Infection of Glial Cells by the Human Polyomavirus JC Is Mediated by an N-Linked Glycoprotein Containing Terminal α(2-6)-Linked Sialic Acids

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49–58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both α(2-3)- and α(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the α(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the α(2-3) or the α(2-6) linkage revealed that JCV preferentially interacts with α(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl N-acetyl-α-d-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.Approximately 70% of the human population worldwide is seropositive for JC virus (JCV). Like other polyomaviruses, JCV establishes a lifelong latent or persistent infection in its natural host (40, 49, 50, 68, 72). Reactivation of JCV in the setting of an underlying immunosuppressive illness, such as AIDS, is thought to lead to virus dissemination to the central nervous system (CNS) and subsequent infection of oligodendrocytes (37, 40, 66, 68). Reactivation of latent JCV genomes already present in the CNS has also been postulated to contribute to the development of progressive multifocal leukoencephalopathy (PML) following immunosuppression (19, 48, 55, 70, 75). Approximately 4 to 6% of AIDS patients will develop PML during the course of their illness (10). In the CNS, JCV specifically infects oligodendrocytes and astrocytes. Outside the CNS, JCV genomes have been identified in the urogenital system, in the lymphoid system, and in B lymphocytes (2, 17, 18, 30, 47, 59). In vitro, JCV infects human glial cells and, to a limited extent, human B lymphocytes (3, 4, 39, 41, 42). Recently, JCV infection of tonsillar stromal cells and CD34⁺ B-cell precursors has been described (47). These observations have led to the suggestion that JCV may persist in a lymphoid compartment and that B cells may play a role in trafficking of JCV to the CNS (4, 30, 47).Virus-receptor interactions play a major role in determining virus tropism and tissue-specific pathology associated with virus infection. Viruses that have a very narrow host range and tissue tropism, such as JCV, are often shown to interact with high affinity to a limited number of specific receptors present on susceptible cells (26, 44). In some instances, virus tropism is strictly determined by the presence of specific receptors that mediate binding and entry (7, 16, 27, 35, 46, 53, 56, 67, 73, 74, 76). In other instances, however, successful entry into a cell is necessary but not sufficient for virus growth (5, 8, 45, 57). In these cases, additional permissive factors that interact with viral regulatory elements are required.The receptor binding characteristics of several polyomaviruses have been described. The mouse polyomavirus (PyV) receptor is an N-linked glycoprotein containing terminal α(2-3)-linked sialic acid (12–14, 22, 28). Both the large and small plaque strains of PyV recognize α(2-3)-linked sialic acid. The small-plaque strain also recognizes a branched disialyl structure containing α(2-3)- and α(2-6)-linked sialic acids. Neither strain recognizes straight-chain α(2-6)-linked sialic acid. The ability of the large- and small-plaque strains of PyV to differentially recognize these sialic acid structures has been precisely mapped to a single amino acid in the major virus capsid protein VP1 (21). The large-plaque strains all contain a glycine at amino acid position 92 in VP1, and the small-plaque strains all contain a negatively charged glutamic acid at this position (21). In addition to forming small or large plaques, these strains also differ in the ability to induce tumors in mice (20). This finding suggests that receptor recognition plays an important role in the pathogenesis of PyV.The cell surface receptor for lymphotropic papovavirus (LPV) is an O-linked glycoprotein containing terminal α(2-6)-linked sialic acid (26, 33, 34). Infection with LPV is restricted to a subset of human B-cell lines, and recognition of specific receptors is a major determinant of the tropism of LPV for these cells (26).Unlike the other members of the polyomavirus family, infection of cells by simian virus 40 (SV40) is independent of cell surface sialic acids. Instead, SV40 infection is mediated by major histocompatibility complex (MHC)-encoded class I proteins (5, 11). MHC class I proteins also play a role in mediating the association of SV40 with caveolae, a prerequisite for successful targeting of the SV40 genome to the nucleus of a cell (1, 63). Not surprisingly, SV40 has been shown not to compete with the sialic acid-dependent polyomaviruses for binding to host cells (15, 26, 38, 58).Very little is known about the early steps of JCV binding to and infection of glial cells. Like other members of the polyomavirus family, JCV is known to interact with cell surface sialic acids (51, 52). A role for sialic acids in mediating infection of glial cells has not been described. It is also not known whether the sialic acid is linked to a glycoprotein or a glycolipid. In a previous report, we demonstrated that JCV bound to a limited number of cell surface receptors on SVG cells that were not shared by the related polyomavirus SV40 (38). In this report, we demonstrate that virus binding to and infection of SVG cells is dependent on an N-linked glycoprotein containing terminal α(2-3)- and α(2-6)-linked sialic acids. Competitive binding assays with sialic acid-specific lectins suggest that the virus preferentially interacts with α(2-6)-linked sialic acids. We are currently evaluating the role of this receptor in determining the tropism of JCV for glial cells and B cells.     

