MHV infection of the CNS: mechanisms of immune-mediated control.

Mice infected with neurotropic strains of mouse hepatitis virus (MHV) clear infectious virus; nevertheless, viral persistence in the central nervous system (CNS) is associated with ongoing primary demyelination. Acute infection induces a potent regional CD8+ T-cell response. The high prevalence of virus specific T cells correlates with ex vivo cytolytic activity, interferon-gamma (IFN-gamma) secretion and efficient reduction in virus. Viral clearance from most cell types is controlled by a perforin dependent mechanism. However, IFN-gamma is essential for controlling virus replication in oligodendrocytes. Furthermore, CD4+ T cells enhance CD8+ T-cell survival and effectiveness. Clearance of infectious virus is associated with a gradual decline of CNS T cells; nevertheless, activated T cells are retained within the CNS. The loss of cytolytic activity, but retention of IFN-gamma secretion during viral clearance suggests stringent regulation of CD8+ T-cell effector function, possibly as a means to minimize CNS damage. However, similar CD8+ T-cell responses to demyelinating and non demyelinating JHMV variants support the notion that CD8+ T cells do not contribute to the demyelinating process. Although T-cell retention is tightly linked to the presence of persisting virus, contributions to regulating the latent state are unknown. Studies in B-cell-deficient mice suggest that antibodies are required to prevent virus recrudescence. Although acute JHMV infection is thus primarily controlled by CD8+ T cells, both CD4+ T cells and B cells make significant contributions in maintaining the balance between viral replication and immune control, thus allowing host and pathogen survival.

Role of viral persistence in retaining CD8(+) T cells within the central nervous system.

The continued presence of virus-specific CD8(+) T cells within the central nervous system (CNS) following resolution of acute viral encephalomyelitis implicates organ-specific retention. The role of viral persistence in locally maintaining T cells was investigated by infecting mice with either a demyelinating, paralytic (V-1) or nonpathogenic (V-2) variant of a neurotropic mouse hepatitis virus, which differ in the ability to persist within the CNS. Class I tetramer technology revealed more infiltrating virus-specific CD8(+) T cells during acute V-1 compared to V-2 infection. However, both total and virus-specific CD8(+) T cells accumulated at similar peak levels in spinal cords by day 10 postinfection (p.i.). Decreasing viral RNA levels in both brains and spinal cords following initial virus clearance coincided with an overall progressive loss of both total and virus-specific CD8(+) T cells. By 9 weeks p.i., T cells had largely disappeared from brains of both infected groups, consistent with the decline of viral RNA. T cells also completely disappeared from V-2-infected spinal cords coincident with the absence of viral RNA. By contrast, a significant number of CD8(+) T cells which contained detectable viral RNA were recovered from spinal cords of V-1-infected mice. The data indicate that residual virus from a primary CNS infection is a vital component in mediating local retention of both CD8(+) and CD4(+) T cells and that once minimal thresholds of stimuli are lost, T cells within the CNS cannot survive in an autonomous fashion.

Contributions of CD8+ T cells and viral spread to demyelinating disease.

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-gamma-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-gamma ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.

Functional activity of hepatocyte nuclear factor-1 is specifically decreased in amino acid-limited hepatoma cells.

Limitation of cultured rat hepatoma cells for an essential amino acid results in a specific decrease in expression of several genes that are preferentially expressed in the liver, including the serum albumin and transthyretin genes. In the work presented here, we examined whether the coordinate repression of these genes is caused by decreased activity of one or more of the liver-enriched transcription factors, hepatocyte nuclear factor-1 (HNF-1), HNF-3, HNF-4 or C/EBP. To address this question, HepG2 human hepatoma cells were transiently transfected with luciferase reporter constructs containing multiple copies of individual transcription factor binding sites. Limitation for an essential amino acid resulted in specific repression of a construct in which luciferase expression was directed by HNF-1. A single HNF-1 binding site located adjacent to the TATA box plays a major role in transcription directed by the serum albumin promoter in transient transfection assays. Amino acid limitation of cells transfected with an albumin promoter/luciferase reporter construct resulted in specific repression of promoter activity. In addition, bacterial methylation or site-directed mutagenesis of the HNF-1 binding site in the albumin proximal promoter region eliminated the regulation of an albumin promoter-luciferase reporter construct under conditions of amino acid limitation. These results demonstrated that the HNF-1 binding site played a major role in regulation of the albumin promoter by amino acid availability. Deletion analysis of the albumin promoter confirmed regulation through the HNF-1 binding site and also identified a second amino acid regulatory element in the upstream region of the albumin promoter, which has been shown previously to contain a functional binding site for HNF-3. The repression of albumin promoter and HNF-1 reporter constructs in amino acid-limited cells occurred without a change in the DNA binding activity of HNF-1. Moreover, HNF-3 DNA binding activity was also not decreased in amino acid-limited cells. These results suggest that the regulation of transcription by amino acids occurs at the level of transcriptional activation by HNF-1 and HNF-3, rather than by alteration of the DNA binding activity of either factor.

