LCMV and the central nervous system: uncovering basic principles of CNS physiology and virus-induced disease.
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Animals↗
Biomedical subjects
G F Rall
Publications and source records attributed to G F Rall.
Model Systems: transgenic mouse models for measles pathogenesis.
Studies of the diseases caused by measles virus (MV) in humans have been restricted owing to the lack of suitable animal models. The discovery of cellular receptors for MV entry has facilitated the development of transgenic mice that are susceptible to MV infection, and that mimic certain aspects of the central nervous system diseases and immunosuppression that can occur in infected humans. Moreover, such mouse models have allowed a clearer understanding of the contributions of the innate and adaptive immune response following infection, and will no doubt be important tools in the future for the development of new antiviral and vaccine reagents.
Acute Disease↗
The application of transgenic and knockout mouse technology for the study of viral pathogenesis.
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Animals↗
Protection of CD3 delta knockout mice from lymphocytic choriomeningitis virus-induced immunopathology: implications for viral neuroinvasion.
For a virus to establish a neuronal infection, it must spread from its primary site of infection to the central nervous system (CNS) before immune-mediated clearance occurs. Lymphocytic choriomeningitis virus (LCMV) is a murine pathogen that can result in persistent neuronal infection in newborn mice and in adults that lack CD8(+) T cells. To determine the neuroinvasive capacity of LCMV in the presence of an existent, but compromised, cytotoxic T lymphocyte response, the course of LCMV infection was examined in mice that possess 10% of the normal complement of T lymphocytes, due to the lack of the CD3 delta (delta) subunit of the T cell receptor complex (CD3 delta KO mice). Unlike immunocompetent mice that produced a massive immune response that caused death by 6-7 days postinfection, CD3 delta KO mice mounted a weak response and survived. The presence of viral antigen gradually shifted from the class I MHC-positive meninges and ependyma to class I MHC-deficient CNS neurons 10-30 days postinoculation. The infected CD3 delta KO mice developed a delayed T cell response that suppressed virus replication in peripheral tissues but not in the CNS; subsequent adoptive transfer experiments supported the hypothesis that the lack of clearance from neurons was due to sequestration of LCMV in an immune-privileged cell type. Based on these results, we propose that a critical parameter in the pathogenesis of neurotropic viruses is the rate of immune activation; individuals with impaired T cell responses may be more vulnerable to persisting CNS infections.
Adoptive Transfer↗
Measles virus spread between neurons requires cell contact but not CD46 expression, syncytium formation, or extracellular virus production.
In patients with subacute sclerosing panencephalitis (SSPE), which is associated with persistent measles virus (MV) infection in the brain, little infectious virus can be recovered despite the presence of viral RNA and protein. Based on studies of brain tissue from SSPE patients and our work with MV-infected NSE-CD46(+) mice, which express the measles receptor CD46 on neurons, several lines of evidence suggest that the mechanism of viral spread in the central nervous system differs from that in nonneuronal cells. To examine this alternate mechanism of viral spread, as well as the basis for the loss of normal transmission mechanisms, infection and spread of MV Edmonston was evaluated in primary CD46(+) neurons from transgenic mice and differentiated human NT2 neurons. As expected, unlike that between fibroblasts, viral spread between neurons occurred in the absence of syncytium formation and with minimal extracellular virus. Electron microscopy analysis showed that viral budding did not occur from the neuronal surface, although nucleocapsids were present in the cytoplasm and aligned at the cell membrane. We observed many examples of nucleocapsids present in the neuronal processes and aligned at presynaptic neuronal membranes. Cocultures of CD46(+) and CD46(-) neurons showed that cell contact but not CD46 expression is required for MV spread between neurons. Collectively, these results suggest that the neuronal environment prevents the normal mechanisms of MV spread between neurons at the level of viral assembly but allows an alternate, CD46-independent mechanism of viral transmission, possibly through the synapse.
Animals↗
Immune response-mediated protection of adult but not neonatal mice from neuron-restricted measles virus infection and central nervous system disease.
In many cases of neurological disease associated with viral infection, such as measles virus (MV)-induced subacute sclerosing panencephalitis in children, it is unclear whether the virus or the antiviral immune response within the brain is the cause of disease. MV inoculation of transgenic mice expressing the human MV receptor, CD46, exclusively in neurons resulted in neuronal infection and fatal encephalitis within 2 weeks in neonates, while mice older than 3 weeks of age were resistant to both infection and disease. At all ages, T lymphocytes infiltrated the brain in response to inoculation. To determine the role of lymphocytes in disease progression, CD46(+) mice were back-crossed to T- and B-cell-deficient RAG-2 knockout mice. The lymphocyte deficiency did not affect the outcome of disease in neonates, but adult CD46(+) RAG-2(-) mice were much more susceptible to both neuronal infection and central nervous system disease than their immunocompetent littermates. These results indicate that CD46-dependent MV infection of neurons, rather than the antiviral immune response in the brain, produces neurological disease in this model system and that immunocompetent adult mice, but not immunologically compromised or immature mice, are protected from infection.
