Modeling subacute sclerosing panencephalitis in a transgenic mouse system: uncoding pathogenesis of disease and illuminating components of immune control.

Subacute sclerosing panencephalitis (SSPE) is a chronic neurodegenerative disease of the central nervous system (CNS) that afflicts eight to 20 individuals per one million of those who become infected with measles virus (MV). The six cardinal elements of SSPE are: (1) progressive fatal CNS disease developing several years after MV infection begins; (2) replication of MV in neurons; (3) defective nonreplicating MV in the CNS that is recoverable by co-cultivation with permissive tissue culture cells; (4) biased hypermutation of the MV recovered from the CNS with massive A to G (U to C) base changes primarily in the M gene of the virus; (5) high titers of antibody to MV; and (6) infiltration of B and T cells into the CNS. All these parameters can be mimicked in a transgenic (tg) mouse model that expresses the MV receptor, thus enabling infection of a usually uninfectable mouse in which the immune system is or is not manipulated. Utilization and analysis of such mice have illuminated how chronic measles virus infection of neurons can be initiated and maintained, leading to the SSPE phenotype. Further, an active role in prolonging MV replication while inhibiting its spread in the CNS can be mapped to a direct affect of the biased hypermutations (A to G changes) of the MV M gene in vivo.

Type I interferon during viral infections: multiple triggers for a multifunctional mediator.

Type I interferons (IFN-I) orchestrate numerous biological and cellular processes and are essential elements during host antiviral defense. After recognition of highly conserved virus signatures, a complex network of signaling events is rapidly initiated and leads to IFN-I synthesis. These cytokines directly induce a strong antiviral state and exert several immune-regulatory actions aimed at preventing virus spread. On the other hand, viruses evolved to evade or subvert the IFN-I system for their own benefit. In the present article, we review selective aspects of IFN-I induction and functions during several viral infections and discuss the beneficial and detrimental roles of IFN-I illustrated during lymphocytic choriomeningitis virus (LCMV) infection in its natural host, the mouse.

Molecular mimicry, microbial infection, and autoimmune disease: evolution of the concept.

Molecular mimicry is defined as similar structures shared by molecules from dissimilar genes or by their protein products. Either several linear amino acids or their conformational fit may be shared, even though their origins are separate. Hence, during a viral or microbe infection, if that organism shares cross-reactive epitopes for B or T cells with the host, then the response to the infecting agent will also attack the host, causing autoimmune disease. A variation on this theme is when a second, third, or repeated infection(s) shares cross-reactive B or T cell epitopes with the first (initiating) virus but not necessarily the host. In this instance, the secondary infectious agents increase the number of antiviral/antihost effector antibodies or T cells that potentiate or precipitate the autoimmune assault. The formation of this concept initially via study of monoclonal antibody or clone T cell cross-recognition in vitro through its evolution to in vivo animal models and to selected human diseases is explored in this mini-review.

Molecular and cellular mechanisms, pathogenesis, and treatment of insulin-dependent diabetes obtained through study of a transgenic model of molecular mimicry.

The portrait of autoimmune diabetes mellitus or type I diabetes can be copied by a transgenic model in which either the nucleoprotein (NP) or glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is expressed in beta cells of the islets of Langerhans. In the absence of further environmental insult, diabetes does not occur. However, when LCMV or a dissimilar virus that shares cross-reactive T cell epitopes with LCMV initiates infection, diabetes ensues. If the self "viral" transgene is expressed only in the beta cells, then diabetes occurs acutely within 8 to 12 days. Specific antiviral (self) CD8 T cells are mandatory for disease, but CD4 T cells are not. In this instance, diabetes can occur in the absence of infection if interferon gamma or B7.1 molecules are also expressed in the islets but not when IL-2, IL-4, IL-10, or IL-12 is similarly expressed. In contrast, both CD8 and CD4 antiviral (self) specific T cells are required when the self "viral" transgene is expressed concomitantly in beta cells and in the thymus. In this instance, infection by LCMV or cross-reacting virus is essential to cause diabetes. Further, the time from onset of infection until disease depends, in part, on the host's MHC background and its quantitative influence on negative selection of high-avidity antiviral (self) T cells. Knowledge of the cells, their numbers, and the molecules required to cause diabetes allows the design of successful strategies to treat and prevent the autoimmune disease.