Antibody prevents virus reactivation within the central nervous system.

The neurotropic JHM strain of mouse hepatitis virus (JHMV) produces an acute CNS infection characterized by encephalomyelitis and demyelination. The immune response cannot completely eliminate virus, resulting in persistence associated with chronic ongoing CNS demyelination. The contribution of humoral immunity to viral clearance and persistent infection was investigated in mice homozygous for disruption of the Ig mu gene (IgM-/-). Acute disease developed with equal kinetics and severity in IgM-/- and syngeneic C57BL/6 (wt) mice. However, clinical disease progressed in IgM-/- mice, while wt mice recovered. Viral clearance during acute infection was similar in both groups, supporting a primary role of cell-mediated immunity in viral clearance. In contrast to wt mice, in which infectious virus was reduced to below detection following acute infection, increasing infectious virus was recovered from the CNS of the IgM-/- mice following initial clearance. No evidence was obtained for selection of variant viruses nor was there an apparent loss of cell-mediated immunity in the absence of Ab. Passive transfer of anti-JHMV Ab following initial clearance prevented reactivation of infectious virus within the CNS of IgM-/- mice. These data demonstrate the clearance of infectious virus during acute disease by cell-mediated immunity. However, immunologic control is not maintained in the absence of anti-viral Ab, resulting in recrudescence of infectious virus. These data suggest that humoral immunity plays no role in controlling virus during acute infection, but plays an important role in establishing and maintaining CNS viral persistence.

Selection of CD8+ T cells with highly focused specificity during viral persistence in the central nervous system.

The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR alpha/beta-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-gamma enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.

IFN-gamma is required for viral clearance from central nervous system oligodendroglia.

Infection of the central nervous system (CNS) by the JHM strain of mouse hepatitis virus (JHMV) is a rodent model of the human demyelinating disease multiple sclerosis. The inability of effective host immune responses to eliminate virus from the CNS results in a chronic infection associated with ongoing recurrent demyelination. JHMV infects a variety of CNS cell types during the acute phase of infection including ependymal cells, astrocytes, microglia, oligodendroglia, and rarely in neurons. Replication within the majority of CNS cell types is controlled by perforin-dependent virus-specific CTL. However, inhibition of viral replication in oligodendroglia occurs via a perforin-independent mechanism(s). The potential role for IFN-gamma as mediator controlling JHMV replication in oligodendroglia was examined in mice deficient in IFN-gamma secretion (IFN-gamma0/0 mice). IFN-gamma0/0 mice exhibited increased clinical symptoms and mortality associated with persistent virus, demonstrating an inability to control replication. Neither antiviral Ab nor CTL responses were diminished in the absence of IFN-gamma, although increased IgG1 was detected in IFN-gamma0/0 mice. Increased virus Ag in the absence of IFN-gamma localized almost exclusively to oligodendroglia and was associated with increased CD8+ T cells localized within white matter. These data suggest that although perforin-dependent CTL control virus replication within astrocytes and microglia, which constitute the majority of infected CNS cells, IFN-gamma is critical for control of viral replication in oligodendroglia. Therefore, different mechanisms are used by the host defenses to control virus replication within the CNS, dependent upon the phenotype of the targets of virus replication.

Upstream region of rat serum albumin gene promoter contributes to promoter activity: presence of functional binding site for hepatocyte nuclear factor-3.