Dendritic Cells Loaded with Tumor B Cells Elicit Broad Immunity against Murine Gammaherpesvirus 68 but Fail To Prevent Long-Term Latency

It is still unknown whether a noninfectious gammaherpesvirus vaccine is able to prevent or reduce virus persistence. This led us to use dendritic cells loaded with tumor B cells as a vaccine approach for the murine gammaherpesvirus 68 (γHV68) model of infection. Dendritic cells loaded with UV-irradiated latently infected tumor B cells induce broad, strong, and long-lasting immunity against γHV68. Dendritic cell vaccination prevents the enlargement of lymph nodes and severely limits acute infection and early latency but does not prevent γHV68 from establishing long-term latency. Our findings support the concept that attenuated viruses may be the best vaccine option for preventing gammaherpesvirus persistence.Gammaherpesviruses have very high prevalence, infecting 95% of the world population. Natural infection does not induce sterilizing immunity (21, 30). Murine gammaherpesvirus 68 (γHV68) has important biological similarities to its human counterparts and is a good model for characterizing the immune response and for testing vaccine strategies (11, 33). Gammaherpesvirus vaccines designed to induce neutralizing antibodies reduce the incidence and symptoms of infectious mononucleosis (26) but are only minimally protective (1, 7, 22, 28). Peptide- or epitope-based vaccines that induce T-cell responses affect the early phase of infection but do not alter long-term latency (9, 17, 19, 29, 32). Infection with latency-attenuated viruses induces protection against a challenge with wild-type γHV68, although the vaccine virus persists in the host (6, 25, 30) except in the case of γHV68 AC-RTA (16). These findings with live-attenuated viruses reflect the ability of latency-defective viruses to elicit a wide range of humoral and cell-mediated immune responses and suggest that optimal broad immunity may achieve protection. Dendritic cells (DC) are at the core of the immune response, and they are also the main target of adjuvants. Ex vivo-loaded dendritic cells can induce humoral immunity and strong T-cell immunity (3) and accelerated generation of memory T cells (2). Dendritic cells loaded with multiple antigens could circumvent the narrow antigen specificity of peptide- or epitope-based vaccines and lack the safety concerns associated with live-attenuated herpesviruses. Thus, dendritic cell vaccination can be attractive where other approaches have failed or as a tool for elucidating mechanisms of immune protection. Here, we wanted to test whether dendritic cells loaded ex vivo with a broad range of viral antigens would ameliorate disease and confer protection to gammaherpesvirus infection by inducing strong and broad cellular and humoral immunity.     

Non-Antigen-Specific B-Cell Activation following Murine Gammaherpesvirus Infection Is CD4 Independent In Vitro but CD4 Dependent In Vivo