Infection of dendritic cells by lymphocytic choriomeningitis virus.

Dendritic cells (DCs) comprise the major antigen-presenting cells (APCs) of the host, uniquely programmed to stimulate immunologically naïve T lymphocytes. Viruses that can target and disorder the function of these cells enjoy a selective advantage. The cellular receptor for lymphocytic choriomeningitis virus (LCMV), Lassa fever virus (LFV), and several other arenaviruses is alpha-dystroglycan (alpha-DG). Among cells of the immune system, CD11c+ and DEC-205+ DCs primarily and preferentially express alpha-DG. By selection, strains and variants of LCMV generated as quasi-species that bind alpha-DG with high affinity replicate in the majority of CD11c+ and DEC-205+ (>75%) DCs, causing a generalized immunosuppression, and establish a persistent infection. In contrast, viral strains and variants that bind with low affinity to alpha-DG display minimal replication in CD11c+ and DEC-205+ DCs (<10%), rarely replicate in the white pulp, and generate a robust anti-LCMV CTL response that clears the virus infection. Hence, receptor-virus interaction on DCs in vivo is an essential step in the initiation of virus-induced immunosuppression and viral persistence. Investigation into the mechanism of how virus-infected DCs cause immunosuppression reveals loss of MHC class II surface expression and costimulatory molecules on surface of such DCs. As a consequence DCs are unable to act as APCs, initiate immune responses, and have a defect in migration into the T cell area. These data indicate that LCMV infection influences DC maturation and migration, leading to decreased T cell stimulatory capacity of DCs, events essential for the initiation of immune responses. Because several other viruses known to cause immunosuppression (HIV, measles) interact with DCs, the observations noted here are likely a common selective mechanism by which viruses also are able to evade the host's immune system.

Constitutive beta cell expression of IL-12 does not perturb self-tolerance but intensifies established autoimmune diabetes.

To analyze the function of the Th1-promoting cytokine IL-12 in vivo, we generated transgenic (tg) mice (RIP-IL12 mice) whose pancreatic beta cells constitutively express bioactive IL-12 or one of its components, p35 or p40. In contrast to non-tg littermates or single-tg RIP-p35 and RIP-p40 mice, RIP-IL12 mice developed a marked pancreatic infiltration of lymphocytes and macrophages, mainly around islets. Expression of bioactive IL-12 primarily upregulated transcript levels of IFN-inducible protein-10 (IP-10), RANTES, IFN-gamma, and TNF-alpha in the pancreas. Despite the substantial recruitment of mononuclear cells, no biochemical or clinical disease was evident in the exocrine or endocrine pancreas. Coexpression of lymphocytic choriomeningitis virus (LCMV) proteins with IL-12 in the beta cells failed to spontaneously activate or expand antigen-specific anti-self/viral T cells in uninfected tg animals. However, when RIP-IL12 x RIP-LCMV tg mice were infected with LCMV, antigen-specific anti-self/viral T cells were induced, which led to an acceleration in the kinetics and severity of insulin-dependent diabetes mellitus (IDDM). Thus, the ectopic expression of IL-12 does not spontaneously break tolerance and activate antigen-specific T cells in the periphery, but it does worsen ongoing autoimmune disease.

Molecular analysis of the interaction of LCMV with its cellular receptor [alpha]-dystroglycan.

alpha-Dystroglycan (DG) has been identified as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). This subunit of DG is a highly versatile cell surface molecule that provides a molecular link between the extracellular matrix (ECM) and a beta-DG transmembrane component, which interacts with the actin-based cytoskeleton. In addition, DG exhibits a complex pattern of interaction with a wide variety of ECM and cellular proteins. In the present study, we characterized the binding of LCMV to alpha-DG and addressed the role of alpha-DG-associated host-derived proteins in virus infection. We found that the COOH-terminal region of alpha-DG's first globular domain and the NH2-terminal region of the mucin-related structures of alpha-DG together form the binding site for LCMV. The virus-alpha-DG binding unlike ECM alpha-DG interactions was not dependent on divalent cations. Despite such differences in binding, LCMV and laminin-1 use, in part, an overlapping binding site on alpha-DG, and the ability of an LCMV isolate to compete with laminin-1 for receptor binding is determined by its binding affinity to alpha-DG. This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to alpha-DG, tissue tropism, and pathological potential. LCMV strains and variants with high binding affinity to alpha-DG but not low affinity binders are able to infect CD11c+ dendritic cells, which express alpha-DG at their surface. Infection followed by dysfunction of these antigen-presenting cells contributes to immunosuppression and persistent viral infection in vivo.

Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory.

Emerging evidence indicates that CD8+ and CD4+ T-cell immunity is differentially regulated. Here we have delineated differences and commonalities among antiviral T-cell responses by enumeration and functional profiling of eight specific CD8+ and CD4+ T-cell populations during primary, memory and recall responses. A high degree of coordinate regulation among all specific T-cell populations stood out against an approximately 20-fold lower peak expansion and prolonged contraction phase of specific CD4+ T-cell populations. Surprisingly, although CD8+ T-cell memory was stably maintained for life, levels of specific CD4+ memory T cells gradually declined. However, this decay, which seemed to result from less efficient rescue from apoptosis, did not affect functionality of surviving virus-specific CD4+ T cells. Our results indicate that CD4+ T-cell memory might become limiting under physiological conditions and that conditions precipitating CD4+ T-cell loss might compromise protective immunity even in the presence of unimpaired CD8+ T-cell responses.

Common antiviral cytotoxic t-lymphocyte epitope for diverse arenaviruses.

Members of the Arenaviridae family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521-1551, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505-1519, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups-LFV, Junin, Machupo, and Sabia viruses-cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for H-2(d) mice (32). This L(d)-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of H-2(d) BALB mice. NP 118-126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L(d). The primary H-2(d) CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118-126 recognized target cells coated with NP 118-126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.

Disease model: dissecting the pathogenesis of the measles virus.

Host-pathogen interactions of measles virus (MV), a leading cause of childhood mortality worldwide, are still poorly understood. Using transgenic mice that express the human MV receptor CD46, we generated models to study the pathogenesis of MV infection of the central nervous system (CNS) and immune system. CNS infection in CD46 transgenic mice allows replication and spread throughout neurons, inflammation, and ultimately death of the animals. CD46-transgenic mice can also be used to study immunosuppression, a hallmark of measles. Together with mouse knockout technology and a system for generating recombinant MVs, CD46 transgenic mice will ultimately lead to a better understanding of both viral and host factors contributing to disease.

Structural and functional identification of major histocompatibility complex class I-restricted self-peptides as naturally occurring molecular mimics of viral antigens. Possible role in CD8+ T cell-mediated, virus-induced autoimmune disease.

Structural similarity (molecular mimicry) between viral epitopes and self-peptides can lead to the induction of autoaggressive CD4(+) as well as CD8(+) T cell responses. Based on the flexibility of T cell receptor/antigen/major histocompatibility complex recognition, it has been proposed that a self-peptide could replace a viral epitope for T cell recognition and therefore participate in pathophysiological processes in which T cells are involved. To address this issue, we used, as a molecular model of viral antigen, the H-2D(b)-restricted immunodominant epitope nucleoprotein (NP)-(396-404) (FQPQNGQFI) of lymphocytic choriomeningitis virus (LCMV). We identified peptide sequences from murine self-proteins that share structural and functional homology with LCMV NP-(396-404) and that bound to H-2D(b) with high affinity. One of these self-peptides, derived from tumor necrosis factor receptor I (FGPSNWHFM, amino acids 302-310), maintained LCMV-specific CD8(+) T cells in an active state as observed both in vitro in cytotoxic assays and in vivo in a model of virus-induced autoimmune diabetes, the rat insulin promoter-LCMV NP transgenic mouse. The natural occurrence and molecular concentration at the surface of H-2(b) spleen cells of tumor necrosis factor receptor I-(302-310) were determined by on-line micro-high pressure liquid chromatography/mass spectrometry and supported its biological relevance.