Transcription of the serum albumin gene occurs almost exclusively in the liver and is controlled in part by a strong liver-specific promoter. The upstream region of the serum albumin gene promoter is highly conserved among species and is footprinted in vitro by a number of nuclear proteins. However, the role of the upstream promoter region in regulating transcription and the identity of the transcription factors that bind to this region have not been established. In the present study, deletion analysis of the rat serum albumin promoter in transiently transfected HepG2 cells demonstrated that elimination of the region between -207 and -153 bp caused a two-fold decrease in promoter activity (P<0.05). Additional analysis of the -207 to -124 bp promoter interval led to the identification of two potential binding sites for hepatocyte nuclear factor-3 (HNF-3) located at -168 to -157 bp (site X) and -145 to -134 bp (site Y). Electrophoretic mobility-shift assays performed with the HNF-3 X and Y sites demonstrated that both sites are capable of binding HNF-3alpha and HNF-3beta. Placement of a single copy of the HNF-3 X site upstream from a minimal promoter increased promoter activity by about four-fold in HepG2 cells, and the reporter construct containing this site could be transactivated if co-transfected with an HNF-3 expression construct. Furthermore, inactivation of the HNF-3 X site by site-directed mutagenesis within the context of the -261 bp albumin promoter construct resulted in a 40% decrease in transcription (P<0.05). These results indicate that the positive effect of the -207 to -153 bp promoter interval is attributable to the presence of the HNF-3 X site within this interval. Additional results obtained with transfected HepG2 cells suggest that the HNF-3 Y site plays a lesser role in activation of transcription than the X site.

LDL induces transcription factor activator protein-1 in human endothelial cells.

Low density lipoprotein (LDL) has been shown to perturb endothelial cells, with manifestations ranging from alterations in free radicals and arachidonate metabolism to stress fiber formation and monocyte recruitment. Some of these changes are regulated by LDL at the transcriptional level. Using mobility shift assays with consensus sequences for various transcription factors, we have detected an increase in activator protein 1 (AP-1), but not nuclear factor-kappaB (NF-kappaB), binding in human umbilical vein endothelial cells exposed to LDL. Following transfection, AP-1-driven chloramphenicol acetyltransferase and AP-1-driven-luciferase are upregulated by LDL. In contrast, there is no effect on NF-kappaB-driven chloramphenicol acetyltransferase. AP-1 increases in a biphasic fashion, with the first peak occurring 6 hours after and the second 48 hours after exposure to LDL. This AP-1 binding increase involves c-Jun, but not c-Fos, as shown by gel supershift, Northern hybridization, and Western blotting analyses. c-Jun mRNA levels are elevated by 9 hours after and remain so until at least 24 hours after exposure to LDL. c-Jun protein levels increase at 12 hours and continue to rise for 24 hours after exposure to LDL. Moreover, this LDL-increased AP-1 binding is suppressed by several protein kinase (PK) inhibitors: the PKC inhibitor calphostin C, the cAMP-dependent PK inhibitor H89, and the tyrosine PK inhibitors genistein and lavendustin A. This study demonstrates that (1) LDL is an endothelial agonist distinct from other cell stimulators, such as cytokines, endotoxin, and phorbol 12-myristate 13-acetate, because LDL appears to activate human umbilical vein endothelial cells predominantly through the transcription factor AP-1 and not NF-kappaB; and (2) LDL increases AP-1 via mechanisms involving multiple kinase activities and c-Jun transcription.

Effect of dietary protein restriction on liver transcription factors.

The transcription of several genes that are preferentially expressed in the liver, including the serum albumin, transthyretin and carbamyl phosphate synthetase-I genes, is specifically decreased in animals consuming inadequate amounts of dietary protein. The high level of transcription of these genes in the liver is directed in part by a number of liver-enriched transcription factors, including hepatocyte nuclear factors (HNF)-1, -3, and -4, and proteins of the CCAAT/enhancer-binding protein (C/EBP) family. In the present study, we investigated the possibility that the co-ordinate decrease in transcription of the nutritionally sensitive genes in protein-deprived rats results from altered activity of one or more of the liver-enriched transcription factors. For HNF-4, Western blots indicated no change in the level of nuclear HNF-4 protein in liver of protein-deprived animals, whereas we observed a 40% reduction in the DNA binding activity of HNF-4 as measured by electrophoretic mobility shift assay (EMSA). Furthermore, the binding affinity of HNF-4 for DNA was unaltered by dietary protein deprivation, while the number of HNF-4 molecules able to bind to DNA (Bmax) was reduced, as determined by Scatchard analysis. This indicates that in the protein-restricted rats a portion of the pool of HNF-4 protein is inactivated or otherwise prevented from binding to DNA. The overall DNA binding activity of C/EBP alpha and beta was increased in protein-restricted animals. This change occurred in the absence of a change in the amount of the full-length forms of these two proteins, quantified by Western blotting. Interestingly, dietary protein restriction specifically increased the level of a truncated form of C/EBP beta (liver-enriched transcriptional inhibitory protein, LIP), which is a protein dominant negative inhibitor of C/EBP function. Analysis of HNF-3 DNA-binding activity by EMSA revealed that HNF-3 alpha and beta DNA binding was increased and that HNF-3 gamma DNA-binding activity was unchanged in protein-restricted animals. We also detected two apparently novel shift complexes with the HNF-3 probe by EMSA, both of which were decreased in protein-restricted animals. HNF-1 DNA-binding activity was increased by dietary protein restriction. We also examined the effect of protein restriction on the DNA-binding activity of two ubiquitous transcription factors, NF1 and Sp1. The DNA binding activity of the major NF1 isoforms was unchanged whereas the binding activity of Sp1 was increased in the protein-restricted animals. In summary, restriction of dietary protein resulted in a number of specific changes in the DNA-binding activity of various transcription factors. Because transcriptional activation typically involves the synergistic action of more than one transcription factor, small changes in the amount/activity of several factors, could have a strong net effect on the transcription of many genes.

