prior to nuclear entry, a process that can be mimicked in vitro by depletion of divalent cations. [ABSTRACT FROM AUTHOR]

Viruses are evolutionarily developed cell-entering nanomachines, which are frequently used as gene or drug delivery systems. Parvovirus B19 (B19V) shows a remarkably restricted tropism for erythropoietin-dependent erythroid differentiation stages, and thus this virus provides an opportunity to deliver cargo to these intermediate differentiated cells. Here we report the construction of a delivery system from B19V subunits that maintains the highly selective cell-entry of the native virus and offers versatile cargo transport. To obtain this specific carrier, we conjugated the cell-targeting VP1u region of B19V to NeutrAvidin as a loading platform for biotinylated cargos. The VP1u-NeutrAvidin conjugate delivered fluorophores, DNA, and toxic payloads specifically to erythroid cells around the proerythroblast differentiation stage, including erythroleukemic cells. The VP1u-NeutrAvidin represents a unique cell surface marker which exclusively detects intermediate erythroid differentiation stages. Furthermore, the cell-entering property of this viral-based targeting system offers opportunities for erythroid-specific drug delivery or gene therapy.