Role of glycosphingolipid microdomains in CD4-dependent HIV-1 fusion

The fusion of HIV-1 with the plasma membrane of CD4+ cells is triggered by the interaction of HIV-1 surface envelope glycoprotein gp120 with the CD4 receptor, and requires coreceptors (CCR5 and CXCR4). Recent advances in the study of HIV-1 entry into CD4+ cells suggest that glycosphingolipids (GSL) may also participate in the fusion process. GSL are organized in functional microdomains which are associated with specific membrane proteins such as CD4. GSL-enriched microdomains were purified from human lymphocytes and reconstituted as a monomolecular film at the air-water interface of a Langmuir film balance. Surface pressure measurements allowed to characterize the sequential interaction of GSL with CD4 and with gp120. Using this approach, we identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main lymphocyte GSL recognized by gp120. In both cases, the interaction was saturable and dramatically increased by CD4. We propose that GSL microdomains behave as moving platforms allowing the recruitment of HIV-1 coreceptors after the initial interaction between the viral particle and CD4. According to this model, the GSL microdomain may: i) stabilize the attachment of the virus with the cell surface through multiple low affinity interactions between the V3 domain of gp120 and the carbohydrate moiety of GSL, and ii) convey the virus to an appropriate coreceptor by moving freely in the outer leaflet of the plasma membrane. This model can be extrapolated to all envelope viruses (e.g. influenza virus) that use cell surface GSL of the host cells as receptors or coreceptors.     

Role of glycosphingolipid microdomains in CD4-dependent HIV-1 fusion

The fusion of HIV-1 with the plasma membrane of CD4⁺ cells is triggered by the interaction of HIV-1 surface envelope glycoprotein gp120 with the CD4 receptor, and requires coreceptors (CCR5 and CXCR4). Recent advances in the study of HIV-1 entry into CD4⁺ cells suggest that glycosphingolipids (GSL) may also participate in the fusion process. GSL are organized in functional microdomains which are associated with specific membrane proteins such as CD4. GSL-enriched microdomains were purified from human lymphocytes and reconstituted as a monomolecular film at the air–water interface of a Langmuir film balance. Surface pressure measurements allowed to characterize the sequential interaction of GSL with CD4 and with gp120. Using this approach, we identified globotriaosylceramide (Gb3) and ganglioside GM3 as the main lymphocyte GSL recognized by gp120. In both cases, the interaction was saturable and dramatically increased by CD4. We propose that GSL microdomains behave as moving platforms allowing the recruitment of HIV-1 coreceptors after the initial interaction between the viral particle and CD4. According to this model, the GSL microdomain may : i) stabilize the attachment of the virus with the cell surface through multiple low affinity interactions between the V3 domain of gp120 and the carbohydrate moiety of GSL, and ii) convey the virus to an appropriate coreceptor by moving freely in the outer leaflet of the plasma membrane. This model can be extrapolated to all envelope viruses (e.g. influenza virus) that use cell surface GSL of the host cells as receptors or coreceptors.     

Parvovirus B19 Uptake Is a Highly Selective Process Controlled by VP1u,a Novel Determinant of Viral Tropism

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.     

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02.

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.     

The Globoside Receptor Triggers Structural Changes in the B19 Virus Capsid That Facilitate Virus Internalization