Protein restriction specifically decreases the abundance of serum albumin and transthyretin nuclear transcripts in rat liver.

The expression of the genes for serum albumin and several other plasma proteins is decreased in animals consuming inadequate amounts of dietary protein. To define the specificity of this phenomenon, we examined the effect of dietary protein restriction on the abundance of the mRNA for nine genes in rat liver. The results of this and previous studies indicate that genes in liver can be divided into two classes based on their response to protein restriction. Group I genes (albumin, transthyretin, carbamyl phosphate synthetase-I, class I alcohol dehydrogenase, insulin-like growth factor-I) exhibit decreased expression in response to protein restriction. In contrast, the expression of group II genes (hypoxanthine-guanine phosphoribosyl transferase, ubiquitin, H-ferritin, insulin-like growth factor binding proteins-1, -2 and -4) is either unchanged or increased in response to protein restriction. To investigate the molecular mechanism(s) leading to the decreased level of albumin and transthyretin mRNA in protein-restricted animals, the effect of protein restriction on the abundance of albumin and transthyretin nuclear transcripts was examined. The results demonstrated that protein restriction specifically decreased the abundance of albumin and transthyretin nuclear transcripts, indicating that the reduction in mRNA levels is caused at least partly by a decrease in gene transcription.

Effect of amino acid limitation on the expression of 19 genes in rat hepatoma cells.

We showed previously that the abundance of serum albumin mRNA is decreased in H4-II-E rat hepatoma cells limited for a single essential amino acid (phenylalanine, methionine, leucine, or tryptophan). To define the specificity of this phenomenon, we examined the effect of amino acid limitation on the abundance of mRNAs for 19 genes in the H4-II-E cells. These genes included six genes whose expression is either completely liver-specific or highly enriched in the liver compared with other tissues [albumin, transthyretin (TTR), transferrin, carbamyl phosphate synthetase-I, urate oxidase, class I alcohol dehydrogenase], as well as a number of ubiquitously expressed "housekeeping" genes. The results indicated that the 19 genes could be divided into three classes based on their response to amino acid limitation. Class I genes (the six liver-specific genes and alpha-tubulin) exhibit decreased expression in response to amino acid limitation. The expression of class II genes [beta 2-microglobulin, hypoxanthine-guanine phosphoribosyl transferase (HPRT), H-ferritin, ubiquitin (UbB), insulin-like growth factor binding protein-4, HNF-1 alpha] is not significantly affected by amino acid limitation. Class III genes [gadd153, beta-actin, ubiquitin (UbC), phosphoglycerate kinase-1, C/EBP alpha, C/EBP beta] exhibit increased expression in response to amino acid limitation. Thus, specific inductive as well as repressive effects on gene expression are quite common in amino acid-limited cells. The observation that all six genes whose expression is liver-specific exhibited decreased expression in amino acid-limited cells suggests a common mode of regulation of these genes by amino acid availability. The strong induction by amino acid limitation of the C/EBP inhibitor gadd153 is of interest in this regard, as increased levels of gadd153 could interfere with C/EBP, which is required for high expression of most liver-specific genes. To investigate further the molecular mechanism for the decrease in albumin mRNA abundance, albumin nuclear transcript levels were quantified in control and tryptophan-limited cells. Tryptophan limitation caused a decrease in albumin nuclear transcript abundance, and this decrease preceded the decrease in albumin mRNA, suggesting that the decrease in albumin mRNA was caused at least partly by a decrease in albumin gene transcription. Additional experiments with actinomycin D indicated that albumin mRNA was also destabilized in the tryptophan-limited cells. Thus, the overall results indicate that the decrease in albumin mRNA in the tryptophan-limited cells is caused by a specific decrease in albumin nuclear transcript abundance and destabilization of albumin mRNA.