The murine gammaherpesvirus MHV-68 multiplies in the respiratory epithelium after intranasal inoculation, then spreads to infect B cells in lymphoid germinal centers. Exposing B cells to MHV-68 in vitro caused an increase in cell size, up-regulation of the CD69 activation marker, and immunoglobulin M (IgM) production. The infectious process in vivo was also associated with increased CD69 expression on B cells in the draining lymph nodes and spleen, together with a rise in total serum Ig. However, whereas the in vitro effect on B cells was entirely T-cell independent, evidence of in vivo B-cell activation was minimal in CD4⁺ T-cell-deficient (I-A^b−/−) or CD4⁺ T-cell-depleted mice. Furthermore, the Ig present at high levels in serum was predominantly of the IgG class. Surprisingly, the titer of influenza virus-specific serum IgG in previously immunized mice fell following MHV-68 infection, suggesting that there was relatively little activation of memory B cells. Thus, CD4⁺ T cells seemed both to amplify a direct viral activation of B cells in lymphoid tissue and to promote new Ig class switching despite a lack of obvious cognate antigen.Herpesvirus (HV) infections are often associated with non-antigen-specific B-cell activation (13, 14, 16, 21, 22). Although no definite role has been established for this process in viral pathogenesis, it is of particular interest in gammaherpesvirus (γ-HV) infections, since chronic B-cell stimulation may contribute to the oncogenesis (9, 15) associated with Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8) infections. Infection with EBV activates B cells expressing the immunoglobulin (Ig) V_4-34 gene (4), which is also overrepresented in certain lymphomas (6, 25). EBV-activated V_4-34-expressing B cells can undergo somatic mutation and isotype switching, indicating a participation in normal germinal-center interactions (5). The latent membrane protein 1 (LMP-1) of EBV, which has intracellular signaling substrates similar to those of CD40 (12), and LMP-2A, which can trigger lymphocyte activation (2), may both contribute to this process. However, analysis of lymphocyte interactions in vivo has not been possible with the human γ-HVs.The murine γ-HV-68 (MHV-68) is a natural γ-HV of small rodents that is related to EBV (8) and to HHV-8 (33). After intranasal (i.n.) infection of conventional mice, the virus spreads from the lung to the lymphoid tissue (29) and then persists in B lymphocytes (28) and in epithelial cells (27). This persistent infection is associated with an infectious mononucleosis-like illness (7, 20) characterized by a CD4-dependent splenomegaly and an increase in viral load (31). In BALB/c mice, MHV-68 causes an acute and apparently non-antigen-specific rise in total serum IgG (26). The virus-specific serum antibody response is, in contrast, relatively slow in onset and does not reach plateau levels until 2 to 3 months after infection (26). MHV-68-infected C57BL/6J (B6) mice have more IgG⁺ cells and fewer IgM⁺ cells in the spleen (18) than uninfected controls, but to what extent this represents normal immunity is unclear.There is evidence (3) of MHV-68 infection in splenic germinal centers, and both the non-antigen-specific B-cell activation and the CD4-dependent increase in viral load may reflect an exploitation by the virus of normal germinal-center function. The present analysis defines the need, or lack thereof, for CD4⁺ T-cell help to drive B-cell activation following in vitro or in vivo exposure to MHV-68.     

Distinct Pathways for Tumor Necrosis Factor Alpha and Ceramides in Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infection can be fatal to immunocompromised individuals. We have previously reported that gamma interferon and tumor necrosis factor alpha (TNF-α) synergistically inhibit HCMV replication in vitro. Ceramides have been described as second messengers induced by TNF-α. To investigate the mechanisms involved in the inhibition of HCMV by TNF-α, in the present study we have analyzed ceramide production by U373 MG astrocytoma cells and the effects of TNF-α versus ceramides on HCMV replication. Our results show that U373 MG cells did not produce ceramides upon incubation with TNF-α. Moreover, long-chain ceramides induced by treatment with exogenous bacterial sphingomyelinase inhibited HCMV replication in synergy with TNF-α. Surprisingly, short-chain permeant C6-ceramide increased viral replication. Our results show that the anti-HCMV activity of TNF-α is independent of ceramides. In addition, our results suggest that TNF-α and endogenous long-chain ceramides use separate pathways of cell signalling to inhibit HCMV replication, while permeant C6-ceramide appears to activate a third pathway leading to an opposite effect.Human cytomegalovirus (HCMV) infections are well controlled in the immunocompetent host. Cellular immune responses (CD4⁺ and CD8⁺ T cells and NK cells) which accompany both acute and latent infections (for a review, see reference 4) are thought to be the main components of this control. HCMV infection during immunosuppression such as in cancer, transplantations, or AIDS results in severe pathology (4). We have previously shown that tumor necrosis factor alpha (TNF-α), in synergy with gamma interferon (IFN-γ), inhibits the replication of HCMV (7). In mice, TNF-α is involved in the clearance of CMV infection (25). TNF-α is a cytokine with multiple effects which is produced by many cell types, including macrophages and CD8⁺ and CD4⁺ T lymphocytes (for a review, see reference 40), and is known to possess antiviral effects (20, 47). The molecular mechanisms involved in the signalling by TNF-α depend on the type of receptor, p55 (TNF-R1) or p75 (TNF-R2) (5), to which it binds. Some cells express only one type of TNF-α receptor; however, expression of these receptors is not always mutually exclusive (5). The cytotoxicity of TNF-α has been reported to be mediated by TNF-R1 (38), whose intracellular region carries a death domain which signals for programmed cell death (39). Signalling through TNF-R1 with specific antibodies can also protect Hep-G2 cells from vesicular stomatitis virus-mediated cytopathic effects (48). Ceramide production after TNF-α treatment has been widely reported (16, 19, 31) and has also been shown to depend on signalling through the TNF-R1 receptor (45). In these experiments concerning myeloid cells, TNF-α induced the activation of a sphingomyelinase, which cleaved sphingomyelin to release ceramide and phosphocholine. The production of ceramides can lead to cell apoptosis (11, 14, 23) or cell cycle arrest (13). Induction of apoptosis by TNF-α has been mimicked by exogenous sphingomyelinase and by synthetic, short-chain, permeant ceramides, which suggests that ceramides, as second messengers, are sufficient to induce the cytotoxic effects of TNF-α (11, 23). Acidic and neutral sphingomyelinases activated in different cell compartments may be responsible for the diverse effects of TNF-α (46), with the former being involved in signalling through NF-κB (34) and the latter being involved in signalling through a ceramide-activated protein kinase and phospholipase A₂ (46).One of the characteristics of HCMV infection is the increase in the content of intracellular DNA, reported to be of viral (3, 8, 18) or cellular (12, 37) origin. Since TNF-α has been known to display antiproliferative properties and to block cells in the G₁ phase (29), we initially tested its effects on the cell cycle of infected cells. Then, based on studies reporting that TNF-α induces ceramides in cells (16, 19, 31) and on a study showing the role of ceramide in cell cycle blockade (13), we originally postulated that ceramide was responsible for the antiviral effect of TNF-α. In the present study, we used astrocytoma cells (U373 MG) as a model for brain cells, which are important targets of HCMV in vivo (22). In contrast to fibroblasts, infected U373 MG cells release smaller quantities of virus particles even though all the cells were infected in our experiments. We believe that the U373 MG model is closer to HCMV infection in vivo. We show that ceramides are not produced by U373 MG cells upon incubation with even high concentrations of TNF-α. In addition, we demonstrate that exogenously added sphingomyelinase induces anti-HCMV effects whereas permeant C6-ceramide increases HCMV proliferation in U373 MG cells. This suggests that lipid second messengers can modulate HCMV infection and that TNF-α and ceramides use distinct signalling pathways in the control of HCMV infection. This is supported by our observation that the protein kinase JNK1 is activated exclusively by TNF-α in U373 MG cells and that TNF-α and exogenous sphingomyelinase act in synergy on HCMV infection.     