Globoside (Gb4Cer), Ku80 autoantigen, and α5β1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections.Human parvovirus B19 (B19V) belongs to the Erythrovirus genus of the Parvoviridae family. The virus has a worldwide distribution and typically causes a mild childhood febrile illness known as erythema infectiosum or fifth disease. In patients with underlying immunologic and hematologic disorders, B19V has been associated with more severe manifestations, such as arthropathies, aplastic anemia, hydrops fetalis, and fetal death (4).B19V has a single-stranded DNA genome encapsidated in a T=1 nonenveloped icosahedral capsid. The capsid is assembled from two structural proteins, VP1 (83 kDa) and VP2 (58 kDa). VP1 is identical to VP2, with the exception of 227 amino acids (aa) in the N-terminal part, the so-called VP1 unique region (VP1u) (9, 26). Despite VP1u being the minor component of the capsid, the dominant immune response against B19V is elicited by the VP1u region, which harbors strong neutralizing epitopes (2, 31, 41). A secreted phospholipase A₂ (PLA₂)-like activity has been located in the VP1 unique region of B19V (12), which is required for infection (13, 17, 40). Despite all these properties, we recently showed that VP1u is not accessible to antibodies. However, brief exposure to mild temperatures or low pH can render this region accessible (30). In this sense, B19V is similar to other parvoviruses in which VP1u is not accessible but can become exposed in vitro by mild heat or low-pH treatment (10, 21). In all parvoviruses tested so far, VP1u becomes exposed during the intracellular trafficking of the virus (18, 23, 28, 32, 33). However, B19V VP1u harbors strong neutralizing epitopes, meaning that its accessibility to antibody binding should occur prior to uptake by cells. In line with this hypothesis, we have demonstrated that incubation of B19V with red blood cells, which allow virus binding but not virus internalization, can trigger the externalization of VP1u in a proportion of the bound particles (3).The glycosphingolipid globoside (globotetraosylceramide [Gb4Cer]) is the cellular receptor of B19V (5, 6). Gb4Cer is largely expressed in human erythroid progenitor cells in the bone marrow, which are the main target cells for the virus. However, the pathogenicity and tropism of B19V cannot be explained if Gb4Cer is the only receptor. Previous studies have suggested that Gb4Cer is necessary for B19V to bind to cells but is not sufficient for cell entry (35). Subsequently, α5β1 integrin (36, 37) and the Ku80 autoantigen (25) were identified as coreceptors for B19V infection. While Ku80 might assist in virus attachment (25), α5β1 integrin is thought to be required for internalization (36, 37). In line with a complex mechanism of internalization based on multiple receptors is the observation that B19V does not stably bind membrane-associated globoside in vitro (20), indicating that B19V probably binds globoside jointly with other molecular structures present on cell membranes.In the present studies, the interaction of B19V with cell surface receptors and the implication of this interaction for the capsid structure were examined. The cells chosen for this study were of the erythropoietin (Epo)-dependent bone marrow megakaryoblastic leukemia UT7/Epo cell line, which is commonly used to study B19V infection. UT7/Epo cells support viral DNA replication and protein expression; however, intracellular factors limit the production of infectious progeny virus. The results indicate that B19V interacts dynamically with cell surface receptors and that internalization is a complex process involving sequential steps. B19V binds initially to Gb4Cer, which triggers the externalization of VP1u. The modified capsid is then ready for a subsequent interaction. Whenever the second interaction is not possible, the virus detaches from Gb4Cer and is ready for another attempt. This mechanism of “detachment-reattachment” is repeated until the required second interaction occurs, after which the virus is internalized.     

Characterization of Parvovirus B19 genotype 2 in KU812Ep6 cells

An infectious parvovirus B19 (B19V) genotype 2 variant was identified as a high-titer contaminant in a human plasma donation. Genome analysis revealed a 138-bp insertion within the p6 promoter. The inserted sequence was represented by an additional 30 bp from the end of the inverted terminal repeat adjacent to a 108-bp element found also, in inverted orientation, at the extreme right end of the unique sequence of the genome. However, despite the profound variations in the promoter region, the pattern of gene expression and DNA replication did not differ between genotype 1 and genotype 2 in permissive erythroid KU812Ep6 cells. Capsid proteins of both genotypes differ in their amino acid sequences. However, equivalent kinetics of virus inactivation at 56 degrees C or pH 4 indicated a comparable physicochemical stability of virus capsids. Sera from six individuals infected by B19V genotype 1 were investigated on cross-neutralization of B19V genotype 2 in vitro. Similar neutralization of both B19V genotypes was observed in sera from three individuals, while the sera from three other individuals showed weaker cross-neutralization for genotype 2. In conclusion, the in vitro replication characteristics and physical stability of B19V capsids are very similar between human parvovirus B19 genotypes 1 and 2, and cross-neutralization indicates a close antigenic relation of genotypes 1 and 2.     

