Reversal of virus-induced systemic shock and respiratory failure by blockade of the lymphotoxin pathway.

At present, little is known about the pathogenesis of acute virus-induced shock and pulmonary failure. A chief impediment in understanding the underlying disease mechanisms and developing treatment strategies has been the lack of a suitable animal model. This study describes a mouse model of virus-induced systemic shock and respiratory distress, and shows that blockade of the lymphotoxin beta receptor pathway reverses the disease.

Regulation of EBNA gene transcription in lymphoblastoid cell lines: characterization of sequences downstream of BCR2 (Cp).

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six EBV nuclear antigens (EBNAs) are expressed from one of two promoters, Cp and Wp, whose activities are mutually exclusive. Upon infection, Wp is initially active, followed by a switch to Cp for the duration of latency. In this study, the impact on Cp and Wp activity of sequences downstream of the distal EBNA gene promoter, Cp, was assessed in two lymphoblastoid cell lines. Cp activity was detected in constructs extending from just upstream of oriP to the first W1 exon. In contrast, Wp activity required the presence of the next downstream exon, W2. Viral sequences from -2199 to +2680 bp, relative to the Cp transcription start site, were dispensable for Wp activity. Sequences from +155 to +2680 bp, relative to the Cp transcription start site, were dispensable for Cp activity. Deletion of a 200-bp region from +2680 to +2880 bp downstream of Cp decreased both Cp and Wp activity two- to fivefold. Wp activity was also significantly diminished by deletion of the sequences from +2880 to +3000 bp downstream of the Cp transcription initiation site, which encompassed the Wp CCATT box. Based on deletion analyses of 10.3 kb of viral genomic sequence extending from just upstream of oriP to the first Wp, the only deletions which significantly upregulated Wp activity were those which abrogated Cp activity. However, reporter constructs in which the orientation of Cp was reversed displayed Wp activity comparable to that of reporter constructs in which Cp was deleted, even though the steady-state level of Cp-initiated transcripts from the inverted promoter was indistinguishable from that observed with Cp in normal orientation. This is the first direct evidence to support transcriptional interference as the mechanism for the mutually exclusive behavior of Cp and Wp.

oriP is essential for EBNA gene promoter activity in Epstein-Barr virus-immortalized lymphoblastoid cell lines.

During Epstein-Barr virus latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities are mutually exclusive. Upon infection, Wp is initially active, followed by a switch to Cp for the duration of latency. In this study, the region upstream of Cp was analyzed for the presence of cis elements involved in regulating the activities of the EBNA gene promoters in established in vitro immortalized lymphoblastoid cell lines (LCLs). It was determined that oriP, the origin for episomal maintenance during latency, is essential for efficient transcription initiation from either Cp or Wp in LCLs, as well as in some Burkitt's lymphoma cell lines. Deletion of the EBNA2-dependent enhancer located upstream of Cp resulted in a ca. two- to fivefold reduction in Cp activity in the LCLs assayed. More extensive deletion of sequences upstream of Cp, including the EBNA2-dependent enhancer, resulted in nearly complete loss of Cp activity. This loss of activity was shown to correlate with deletion of two CCAAT boxes, a proximal CCAAT box located at bp -61 to -65 and a distal CCAAT box located at bp -253 to -257, upstream of Cp. Site-directed mutagenesis of these cis elements demonstrated that Cp activity is highly dependent on the presence of a properly positioned CCAAT box, with the dependence on the distal CCAAT box apparent only when the proximal CCAAT box was deleted or mutated. Deletion of the glucocorticoid response elements located at ca. bp -850 upstream of Cp did not result in a significant loss in activity. In general, deletions which diminished Cp activity resulted in induction of Wp activity, consistent with suppression of Wp activity by transcriptional interference from Cp. The identification of oriP and the EBNA2-dependent enhancer as the major positive cis elements involved in regulating Cp activity in LCL suggests that EBNA gene transcription is largely autoregulated by EBNA 1 and EBNA 2.

B-cell lines immortalized with an Epstein-Barr virus mutant lacking the Cp EBNA2 enhancer are biased toward utilization of the oriP-proximal EBNA gene promoter Wp1.

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities have previously been shown to be mutually exclusive in established lymphoblastoid cell lines. Initially after infection, the EBNA genes are transcribed from Wp, which is present in multiples copies within the major internal repeat of EBV. Approximately 48 to 72 h postinfection, Wp is downregulated, with a corresponding increase in transcription from Cp. An EBNA2-responsive enhancer exists upstream of Cp, and a role for EBNA2 in the induction of Cp activity during the establishment of viral latency has previously been proposed (Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 87:1725-1729, 1991). To critically assess the potential role for this enhancer region in determining relative usage of Cp and Wp, an EBNA2 enhancer deletion mutant virus was generated. Lymphoblastoid cell lines were screened by PCR and Southern blotting for the presence of mutant virus harboring the EBNA2 enhancer deletion. A quantitative S1 nuclease protection assay was developed to allow comparison of relative Cp and Wp activities for the cell lines containing mutant virus and those of the wild-type recombinants which lacked the enhancer deletion. In general, the wild-type recombinants had higher levels of Cp-initiated transcripts than Wp-initiated transcripts. In contrast, the Cp EBNA2 enhancer deletion mutants exhibited a strong bias toward Wp activity. Notably, only the first Wp (oriP-proximal Wp; Wp1) appears active in these mutants. S1 nuclease protection assays using a probe which hybridizes to the W2 exon, contained in both Cp- and Wp-initiated transcripts, indicated that the total level of transcription from Cp and Wp remained the same in wild-type and EBNA2 enhancer mutant cell lines. The presence of both Cp and Wp activity in the wild-type recombinants, as well as in newly derived lymphoblastoid cell lines established with the prototype B95.8 virus, demonstrated that Cp and Wp activities are not always mutually exclusive.

In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb.

CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.