The effect of fasting on insulin-like growth factor-I nuclear transcript abundance in rat liver.

The abundance of insulin-like growth factor I (IGF-I) messenger RNA (mRNA) is decreased in the liver of fasting, protein-restricted, and energy-restricted rats. The extent to which this decrease in steady state mRNA abundance may be attributed to a decrease in IGF-I gene transcription remains unresolved. In the present study, we used an RNase protection assay to quantify IGF-I nuclear transcript (pre-mRNA) and mRNA abundance in whole cellular RNA isolated from liver of fasted and nonfasted male rats (4-6 weeks of age). The results of the RNase protection assay of IGF-I nuclear transcripts were strongly correlated with the results of nuclear transcription elongation (run-on) assays (r > 0.90; P < 0.001). In addition, the RNase protection assay allows for a greater capability for sensitively monitoring gene transcription in a large number of samples. In four different experiments, a consistent decrease in the quantity of IGF-I nuclear transcripts was observed in liver of animals fasted for 72 h, whereas IGF-I pre-mRNA abundance in animals fed ad libitum was highly variable (average intraassay coefficient of variation = 74% vs. 34% for nonfasted and fasted groups). When data from the four experiments were pooled, fasting reduced IGF-I pre-mRNA and mRNA levels by 78% and 70% (P < 0.001), respectively. Fasting also caused a significant decrease in mRNA and nuclear transcript abundance for another nutritionally sensitive gene, the gene encoding transthyretin (TTR). To determine whether the decrease in IGF-I and TTR nuclear transcripts was gene specific, levels of nuclear transcripts for serum albumin, H-ferritin, and ribosomal RNA were also quantified. The results indicated that serum albumin, H-ferritin, and ribosomal RNA nuclear transcripts were not decreased by fasting, demonstrating that the negative effect of fasting was specific for IGF-I and TTR. In summary, these results indicate that IGF-I and TTR nuclear transcripts are specifically decreased by fasting. The decrease in IGF-I mRNA is matched by a similar decrease in IGF-I nuclear transcripts, suggesting that fasting controls IGF-I gene expression primarily at the transcriptional level.

Induction of insulin-like growth factor binding protein-1 gene expression in liver of protein-restricted rats and in rat hepatoma cells limited for a single amino acid.

Restriction of the dietary protein intake of young growing animals results in a rapid cessation of growth. In order to gain further insight into the molecular mechanisms for metabolic adaptation to protein restriction, the expression of the insulin-like growth factor binding protein-1 (IGFBP-1) gene was examined in 4-week-old male rats fed isocaloric diets containing 20%, 8%, or 4% protein over a 10-day period. Expression of the IGFBP-1 gene was strongly induced in the protein-restricted animals. Animals on the 8% protein diet exhibited a 14-fold increase, and animals on the 4% protein diet exhibited a 33-fold increase in hepatic IGFBP-1 messenger RNA (mRNA) abundance relative to the abundance of IGFBP-1 mRNA in animals on the 20% protein diet. Expression of the IGFBP-1 gene was also strongly increased by severe energy restriction: IGFBP-1 mRNA abundance was increased 15-fold in animals maintained for 10 days on a diet with energy restricted to 50% of the ad libitum intake rate. In animals fasted for 24 h there was a 6-fold increase in IGFBP-1 mRNA abundance, a lower induction than was observed in either of the two chronic nutritional restriction models. To determine whether limitation for substrate (i.e. amino acids) might have a direct effect on IGFBP-1 gene expression, we examined the effect on IGFBP-1 gene expression of limitation of H4-II-E rat hepatoma cells for a single essential amino acid (phenylalanine, methionine, leucine, or tryptophan) for a period of 24 h. The abundance of IGFBP-1 mRNA was increased by approximately 4- to 5-fold in cultures limited for any of these four amino acids as compared with its abundance in cells incubated in medium containing all essential amino acids. To study further the molecular mechanism for induction of IGFBP-1 gene expression by nutritional restriction, probes specific for intron 3 or intron 1 of the rat IGFBP-1 gene were used to quantify levels of the IGFBP-1 primary nuclear transcript in protein-restricted rats and amino acid-limited cultured cells. The level of the IGFBP-1 primary transcript was increased by 8-fold in animals on the 8% protein diet and 14-fold in animals on the 4% protein diet, suggesting that the induction of IGFBP-1 mRNA was caused largely by an increase in transcription.(ABSTRACT TRUNCATED AT 400 WORDS)