Age Factors↗
CNS neurons: the basis and benefits of low class I major histocompatibility complex expression.
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Animals↗
A transgenic mouse model for measles virus infection of the brain.
In addition to the rash, fever, and upper respiratory tract congestion that are the hallmarks of acute measles virus (MV) infection, invasion of the central nervous system (CNS) can occur, establishing a persistent infection primarily in neurons. The recent identification of the human membrane glycoprotein, CD46, as the MV receptor allowed for the establishment of transgenic mice in which the CD46 gene was transcriptionally regulated by a neuron-specific promoter. Expression of the measles receptor rendered primary CD46-positive neurons permissive to infection with MV-Edmonston. Notably, viral transmission within these cultures occurred in the absence of extracellular virus, presumably via neuronal processes. No infection was seen in nontransgenic mice inoculated intracerebrally with MV-Edmonston. In contrast, scattered neurons were infected following inoculation of transgenic adults, and an impressive widespread neuronal infection was established in transgenic neonates. The neonatal infection resulted in severe CNS disease by 3-4 weeks after infection. Illness was characterized initially by awkward gait and a lack of mobility, and in later stages seizures leading to death. These results show that expression of the MV receptor on specific murine cells (neurons) in vivo is absolutely essential to confer both susceptibility to infection and neurologic disease by this human virus. The disparity in clinical findings between neonatal and adult transgenic mice indicates that differences exist between the developing and mature CNS with respect to MV infection and pathogenesis.
Animals↗
Deficits in discriminated learning remain despite clearance of long-term persistent viral infection in mice.
Mice persistently infected with lymphocytic choriomeningitis virus (LCMV) exhibit impaired learning ability. In this report, we determined whether clearance of the virus was associated with restoration of behavioral function. Neonatal Balb/cByJ mice were persistently infected with LCMV and tested as adults in a nonconditional spatial discrimination task. The presence of viral proteins in neurons was confirmed immunohistochemically and infectious virus was quantified in the blood by plaque assay. LCMV-infected adult mice made more errors in a Y-maze avoidance task compared to sham-inoculated controls. After the initial behavioral analysis, infected and control mice received a dose of cytotoxic T-lymphocytes sufficient to clear virus from these mice. Following complete clearance of the virus, mice were re-tested in the behavioral task, 5 months after the original test. No reversal of the learning deficit was seen following viral clearance; mice that had been cleared of the virus and those that remained persistently infected behaved similarly. These data indicate that persistent LCMV infection of the CNS lasting up to 7 months results in discriminated learning impairments that are not reversed by subsequent anti-viral immunocytotherapy.
Aging↗
Consequences of cytotoxic T lymphocyte interaction with major histocompatibility complex class I-expressing neurons in vivo.
Neurons have evolved strategies to evade immune surveillance that include an inability to synthesize the heavy chain of the class I major histocompatibility complex (MHC), proteins that are necessary for cytotoxic T lymphocyte (CTL) recognition of target cells. Multiple viruses have taken advantage of the lack of CTL-mediated recognition and killing of neurons by establishing persistent neuronal infections and thereby escaping attack by antiviral CTL. We have expressed a class I MHC molecule (Db) in neurons of transgenic mice using the neuron-specific enolase (NSE) promoter to determine the pathogenic consequences of CTL recognition of virally infected, MHC-expressing central nervous system (CNS) neurons. The NSE-Db transgene was expressed in H-2b founder mice, and transgene-derived messenger RNA was detected by reverse transcriptase-polymerase chain reaction in transgenic brains from several lines. Purified primary neurons from transgenic but not from nontransgenic mice adhered to coverslips coated with a conformation-dependent monoclonal antibody directed against the Dv molecule and presented viral peptide to CTL in an MHC-restricted manner, indicating that the Db molecule was expressed on transgenic neurons in a functional form. Transgenic mice infected with the neurotropic lymphocytic choriomeningitis virus (LCMV) and given anti-LCMV, MHC-restricted CTL displayed a high morbidity and mortality when compared with controls receiving MHC-mismatched CTL or expressing alternative transgenes. After CTL transfer, transgenic brains showed an increased number of CD8+ cells compared with nontransgenic controls as well as an increased rate of clearance of infectious virus from the CNS. Additionally, an increase in blood-brain barrier permeability was detected during viral clearance in NSE-Db transgenic mice and lasted several months after clearance of virus from neurons. In contrast, LCMV-infected, nontransgenic littermates and mice expressing other gene products from the NSE promoter showed no CNS disease, no increased intraparenchymal CTL, and no blood-brain barrier damage after the adoptive transfer of antiviral CTL. Our study indicates that viral infections and CTL-CNS interactions may induce blood-brain barrier disruptions and neurologic disease by a "hit-and-run" mechanism, triggering a cascade of pathogenic events that proceeds in the absence of continual viral stimulation.