Productive parvovirus B19 infection of primary human erythroid progenitor cells at hypoxia is regulated by STAT5A and MEK signaling but not HIFα

Human parvovirus B19 (B19V) causes a variety of human diseases. Disease outcomes of bone marrow failure in patients with high turnover of red blood cells and immunocompromised conditions, and fetal hydrops in pregnant women are resulted from the targeting and destruction of specifically erythroid progenitors of the human bone marrow by B19V. Although the ex vivo expanded erythroid progenitor cells recently used for studies of B19V infection are highly permissive, they produce progeny viruses inefficiently. In the current study, we aimed to identify the mechanism that underlies productive B19V infection of erythroid progenitor cells cultured in a physiologically relevant environment. Here, we demonstrate an effective reverse genetic system of B19V, and that B19V infection of ex vivo expanded erythroid progenitor cells at 1% O(2) (hypoxia) produces progeny viruses continuously and efficiently at a level of approximately 10 times higher than that seen in the context of normoxia. With regard to mechanism, we show that hypoxia promotes replication of the B19V genome within the nucleus, and that this is independent of the canonical PHD/HIFα pathway, but dependent on STAT5A and MEK/ERK signaling. We further show that simultaneous upregulation of STAT5A signaling and down-regulation of MEK/ERK signaling boosts the level of B19V infection in erythroid progenitor cells under normoxia to that in cells under hypoxia. We conclude that B19V infection of ex vivo expanded erythroid progenitor cells at hypoxia closely mimics native infection of erythroid progenitors in human bone marrow, maintains erythroid progenitors at a stage conducive to efficient production of progeny viruses, and is regulated by the STAT5A and MEK/ERK pathways.     

Adamantyl glycosphingolipids provide a new approach to the selective regulation of cellular glycosphingolipid metabolism

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl- or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 μM adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 μM adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 μM adaGalCer, cell synthesis of only Gb(3) and Gb(4) was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb(2)), was generated, indicating substrate competition for Gb(3) synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb(3) synthesis was inhibited by adaGalCer. Metabolic labeling of Gb(3) in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb(2) was produced. AdaGb(2) in cells was 10-fold more effectively shed into the medium than the more polar Gb(3), providing an easily eliminated "safety valve" alternative to Gb(3) accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients.     

Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody

The structure of virus-like particles of the lymphotropic, immunosuppressive strain of minute virus of mice (MVMi) in complex with the neutralizing Fab fragment of the mouse monoclonal antibody (MAb) B7 was determined by cryo-electron microscopy to 7-A resolution. The Fab molecule recognizes a conformational epitope at the vertex of a three-fold protrusion on the viral surface, thereby simultaneously engaging three symmetry-related viral proteins in binding. The location of the epitope close to the three-fold axis is consistent with the previous analysis of MVMi mutants able to escape from the B7 antibody. The binding site close to the symmetry axes sterically forbids the binding of more than one Fab molecule per spike. MAb as well as the Fab molecules inhibits the binding of the minute virus of mice (MVM) to permissive cells but can also neutralize MVM postattachment. This finding suggests that the interaction of B7 with three symmetry-related viral subunits at each spike hinders structural transitions in the viral capsid essential during viral entry.     

Human parvovirus b19 DNA replication induces a DNA damage response that is dispensable for cell cycle arrest at phase g2/m

Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G(2)/M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G(2)/M significantly, during B19V infection.     

Recombinant human parvovirus B19 vectors: erythrocyte P antigen is necessary but not sufficient for successful transduction of human hematopoietic cells

The blood group P antigen, known to be abundantly expressed on erythroid cells, has been reported to be the cellular receptor for parvovirus B19. We have described the development of recombinant parvovirus B19 vectors with which high-efficiency, erythroid lineage-restricted transduction can be achieved (S. Ponnazhagan, K. A. Weigel, S. P. Raikwar, P. Mukherjee, M. C. Yoder, and A. Srivastava, J. Virol. 72:5224-5230, 1998). However, since a low-level transduction of nonerythroid cells could also be detected and since P antigen is expressed in nonerythroid cells, we reevaluated the role of P antigen in the viral binding and entry into cells. Cell surface expression analyses revealed that approximately 75% of primary human bone marrow mononuclear erythroid cells and approximately 31% of cells in the nonerythroid population were positive for P antigen. Two human erythroleukemia cell lines, HEL and K562, and a human promyelocytic leukemia cell line, HL-60, were also examined for P antigen expression and binding and entry of the vector. HEL and K562 cells showed intermediate levels, whereas HL-60 cells demonstrated high levels of expression of P antigen. However, the efficiency of vector binding to these cells did not correlate with P antigen expression. Moreover, despite P antigen positivity and efficient viral binding, HEL, K562, and HL-60 cells could not be transduced with the vector. Low levels of P antigen expression could also be detected in two primary cell types, human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblasts (NHLF). In addition, vector binding occurred in both cell types and was inhibited by globoside, indicating the involvement of P antigen in virus binding to these cells. These primary cells could be efficiently transduced with the recombinant vector. These data suggest that (i) P antigen is expressed on a variety of cell types and is involved in binding of parvovirus B19 to human cells, (ii) the level of P antigen expression does not correlate with the efficiency of viral binding, (iii) P antigen is necessary but not sufficient for parvovirus B19 entry into cells, and (iv) parvovirus B19 vectors can be used to transduce HUVEC and NHLF. These studies further suggest the existence of a putative cellular coreceptor for efficient entry of parvovirus B19 into human cells.     

