Enhanced Virus Clearance by Early Inducible Lymphocytic Choriomeningitis Virus-Neutralizing Antibodies in Immunoglobulin-Transgenic Mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin μ heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host’s capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin μ heavy and the κ light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.Against many cytopathic viruses such as poliovirus, influenza virus, rabies virus, and vesicular stomatitis virus, protective virus-neutralizing antibodies are generated early, within 1 week after infection (3, 31, 36, 44, 49). In contrast, several noncytopathic viruses (e.g., human immunodeficiency virus and hepatitis viruses B and C in humans or lymphocytic choriomeningitis virus [LCMV] in mice) elicit poor and delayed virus-neutralizing antibody responses (1, 7, 20, 24, 27, 35, 45, 48).In the mouse, the natural host of LCMV, the acute LCMV infection is predominantly controlled by cytotoxic T lymphocytes (CTLs) in an obligatory perforin-dependent manner (13, 18, 28, 50). In addition to the CTL response, LCMV-specific antibodies are generated. Early after infection (by day 8), a strong antibody response specific for the internal viral nucleoprotein (NP) is mounted (7, 19, 23, 28). These early LCMV NP-specific antibodies exhibit no virus-neutralizing capacity (7, 10). Results from studies of B-cell-depleted mice and B-cell-deficient mice implied that the early LCMV NP-specific antibodies are not involved in the clearance of LCMV (8, 11, 12, 40). Late after infection (between days 30 and day 60), LCMV-neutralizing antibodies develop (7, 19, 22, 28, 33); these antibodies are directed against the surface glycoprotein (GP) of LCMV (9, 10). LCMV-neutralizing antibodies have an important function in protection against reinfection (4, 6, 38, 41, 47).In some viral infections, subprotective virus-neutralizing antibody titers can enhance disease rather than promote host recovery (i.e., exhibit antibody-dependent enhancement of disease [ADE] [14, 15, 21, 46]). For example, neutralizing antibodies are involved in the resolution of a primary dengue virus infection and in the protection against reinfection. However, if subprotective neutralizing antibody titers are present at the time of reinfection, a severe form of the disease (dengue hemorrhagic fever/dengue shock syndrome [15, 21]), which might be caused by Fc receptor-mediated uptake of virus-antibody complexes leading to an enhanced infection of monocytes (15, 16, 25, 39), can develop. Similarly, an enhancement of disease after intracerebral (i.c.) LCMV infection was observed in mice which had been treated with virus-neutralizing antibodies before the virus challenge (6). ADE in LCMV-infected mice was either due to an enhanced infection of monocytes by Fc receptor-mediated uptake of antibody-virus complexes or due to CTL-mediated immunopathology caused by an imbalanced virus spread and CTL response.To analyze whether LCMV-neutralizing antibodies generated early after infection improve the host’s capacity to clear the virus or enhance immunopathological disease, immunoglobulin (Ig)-transgenic mice expressing LCMV-neutralizing IgM antibodies were generated. After LCMV infection of transgenic mice expressing the Ig heavy chain (H25 transgenic mice), LCMV-neutralizing serum antibodies were mounted within 8 days, which significantly improved the host’s capacity to eliminate LCMV. H25 transgenic mice did not show any signs of ADE after i.c. LCMV infection.Transgenic mice expressing the Ig heavy and light chains (HL25 transgenic mice) exhibited spontaneous LCMV-neutralizing serum antibodies and confirmed the protective role of preexisting LCMV-neutralizing antibodies, even though the neutralizing serum antibodies were of the IgM isotype. Similar to mice which had been treated with LCMV-neutralizing antibodies, HL25 transgenic mice developed an enhanced disease after i.c. LCMV infection, which indicated that ADE was due to an imbalance between virus spread and CTL response. Thus, the early-inducible LCMV-neutralizing antibody response significantly enhanced clearance of the acute infection without any risk of causing ADE.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Mtv-1 Superantigen Trafficks Independently of Major Histocompatibility Complex Class II Directly to the B-Cell Surface by the Exocytic Pathway

Presentation of the Mtv-1 superantigen (vSag1) to specific V_β-bearing T cells requires association with major histocompatibility complex class II molecules. The intracellular route by which vSag1 trafficks to the cell surface and the site of vSag1-class II complex assembly in antigen-presenting B lymphocytes have not been determined. Here, we show that vSag1 trafficks independently of class II to the plasma membrane by the exocytic secretory pathway. At the surface of B cells, vSag1 associates primarily with mature peptide-bound class II αβ dimers, which are stable in sodium dodecyl sulfate. vSag1 is unstable on the cell surface in the absence of class II, and reagents that alter the surface expression of vSag1 and the conformation of class II molecules affect vSag1 stimulation of superantigen reactive T cells.