Disruption of differentiated functions during viral infection in vivo. V. Mapping of a locus involved in susceptibility of mice to growth hormone deficiency due to persistent lymphocytic choriomeningitis virus infection.

Lymphocytic choriomeningitis virus (LCMV) Armstrong strain selectively and persistently infects the majority of growth hormone (GH) producing cells in the anterior lobe of pituitary glands of C3H/St mice but negligibly infects GH producing cells of BALB/WEHI mice (Oldstone et al., Virology 142, 175--182, 1985; Oldstone et al., Science 218, 1125--1127, 1982). Although infected GH cells remain free of structural damage, disrupted initiation of GH transcription (Klavinskis and Oldstone, J. Gen. Virol. 68, 1867--1873, 1989; Valsamakis et al., Virology 156, 214--220, 1987) occurs with a resultant decrease in the synthesis of GH, leading to a failure of growth and development (Oldstone et al., Science 218, 1125--1127, 1982). Microsatellite mapping of DNA obtained from 101 individual C3H/St x BALB/WEHI F1 x F1 mice shows that the growth failure correlates with host genes linked (P value 0.0008) on chromosome 17 just outside of the H-2D MHC site between D17 Mit24 and D17 Mit51, a distance of 2.5 cM. The genetic mapping done here excludes alpha-dystroglycan (alpha-DG), a known receptor for LCMV (Cao et al., Science 282, 2079--2081, 1998) in pathogenesis of GH disease, as alpha-DG is encoded in the mouse by a gene residing on chromosome 9 (Yotsumoto et al., Hum. Mol. Genet. 5, 1259--1267, 1996).

Evidence that the hypermutated M protein of a subacute sclerosing panencephalitis measles virus actively contributes to the chronic progressive CNS disease.

Subacute sclerosing panencephalitis (SSPE) is a progressive degenerative disease of the brain uniformly leading to death. Although caused by measles virus (MV), the virus recovered from patients with SSPE differs from wild-type MV; biologically SSPE virus is defective and its genome displays a variety of mutations among which biased replacements of many uridine by cytidine resides primarily in the matrix (M) gene. To address the question of whether the SSPE MVs with M mutations are passive in that they are not infectious, cannot spread within the CNS, and basically represent an end-stage result of a progressive infection or alternatively SSPE viruses are infectious, and their mutations enable them to persist and thereby cause a prolonged neurodegenerative disease, we utilized reverse genetics to generate an infectious virus in which the M gene of MV was replaced with the M gene of Biken strain SSPE MV and inoculated the recombinant virus into transgenic mice bearing the MV receptor. Our results indicate that despite biased hypermutations in the M gene, the virus is infectious in vivo and produces a protracted progressive infection with death occurring as long as 30 to 50 days after that caused by MV. In primary neuron cultures, the mutated M protein is not essential for MV replication, prevents colocalization of the viral N with membrane glycoproteins, and is associated with accumulation of nucleocapsids in cells' cytoplasm and nucleus.

Differences in affinity of binding of lymphocytic choriomeningitis virus strains to the cellular receptor alpha-dystroglycan correlate with viral tropism and disease kinetics.

alpha-Dystroglycan (alpha-DG) was recently identified as a receptor for lymphocytic choriomeningitis virus (LCMV) and several other arenaviruses, including Lassa fever virus (W. Cao, M. D. Henry, P. Borrow, H. Yamada, J. H. Elder, E. V. Ravkov, S. T. Nichol, R. W. Compans, K. P. Campbell, and M. B. A. Oldstone, Science 282:2079-2081, 1998). Data presented in this paper indicate that the affinity of binding of LCMV to alpha-DG determines viral tropism and the outcome of infection in mice. To characterize this relationship, we evaluated the interaction between alpha-DG and several LCMV strains, variants, and reassortants. These viruses could be divided into two groups with respect to affinity of binding to alpha-DG, dependence on this protein for cell entry, viral tropism, and disease course. Viruses that exhibited high-affinity binding to alpha-DG displayed a marked dependence on alpha-DG for cell entry and were blocked from infecting mouse 3T6 fibroblasts by 1 to 4 nM soluble alpha-DG. In addition, high-affinity binding to alpha-DG correlated with an ability to infiltrate the white pulp (T-dependent) area of the spleen, cause ablation of the cytotoxic T-lymphocyte (CTL) response by day 7 postinfection, and establish a persistent infection. In contrast, viruses with a lower affinity of binding to alpha-DG were only partially inhibited from infecting alpha-DG(-/-) embryonic stem cells and required a concentration of soluble alpha-DG higher than 100 nM to prevent infection of mouse 3T6 fibroblasts. These viruses that bound at low affinity were mainly restricted to the splenic red pulp, and the host generated an effective CTL response that rapidly cleared the infection. Reassortants of viruses that bound to alpha-DG at high and low affinities were used to map genes responsible for the differences described to the S RNA, containing the virus attachment protein glycoprotein 1.