Stable association of herpes simplex virus with target membranes is triggered by low pH in the presence of the gD receptor, HVEM

Using a liposome-binding assay, we investigated the requirements for activation of herpes simplex virus (HSV) into a state capable of membrane interaction. Virions were mixed with liposomes along with the ectodomain of one of three gD receptors (HVEMt, nectin-1t, or nectin-2t) and incubated under different pH and temperature conditions. Virions failed to associate with liposomes in the presence of nectin-1 or nectin-2 at any temperature or pH tested. In contrast, HVEMt triggered association of HSV with liposomes at pH 5.3 or 5.0 when incubated at 37 degrees C, suggesting that HVEM binding and mildly acidic pH at a physiological temperature provide coactivation signals, allowing virus association with membranes. Virions incubated with HVEMt at 37 degrees C without liposomes rapidly lost infectivity upon exposure to pH 5.0, suggesting that these conditions lead to irreversible virus inactivation in the absence of target membranes. Consistent with the idea that soluble receptor molecules provide a trigger for HSV entry, HVEMt promoted virus entry into receptor-deficient CHO K1 cells. However, in B78H1 cells, HVEMt promoted virus entry with markedly lower efficiency. Interestingly, HSV entry into receptor-bearing CHO K1 cells has been shown to proceed via a pH-dependent manner, whereas HSV entry into receptor-bearing B78H1 cells is pH independent. Based on these observations, we propose that the changes triggered by HVEM and mildly acidic pH that allow liposome association are similar or identical to changes that occur during pH-dependent HSV entry.     

Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells from normal human bone marrow.

The target cell specificity of the B19 parvovirus infection was examined by isolating highly enriched hematopoietic progenitor and stem cells from normal human bone marrow. The efficiency of the B19 parvovirus replication in enriched erythroid progenitor cells was approximately 100-fold greater than that in unseparated bone marrow cells. The more-primitive progenitor cells identical to or closely related to the human pluripotent hematopoietic stem cells, on the other hand, did not support viral replication. The B19 progeny virus produced by the enriched erythroid progenitor cells was infectious and strongly suppressed erythropoiesis in vitro. The susceptibility of both the more-primitive erythroid progenitors (burst-forming units-erythroid) and the more-mature erythroid progenitors (CFU-erythroid) to the cytolytic response of the virus and the lack of effect on the myeloid progenitors (CFU-granulocyte-macrophage) further give evidence to the remarkable tropism of the B19 parvovirus for human hematopoietic cells of erythroid lineage.     

Specific binding sites for a parvovirus, minute virus of mice, on cultured mouse cells.

The early interactions between parvoviruses and host cells have not been extensively described previously. In this study we have characterized some aspects of viral binding to the cell surface and demonstrated the existence of specific cellular receptor sites for minute virus of mice (MVM) on two murine cell lines that are permissive for viral growth. The interaction had a pH optimum of 7.0 to 7.2, and both the rate and extent of the reactions were slightly affected by temperature. Mouse A-9 cells (L-cell derivative) had approximately 5 X 10(5) specific MVM binding sites per cell, and Friend erythroleukemia cells had 1.5 X 10(5) MVM sites per cell. In contrast, the nonpermissive mouse lymphoid cell line L1210 lacked specific viral receptors. Also, cloned lines of A-9 cells resistant to viral infection have been isolated. One of these lines lacked the "specific" virus attachment sites but exhibited low levels of nonsaturable virus binding. Based on these examples, infectivity is correlated with the presence of specific viral receptors on the cell surface.