T lymphocytes respond to peptide antigens presented by either major histocompatibility complex (MHC) class I or class II molecules. Many viruses have evolved sophisticated strategies that interfere with antigen presentation by infected cells in order to escape recognition by T lymphocytes. Most strategies studied rely on disrupting MHC class I presentation, either by affecting components of the processing machinery that generate and transport viral peptides into the endoplasmic reticulum (ER) or by retarding transport or targeting class I molecules into the degradation pathway (for a review, see reference 73).In contrast, mouse mammary tumor virus (MMTV) utilizes T-cell stimulation to promote its life cycle. MMTVs encode within their 3′ long terminal repeat a viral superantigen (vSag), and coexpression of the Sag glycoprotein with MHC class II molecules on the surface of virally infected B cells induces V_β-specific T-cell stimulation, generating an immune response which is critical for amplification of MMTV and ensures vertical transmission of virus to the next generation (13, 29, 30). In the absence of B cells, MHC class II, or Sag-reactive T cells, the infection is short-lived (5, 6, 24, 28). The assembly and functional expression of vSag-class II complexes are therefore essential to the viral life cycle. When inherited as germ line elements, Mtv proviruses expressing vSags during ontogeny trigger V_β-specific clonal elimination of immature T cells and profoundly shape the T-cell repertoire (for a review, see reference 1).vSags are type II integral membrane glycoproteins (14, 36). They possess up to six potential N-linked glycosylation sites, and carbohydrate addition is essential for vSag stability and activity (45). Their protein sequence is highly conserved among all MMTV strains except at the C-terminal 29 to 32 residues, which vary and confer T-cell V_β specificity (77). Biochemical analyses of vSag7 (minor lymphocyte stimulating locus 1, Mls-1^a) molecular forms after transfection into a murine B-cell line have identified a predominant 45-kDa endo-β-N-acetylglucosaminidase H (endo H)-sensitive ER-resident glycoprotein, as well as multiple highly glycosylated forms (74). It is thought that an 18-kDa C-terminal fragment binds MHC class II products (75). It has also been suggested that vSags associate weakly with class II in the ER and that proteolytic processing is required for the efficient assembly of vSag-class II complexes for presentation to T cells (46, 49, 75). As yet, the intracellular route that vSags take to the cell surface, the compartment in which they bind class II, and whether they associate with peptide-loaded class II dimers have been enigmatic.Newly synthesized MHC class II αβ heterodimers assemble with invariant chain (Ii), a type II integral membrane protein, to form an oligomeric complex in the ER (37). Ii prevents class II heterodimers from binding peptides in the ER and Golgi complex (55), and signals in its cytoplasmic tail sort the complex into the endocytic pathway (4, 42). In this acidic, protease-rich compartment, Ii is degraded and class II binds antigenic peptides. After the formation of peptide-class II dimers, the complexes are exported to the plasma membrane (8, 48). In the absence of Ii, class II αβ heterodimers exhibit defective post-ER transport, and their conversion into functionally mature, sodium dodecyl sulfate (SDS)-stable compact dimers by peptide antigens is affected (7, 16, 22, 70).A specialized endosomal compartment where class II peptide loading occurs, termed the MHC class II-enriched compartment (MIIC or CIIV), has been found recently in antigen-presenting cells (2, 50, 53, 58, 68, 71). Whether nascent Ii-class II complexes traffic directly to the MIIC from the trans-Golgi network (TGN) or transit first to early endosomes, either directly or via the cell surface, before entering late endocytic vesicles and MIIC is still under debate (26, 56, 57). Transport by all these routes most probably occurs to ensure the capture and loading of antigenic peptides throughout the endocytic pathway (12). MIIC vesicles are positive for lysosome-associated membrane proteins (LAMPs) and cathepsin D and are enriched for HLA-DM or H-2M (18, 32, 59), proteins that facilitate the catalytic exchange of class II-associated invariant peptide chain (CLIP) for antigenic peptides (19, 61, 62). The ultrastructural colocalization of DM with intracellular peptide-class II complexes suggests that the MIIC is a main site where class II dimers bind exogenous and endogenous peptide antigens (47, 58).Determining the route by which vSag protein(s) trafficks to the cell surface and the cellular location where vSag1 processing and assembly with class II molecules occurs is central to understanding the mechanism whereby vSags activate T cells to maintain the viral life cycle. It has been unclear whether vSags traffic independently by the constitutive exocytic pathway or with class II and Ii to the MIIC before reaching the cell surface. Reagents that alter class II expression have been shown to affect vSag presentation (43, 46). Furthermore, mice lacking Ii show reduced intrathymic V_β-specific T-cell deletion (70), suggesting that Ii may play a role, either by ensuring proper maturation of class II dimers or by targeting vSag-class II complexes to the MIIC, in promoting efficient vSag-induced immune responses.To investigate these issues, we used immunochemical detection of vSag1 protein in combination with subcellular fractionation and surface reexpression assays. We show that class II is required for stable vSag1 surface expression. vSag1 trafficks directly to the cell surface independently of class II, and reagents that alter the conversion of newly synthesized class II into peptide-loaded SDS-stable dimers affect functional vSag1 surface expression.     

