Theiler's viruses with mutations in loop I of VP1 lead to altered tropism and pathogenesis.
Theiler's murine encephalomyelitis viruses are picornaviruses that can infect the central nervous system. The DA strain produces an acute polioencephalomyelitis followed by a chronic demyelinating disease in its natural host, the mouse. The ability of DA virus to induce a demyelinating disease renders this virus infection a model for human demyelinating diseases such as multiple sclerosis. Here we describe the generation and characterization of DA virus mutants that contain specific mutations in the viral capsid protein VP1 at sites believed to be important contact regions for the cellular receptor(s). A mutant virus with a threonine-to-aspartate (T81D) substitution in VP1 loop I adjacent to the putative virus receptor binding site exhibited a large-plaque phenotype but had a slower replication cycle in vitro. When this mutant virus was injected into susceptible mice, an altered tropism was seen during the acute stage of the disease and the chronic demyelinating disease was not produced. A virus with a threonine-to-valine substitution (T81V) did not cause any changes in the pattern or extent of disease seen in mice, whereas a virus with a tryptophan substitution at this position (T81W) produced a similar acute disease but was attenuated for the development of the chronic disease. A change in amino acids in a hydrophobic patch located in the wall of the pit, VP1 position 91, to a hydrophilic threonine (V91T) resulted in a profound attenuation of the acute and chronic disease without persistence of virus. This report illustrates the importance of the loop I of VP1 and a site in the wall of the pit in pathogenesis and that amino acid substitutions at these sites result in altered virus-host interactions. (+info)
Distinct attenuation phenotypes caused by mutations in the translational starting window of Theiler's murine encephalomyelitis virus.
Upon initiation of translation of picornavirus RNA, the ribosome is believed to bind the internal ribosome entry site of the template and then to form a productive complex with a downstream RNA segment, the starting window. The presence or absence of an AUG triplet within the starting window of the RNA of Theiler's murine encephalomyelitis virus (a picornavirus) is known to modulate its neurovirulence. In this study, mutants of this virus in which the starting windows, lying upstream of the viral polyprotein reading frame, had AUGs with different nonoptimal contexts were engineered. Upon intracerebral inoculation of mice, the mutants proved to be partially attenuated, as judged by a significant increase in the dose causing paralysis in 50% of the animals (PD50). Mutants with similar PD50s might differ from one another by eliciting either a severe, fatal tetraplegy or only mild, recoverable neurologic lesions. Some of the mutants triggered a chronic inflammatory reaction in the white matter of the spinal cord in the absence of detectable viral RNA or antigen. Thus, point mutations changing the context of an AUG within the starting window outside the polyprotein reading frame may differently affect the morbidity and mortality caused by a viral infection and may result in distinct attenuation phenotypes. (+info)
Differentiation of M1 myeloid precursor cells into macrophages results in binding and infection by Theiler's murine encephalomyelitis virus and apoptosis.
Infection of susceptible mouse strains with BeAn, a less virulent strain of Theiler's murine encephalomyelitis virus (TMEV), results in immune system-mediated demyelinating lesions in the central nervous system (CNS) similar to those in multiple sclerosis. Since macrophages appear to carry the major detectable antigen burden in vivo, and purification of sufficient cell numbers from the CNS for detailed analysis is difficult, macrophage-like cell lines provide an accessible system with which to study virus-macrophage interactions. The myeloid precursor cell line M1 differentiates in response to cytokines and expresses many characteristics of tissue macrophages. Incubation of TMEV with undifferentiated M1 cells produced neither infection nor apoptosis, whereas differentiated M1 (M1-D) cells developed a restricted virus infection and changes indicative of apoptosis. Virus binding and RNA replication as well as cellular production of alpha/beta interferons increased with differentiation. Although the amount of infectious virus was highly restricted, BeAn-infected M1-D cells synthesized and appropriately processed virus capsid proteins at levels comparable to those for permissive BHK-21 cells. Analysis of Bcl-2 protein family expression in undifferentiated and differentiated cells suggests that susceptibility of M1-D cells to apoptosis may be controlled, in part, by expression of the proapoptotic alpha isoform of Bax and Bak. These data suggest that macrophage differentiation plays a role in susceptibility to TMEV infection and apoptosis. (+info)
Prevalent class I-restricted T-cell response to the Theiler's virus epitope Db:VP2121-130 in the absence of endogenous CD4 help, tumor necrosis factor alpha, gamma interferon, perforin, or costimulation through CD28.
C57BL/6 mice mount a cytotoxic T-lymphocyte (CTL) response against the Daniel's strain of Theiler's murine encephalomyelitis virus (TMEV) 7 days after infection and do not develop persistent infection or the demyelinating syndrome similar to multiple sclerosis seen in susceptible mice. The TMEV capsid peptide VP2121-130 sensitizes H-2Db+ target cells for killing by central-nervous-system-infiltrating lymphocytes (CNS-ILs) isolated from C57BL/6 mice infected intracranially. Db:VP2121-130 peptide tetramers were used to stain CD8(+) CNS-ILs, revealing that 50 to 63% of these cells bear receptors specific for VP2121-130 presented in the context of Db. No T cells bearing this specificity were found in the cervical lymph nodes or spleens of TMEV-infected mice. H-2(b) mice lacking CD4, class II, gamma interferon, or CD28 expression are susceptible to persistent virus infection but surprisingly still generate high frequencies of CD8(+), Db:VP2121-130-specific T cells. However, CD4-negative mice generate a lower frequency of Db:VP2121-130-specific T cells than do class II negative or normal H-2(b) animals. Resistant tumor necrosis factor alpha receptor I knockout mice also generate a high frequency of CD8(+) CNS-ILs specific for Db:VP2121-130. Furthermore, normally susceptible FVB mice that express a Db transgene generate Db:VP2121-130-specific CD8(+) CNS-ILs at a frequency similar to that of C57BL/6 mice. These results demonstrate that VP2121-130 presented in the context of Db is an immunodominant epitope in TMEV infection and that the frequency of the VP2121-130-specific CTLs appears to be independent of several key inflammatory mediators and genetic background but is regulated in part by the expression of CD4. (+info)
Two loci, Tmevp2 and Tmevp3, located on the telomeric region of chromosome 10, control the persistence of Theiler's virus in the central nervous system of mice.
