Characterization of the viral response element (VRE) of murine Rantes and Crg-2 chemokine genes and examination of the VRE-binding factors with a comparison to the LPS response element (LRE)

Abstract

Induction of cytokine genes occurs during many inflammatory processes including viral and bacterial infections. Depending on the intensity, duration, and site of induction, it can be beneficial or detrimental to the host. Chemokines, potent proinflammatory immune effectors, contribute to viral and bacterial clearance during infections, yet are also involved in a number of disease states. MuRantes and Crg-2 are members of this superfamily. The goal of this study was to understand the induction mechanisms of these genes in response to viral and bacterial infection by delineating; (1) signal pathways of their induction by virus, (2) promoter regions required for this induction, (3) transactivating factors interacting with the viral response element (VRE), and (4) similarities and differences in chemokine gene induction by virus or bacterial endotoxin (LPS). MuRantes and Crg-2 mRNA accumulation was induced by Newcastle disease virus (NDV) and LPS in RAW 264.7, a mouse macrophage cell line. NDV and LPS also induced these genes in peritoneal macrophages of LPS-responder C3HeB/FeJ mice. Only NDV, however, induced these genes in C3H/HeJ, an LPS-non-responder strain. This finding indicates that although both NDV and LPS induce chemokine gene expression, the induction occurs via overlapping, yet distinct mechanisms. Using a transient transfection system, the boundaries of the VRE were localized between {dollar}-{dollar}175 and {dollar}-{dollar}125 for MuRantes and between {dollar}-{dollar}236 and {dollar}-{dollar}185 for Crg-2. Although the MuRantes VRE contains an AP-1 site (6/7) and an NF-kB-like site (8/10), the Crg-2 VRE lacks the potential NF-kB site. Comparison of the two VREs indicated two shared motifs; motif 1 is TCATACTT and motif 2 is TGATTTCAGTTT. Interestingly, both motifs were also seen in the minimal LPS response element of these genes, although the VREs included additional A{dollar}\cdot{dollar}T-rich sequences. Mutational analyses of the MuRantes promoter indicated that NF-kB does not bind directly to the VRE. Examination of the VRE-binding factors in NDV-stimulated nuclear extracts indicated that high mobility group protein-C (HMG-C) is present in the VRE-protein complexes. In addition, c-jun and JunD may bind to motif 1 which overlaps with the AP-1 site. Although the protein(s) binding to motif 2 has not been resolved, it is likely that it functions with HMG-C and AP-1 proteins bound within the VRE in order to achieve NDV-induced transcription.