Immunosuppression and resultant viral persistence by specific viral targeting of dendritic cells.

Among cells of the immune system, CD11c(+) and DEC-205(+) splenic dendritic cells primarily express the cellular receptor (alpha-dystroglycan [alpha-DG]) for lymphocytic choriomeningitis virus (LCMV). By selection, strains and variants of LCMV that bind alpha-DG with high affinity are associated with virus replication in the white pulp, show preferential replication in a majority of CD11c(+) and DEC-205(+) cells, cause immunosuppression, and establish a persistent infection. In contrast, viral strains and variants that bind with low affinity to alpha-DG are associated with viral replication in the red pulp, display minimal replication in CD11c(+) and DEC-205(+) cells, and generate a robust anti-LCMV cytotoxic T lymphocyte response that clears the virus infection. Differences in binding affinities can be mapped to a single amino acid change in the viral glycoprotein 1 ligand that binds to alpha-DG. These findings indicate that receptor-virus interaction on dendritic cells in vivo can be an essential step in the initiation of virus-induced immunosuppression and viral persistence.

Neither B lymphocytes nor antibodies directed against self antigens of the islets of Langerhans are required for development of virus-induced autoimmune diabetes.

We evaluated the role of the humoral arm of the immune response in causing or contributing to virus-induced diabetes. Transgenic mice expressing the nucleoprotein (NP) or glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter (RIP) in pancreatic beta cells (RIP-LCMV) and RIP-LCMV mice with genetic dysfunction of B cells (RIP-LCMV x microMT/microMT) were compared for development of diabetes after challenge with LCMV. After inoculation with LCMV, B and T lymphocytes and macrophages infiltrated into pancreatic islets in RIP-LCMV mice, and over 50% of these mice generated Abs against host insulin or glutamate decarboxylase. However, neither B cells nor the autoantibodies played a direct role in the initiation, kinetics, or severity of the virus-induced diabetes as judged by comparing disease in RIP-LCMV mice to littermates whose functional B cells were genetically eliminated. Furthermore, the quality and quantity of T lymphocyte and macrophage infiltration was similar in the B cell-deficient and non-B cell-deficient RIP-LCMV mice. Although the development of autoantibodies to islet Ags had no direct influence on the pathogenesis of insulin-dependent (type 1) diabetes mellitus, it served as a prediabetes marker, as such autoantibodies were often elevated before the onset of disease. Hence, the RIP-LCMV model is not only useful for understanding the pathogenetic mechanisms of how islets are destroyed and spared but also for evaluating therapeutic strategies before onset of clinical diabetes.

Generalized immunosuppression: how viruses undermine the immune response.

Following infection, a virus must battle against the host's immune response. Viruses have developed many ways to escape immune surveillance and downregulate the host's immune response. Some viruses cause a generalized immunosuppression, thereby inhibiting or depressing the immune response towards themselves as well as towards unrelated pathogens. This review will focus on the mechanisms involved in the three main human viral infections causing immunosuppression: measles, human immunodeficiency virus and cytomegalovirus. We will also discuss what has been learned from the extensively studied mouse models of viral-induced immunosuppression: lymphocytic choriomeningitis virus and Rauscher leukemia virus. All of these viruses that induce generalized immunosuppression appear to do so by very similar mechanisms. They hinder antigen presentation to T cells and/or hematopoiesis. We will highlight the similarities in the viral targets as well as present evidence for alternate mechanisms.