Quantitative Proteomic Analysis Reveals Metabolic Alterations,Calcium Dysregulation,and Increased Expression of Extracellular Matrix Proteins in Laminin α2 Chain–deficient Muscle

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain–deficient dy^3K/dy^3K mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain–deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).Congenital muscular dystrophy with laminin α2 chain deficiency, also known as MDC1A,¹ is a severe muscle wasting disease for which there is no cure. MDC1A is caused by mutations in the LAMA2 gene that lead to complete or partial deficiency of laminin α2 chain (1–3). Although the primary defect in MDC1A is known, the secondary molecular mechanisms eventually leading to muscle degeneration are not fully understood. In normal muscle, laminin α2 chain binds to the cell surface receptors dystroglycan and integrin α7β1, which both indirectly bind the cytoskeleton (4–7). Both of these adhesion complexes are important for normal skeletal muscle function, and laminin α2 chain binding to dystroglycan contributes to the maintenance of sarcolemmal integrity and protects muscles from damage (8), whereas laminin α2 chain binding to integrin α7β1 promotes myofiber survival (9, 10). In MDC1A, laminin α2 chain is absent or severely reduced, and the expression of dystroglycan and α7β1 is also dysregulated in MDC1A (9, 11, 12). Thus, the structural link is broken, and the yet to be determined downstream intracellular signaling pathways are also interrupted. Consequently, laminin α2 chain–deficient muscle fibers undergo degeneration–regeneration cycles, but rather quickly regeneration fails and muscle fibers die by apoptosis/necrosis followed by a major replacement of muscle tissue with connective tissue (3, 7). In order to unravel novel secondary molecular mechanisms, which could indicate new therapeutic targets, we decided to evaluate the protein expression profile in laminin α2 chain–deficient dy^3K/dy^3K muscle. Several proteomic profiling studies of dystrophin-deficient muscles (Duchenne muscular dystrophy) have been performed (13–20), as well as some with dysferlin-deficient muscles (Limb-girdle muscular dystrophy type 2B, Miyoshi myopathy) (21, 22). They all showed a great number of proteins that were differentially expressed in different dystrophic muscles and at different ages (13–22). However, proteomic analyses of laminin α2 chain–deficient muscle have not yet been performed. We here used multidimensional protein identification technology with tandem mass tags (TMT), a powerful shotgun label-based proteomic method that separates peptides in two-dimensional liquid chromatography (23, 24). We identified around 100 proteins that were differentially expressed in laminin α2 chain–deficient gastrocnemius and diaphragm muscles relative to the corresponding wild-type muscles, and the differential expression of selected proteins was verified with Western blot analysis or immunofluorescence.