Amino Acid Sequence↗
Virus-neuron-cytotoxic T lymphocyte interactions.
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Animals↗
Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent.
To study the effect of cell type-restricted hamster PrP expression on susceptibility to the hamster scrapie agent, we generated transgenic mice using a 1 kb hamster cDNA clone containing the 0.76 kb HPrP open reading frame under control of the neuron-specific enolase promoter. In these mice, expression of HPrP was detected only in brain tissue, with highest levels found in neurons of the cerebellum, hippocampus, thalamus, and cerebral cortex. These transgenic mice were susceptible to infection by the 263K strain of hamster scrapie with an average incubation period of 93 days, compared to 72 days in normal hamsters. In contrast, nontransgenic mice were not susceptible to this agent. These results indicate that neuron-specific expression of the 1 kb HPrP minigene including the HPrP open-reading frame is sufficient to mediate susceptibility to hamster scrapie, and that HPrP expression in nonneuronal brain cells is not necessary to overcome the TSE species barrier.
Animals↗
Central nervous system damage produced by expression of the HIV-1 coat protein gp120 in transgenic mice.
Many people infected with human immunodeficiency virus type 1 (HIV-1) develop neurological complications that can culminate in dementia and paralysis. The discrepancy between the severity of impairment and the paucity of detectable HIV-1 within neurons has led to an intense search for diffusible virus- and host-derived factors that might be neurotoxic (see ref. 2 for review). The HIV-1 envelope glycoprotein gp120 is an extracellular protein that is shed from infected cells and so has the potential to diffuse and interact with distant uninfected brain cells. Studies on cultured immature cells suggest that gp120 induces neurotoxicity (reviewed in refs 2, 4), and systemic injection of gp120 in neonatal rats and intracerebroventricular injection in adult rats results in deleterious effects on the brain. To assess the pathogenic potential of gp120 in the intact brain, we have now produced gp120 in the brains of transgenic mice and found a spectrum of neuronal and glial changes resembling abnormalities in brains of HIV-1-infected humans. The severity of damage correlated positively with the brain level of gp120 expression. These results provide in vivo evidence that gp120 plays a key part in HIV-1-associated nervous system impairment. This model should facilitate the evaluation and development of therapeutic strategies aimed at HIV-brain interactions.
AIDS Dementia Complex↗
A transgenic mouse model to assess the interaction of cytotoxic T lymphocytes with virally infected, class I MHC-expressing astrocytes.
Astrocytes provide crucial support for neurons and their impairment by viruses or their interactions with anti-viral or autoimmune responses could contribute to neurological disease. We have developed a transgenic mouse model to assess lymphocyte-astrocyte interactions. The major histocompatibility complex (MHC) class I molecule, Db, was expressed in astrocytes under the transcriptional control of regulatory sequences from the glial fibrillary acidic protein (GFAP) gene. Baseline cerebral MHC class I mRNA levels from transgenic mice were elevated over those of non-transgenic controls, and a prominent increase in cerebral MHC class I expression occurred following focal, injury-induced astroglial activation within transgenic brains but not in non-transgenic controls. FACS analysis of explant astrocyte cultures from established transgenic lines demonstrated astroglial expression of the GFAP-Db fusion gene at the protein level. Functional antigen-presenting capacity was conferred by the Db transgene, as virus-infected primary astrocytes obtained from transgenic BALB/c mice (KdIdDdLd) expressing the Db molecule were lysed by Db-restricted anti-viral CTL.
Animals↗
CD2-deficient mice generate virus-specific cytotoxic T lymphocytes upon infection with lymphocytic choriomeningitis virus.
The major host response to many viral infections is the generation of virus-specific CTL. Many protein molecules on the surfaces of both CTL and target cells interact to mediate adhesion of the cells and generate signals that lead to T cell activation and proliferation of virus-specific CTL that then mediate lysis of infected cells. One such protein, CD2, has been shown to increase the binding affinity of CTL to infected cells, and, in addition, enhance CTL activation signals. To determine whether virus-specific CTL could be generated in the absence of CD2, mice lacking a functional CD2 gene were infected with lymphocytic choriomeningitis virus (LCMV), and the responses to the virus were monitored. CD2-deficient mice infected intracerebrally with LCMV died as a consequence of CTL-mediated choriomeningitis, similar to control littermates. Additionally, CD2-deficient mice inoculated i.p. with LCMV cleared the infection by 2 wk postinfection, as did control mice. Viral clearance in these mice was shown to be due to the generation of a vigorous virus-specific MHC-restricted CTL response. Finally, to determine whether CD2 is essential for the generation of memory CTL, we examined the ability of CD2-deficient mice to generate memory CTL to LCMV and found normal memory CTL responses. Our results indicate that CD2 is not required for the generation of an LCMV-specific CTL response in vivo, nor is CD2 required for the maintenance or activation of memory CTL.
