The investigators have shown that interferons (IFNs) upregulate the functional expression of chemokine receptors (CCR), CCR1, CCR3 and CCR5 in primary monocyte-derived macrophages (MDMs) and in the monocytoid cell line U937. This effect is mediated at least in part at the level of RNA expression, and it is likely to involve the JAK-STAT signaling pathway. Since entry of HIV into target cells is mediated at least in part by chemokine receptors, since the immune abnormalities in AIDS appear to include defects in antigen presentation, and since AIDS patients appear to have elevated serum levels of interferons, an analysis into the mechanisms of interferon-mediated upregulation of these chemokine receptors seems warranted. The investigators plan to identify the promoter regions of CCR1, CCR3 and CCR5 by cloning the regions 5' to the mRNA initiation sites and characterizing them in transient transfection assays for their ability to drive the expression of reporter genes and to respond to IFNa and g by an increase in activity. Since the 5' ends of the mRNAs have not been unambiguously determined, it may be necessary to identify the 5' ends by 5' RACE. Sequence analysis of active promoters may identify consensus transcription factor response elements. The promoter regions may be better defined by testing the activity of 5' and 3'deletion mutants. The binding sites for transcription factors will be identified by electrophoretic mobility shift and supershift assays. Site directed mutagenesis will be used to better define the binding sites of transcriptional factors. This work may provide important clues on how chemokine receptor expression is affected by IFNs and give insights on factors affecting HIV-1 replication in infected people.
| Zella, D; Barabitskaja, O; Casareto, L et al. (1999) Recombinant IFN-alpha (2b) increases the expression of apoptosis receptor CD95 and chemokine receptors CCR1 and CCR3 in monocytoid cells. J Immunol 163:3169-75 |