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.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

In Vivo Identification of Nuclear Factors Interacting with the Conserved Elements of Box C/D Small Nucleolar RNAs

The U16 small nucleolar RNA (snoRNA) is encoded by the third intron of the L1 (L4, according to the novel nomenclature) ribosomal protein gene of Xenopus laevis and originates from processing of the pre-mRNA in which it resides. The U16 snoRNA belongs to the box C/D snoRNA family, whose members are known to assemble in ribonucleoprotein particles (snoRNPs) containing the protein fibrillarin. We have utilized U16 snoRNA in order to characterize the factors that interact with the conserved elements common to the other members of the box C/D class. In this study, we have analyzed the in vivo assembly of U16 snoRNP particles in X. laevis oocytes and identified the proteins which interact with the RNA by label transfer after UV cross-linking. This analysis revealed two proteins, of 40- and 68-kDa apparent molecular size, which require intact boxes C and D together with the conserved 5′,3′-terminal stem for binding. Immunoprecipitation experiments showed that the p40 protein corresponds to fibrillarin, indicating that this protein is intimately associated with the RNA. We propose that fibrillarin and p68 represent the RNA-binding factors common to box C/D snoRNPs and that both proteins are essential for the assembly of snoRNP particles and the stabilization of the snoRNA.One of the most interesting recent findings related to ribosome biogenesis has been the identification of a large number of small RNAs localized in the nucleolus (snoRNAs). So far, more than 60 snoRNAs have been identified in vertebrates (17), and more than 30 have been identified in yeast (2). The total number of snoRNAs is not known, but it is likely to be close to 200 (33, 38). These snoRNAs, with the exception of the mitochondrial RNA processing (MRP) species (38), can be grouped into two major families on the basis of conserved structural and sequence elements. The first group includes molecules referred to as box C/D snoRNAs, whereas the second one comprises the species belonging to the box H/ACA family (2, 15).The two families differ in many aspects. The box C/D snoRNAs are functionally heterogeneous. Most of them function as antisense RNAs in site-specific ribose methylation of the pre-rRNA (1, 10, 17, 26); a minority have been shown to play a direct role in pre-rRNA processing in both yeast and metazoan cells (11, 21). The box C/D snoRNAs play their role by means of unusually long (up to 21 contiguous nucleotides) regions of complementarity to highly conserved sequences of 28S and 18S rRNAs (1). In contrast, several members of the H/ACA RNA family have been shown to direct site-specific isomerization of uridines into pseudouridines and to display shorter regions of complementarity to rRNA (14, 24). Mutational analysis suggests that H/ACA snoRNAs can also play a role as antisense RNAs by base pairing with complementary regions on rRNA (15, 24).Another difference between the two families can be seen by comparison of secondary structures. A Y-shaped motif, where a 5′,3′-terminal stem adjoins the C and D conserved elements, has been proposed for many box C/D snoRNAs (16, 26, 40, 42), whereas box H/ACA snoRNAs have been proposed to fold into two conserved hairpin structures connected by a single-stranded hinge region, followed by a short 3′ tail (15).Despite these differences, analogies have been found in the roles played by the conserved box elements. Mutational analysis and competition experiments indicated that C/D and H/ACA boxes are required both for processing and stable accumulation of the mature snoRNA, suggesting that they represent binding sites for specific trans-acting factors (2, 3, 8, 15, 16, 28, 36, 41).All snoRNAs are associated with proteins to form specific ribonucleoparticles (snoRNPs). The study of these particles began only recently, and so far, very few aspects of their structure and biosynthesis have been clarified. The only detailed analysis performed was on the mammalian U3 (19) and the yeast snR30 (20) snoRNPs. Of the identified components, a few appear to be more general factors: fibrillarin, which was shown to be associated with C/D snoRNPs (3, 4, 8, 13, 28, 31, 39), and the nucleolar protein GAR1, which was found associated with H/ACA snoRNAs in yeast (20). Just as the study of small nuclear RNP (snRNP) particles was crucial to the understanding of the splicing process, a detailed structural and functional analysis of snoRNP particles will be essential to elucidate the complex process of ribosome biosynthesis.In this study, we have analyzed the snoRNP assembly of wild-type and mutant U16 snoRNAs by following the kinetics of complex formation in the in vivo system of the Xenopus laevis oocyte. By a UV cross-linking technique, we have identified two proteins, of 40- and 68-kDa apparent molecular mass, which require intact boxes C and D together with the terminal stem for their binding. The 40-kDa species is specifically recognized by fibrillarin antibodies, indicating that this protein is intimately associated with the RNA.