Animals↗
Mechanism and consequence of viral persistence in cells of the immune system and neurons.
Viral persistence depends on a virus having a non-lytic strategy of replication and the ability to escape immune surveillance. Cells of the immune system (lymphocytes/monocytes/macrophages) and central nervous system (neurons) are most often infected by DNA and RNA viruses that persist. Cytotoxic T lymphocytes (CTL) are the primary host defense that aborts or prevents viral persistence. Viral interaction with these specialized cells and of such infected cells with CTL is explored in this paper.
Animals↗
Cleavage of concatemeric DNA at the internal junction of "translocation" mutants of pseudorabies virus and inversion of their L component appear to be linked.
When pseudorabies virus (PrV) strains are grown in chicken embryo fibroblasts (CEF), variants ("translocation" mutants) arise in which there is a duplication of the leftmost sequences of the genome and their translocation in inverted orientation next to the internal inverted repeat bracketing the S component. In these variants, the UL becomes bracketed by inverted repeats and is found in two orientations relative to the Us. To study the cis-functions involved in cleavage of concatemeric DNA as well as those involved in inversion of the L component and to ascertain whether the two events are linked in the "translocation" mutants, a viral mutant (vLD68) was constructed in which the terminal 64 bp of the L component (that include sequences with homology to the pac 2 site of HSV) and the 4 terminal bp of the S component were deleted from the internal junction. Although revertants that have acquired the 68 bp at the internal junction emerge rapidly in populations of vLD68, analysis of the characteristics of this mutant revealed that: (1) the termini derived from both orientations of the L component include the 64 bp that have been deleted from the internal junction of vLD68; (2) in contrast to other "translocation" mutants, the internal junction of the vLD68 genome is not a good substrate for cleavage; (3) inversion of the L component of true vLD68 DNA does not occur or is rare; a good correlation exists in the populations of vLD68 between the proportion of revertants that have acquired an intact internal junction and the proportion of genomes with an L component that inverts. These results show that an intact internal junction in "translocation" mutants is necessary for both inversion of their L components and cleavage at their alternative internal junction. Since cleavage at the alternative junction will result in inversion of the L component, we conclude that inversion of the L component of "translocation" mutants of PrV can be attributed to cleavage of concatemeric DNA at the internal alternative junction.
Animals↗
Functions of the sequences at the ends of the inverted repeats of pseudorabies virus.
Two mutants were constructed to explore the functions of the sequences at the end of the S terminus of pseudorabies virus (PrV). In mutant vYa, 17 bp from the internal inverted repeat, as well as adjacent sequences from the L component, were deleted. In mutant v135/9, 143 bp from the internal inverted repeat (including sequences with homology to the pac-1 site of herpes simplex virus), as well as adjacent sequences from the L component, were deleted. Our aim in constructing these mutants was to ascertain whether equalization of the terminal regions of the S component would occur, whether genome termini that lack either the terminal 17 or 143 bp would be generated as a result of equalization of the repeats (thereby identifying the terminal nucleotides that may include cleavage signals), and whether inversion of the S component would occur (thereby ascertaining the importance of the deleted sequences in this process). The results obtained show the following (i) The removal of the terminal 17 or 143 bp of the internal S component, including the sequences with homology to the pac-1 site, does not affect the inversion of the Us. (ii) The equalization of both the vYa and the v135/9 inverted repeats occurs at high frequency, the terminal repeats being converted and becoming similar to the mutated internal inverted repeat. (iii) Mutants in which the 17 terminal base pairs (vYa) have been replaced by unrelated sequences are viable. However, the 143 terminal base pairs appear to be essential to virus survival; concatemeric v135/9 DNA with equalized, mutant-type, inverted repeats accumulates, but mature virions with such equalized repeats are not generated at high frequency. Since concatemeric DNA missing the 143 bp at both ends of the S component is not cleaved, the terminal 143 bp that include the sequences with homology to the pac-1 site are necessary for efficient cleavage. (iv) v135/9 intracellular DNA is composed mainly of arrays in which one S component (with two equalized inverted repeats both having the deletion) is bracketed by two L components in opposite orientations and in which two L components are in head-to-head alignment.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