Theiler's virus persistently infects the white matter of the spinal cord in susceptible strains of mice. This infection is associated with inflammation and primary demyelination and is studied as a model of multiple sclerosis. The H-2D gene is the major gene controlling viral persistence. However, the SJL/J strain is more susceptible than predicted by its H-2(s) haplotype. An (SJL/J x B10. S)F1 x B10.S backcross was analyzed, and one quantitative trait locus (QTL) was located in the telomeric region of chromosome 10 close to the Ifng locus. Another one was tentatively mapped to the telomeric region of chromosome 18, close to the Mbp locus. We now report the study of 14 congenic lines that carry different segments of these two chromosomes. Although the presence of a QTL on chromosome 18 was not confirmed, two loci controlling viral persistence were identified on chromosome 10 and named Tmevp2 and Tmevp3. Furthermore, the Ifng gene was excluded from the regions containing Tmevp2 and Tmevp3. Analysis of the mode of inheritance of Tmevp2 and Tmevp3 identified an effect of sex, with males being more infected than females. (+info)
Theiler's murine encephalomyelitis virus infection induces early expression of c-fos in astrocytes.
We have determined whether Theiler's murine encephalomyelitis virus (TMEV), a picornavirus that produces demyelination in genetically susceptible strains of mice, induces c-fos in pure quiescent cultures of mouse brain astrocytes. As observed in Northern blots, the expression of this immediate early gene increases in a dose-dependent manner, with its expression peaking at a multiplicity of infection of 100. The expression of c-fos is transient, peaking after 30 min and disappearing 2 h after infection. The virus is quickly internalized at 37 degrees C upon binding to its specific receptor located at the cell surface and is actively replicated in the cytoplasm of the astrocytes, as demonstrated by FACS flow cytometry. Using the same technique, nuclear translation of c-fos mRNA is also shown. The specificity of viral induction is demonstrated by its neutralization with TMEV-specific antibodies and by the fact that only viral particles and not purified protein components VP1, VP2, and VP3 induced proto-oncogene expression. This rapid induction of c-fos in astrocytes could be the first stage in the infection of these central nervous system cell populations by TMEV. The biological relevance of these findings is assessed by the demonstration of c-fos activation after viral infection in vivo. (+info)
Potential role of CD4+ T cell-mediated apoptosis of activated astrocytes in Theiler's virus-induced demyelination.
Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) into susceptible mouse strains results in a chronic, immune-mediated demyelinating disease similar to human multiple sclerosis. Here, we examined the role of astrocytes as an APC population in TMEV-induced demyelination and assessed the potential consequences of T cell activation following Ag presentation. IFN-gamma-pretreated astrocytes were able to process and present all the predominant T cell epitopes of TMEV to virus-specific T cell hybridomas, clones, as well as bulk T cells. Despite low levels of proliferation of T cells due to prostaglandins produced by astrocytes, such Ag presentation by activated astrocytes induced the production of IFN-gamma, a representative proinflammatory cytokine, in TMEV-specific Th cell clones derived from the CNS of virus-infected mice. Furthermore, these Th cell clones mediate lysis of the astrocytes in vitro in a Fas-dependent mechanism. TUNEL staining of CNS tissue demonstrates the presence of apoptotic GFAP+ cells in the white matter of TMEV-infected mice. These results strongly suggest that astrocytes could play an important role in the pathogenesis of TMEV-induced demyelination by activating T cells, subsequently leading to T cell-mediated apoptosis of astrocytes and thereby compromising the blood-brain barrier. (+info)
Diverse fine specificity and receptor repertoire of T cells reactive to the major VP1 epitope (VP1230-250) of Theiler's virus: V beta restriction correlates with T cell recognition of the c-terminal residue.
Theiler's murine encephalomyelitis virus induces chronic demyelinating disease in genetically susceptible mice. The histopathological and immunological manifestation of the disease closely resembles human multiple sclerosis, and, thus, this system serves as a relevant infectious model for multiple sclerosis. The pathogenesis of demyelination appears to be mediated by the inflammatory Th1 response to viral epitopes. In this study, T cell repertoire reactive to the major pathogenic VP1 epitope region (VP1233-250) was analyzed. Diverse minimal T cell epitopes were found within this region, and yet close to 50% of the VP1-reactive T cell hybridomas used V beta 16. The majority (8/11) of the V beta 16+ T cells required the C-terminal amino acid residue on the epitope, valine at position 245, and every T cell hybridoma recognizing this C-terminal residue expressed V beta 16. However, the complementarity-determining region 3 sequences of the V beta 16+ T cell hybridomas were markedly heterogeneous. In contrast, such a restriction was not found in the V alpha usage. Only restricted residues at this C-terminal position allowed for T cell activation, suggesting that V beta 16 may recognize this terminal residue. Further functional competition analysis for TCR and MHC class II-contacting residues indicate that many different residues can be involved in the class II and/or TCR binding depending on the T cell population, even if they recognize the identical minimal epitope region. Thus, recognition of the C-terminal residue of a minimal T cell epitope may associate with a particular V beta (but not V alpha) subfamily-specific sequence, resulting in a highly restricted V beta repertoire of the epitope-specific T cells. (+info)