Abstract
In vivo, equine infectious anemia virus (EIAV) replicates in tissues rich in macrophages, and it is widely believed that the tissue macrophage is the principal, if not sole, cell within the host that replicates virus. No viral replication has been detected in circulating peripheral blood monocytes. However, proviral DNA can be detected in these cells, and monocytes may serve as a reservoir for the virus. In this study, an in vitro model was developed to clarify the role of monocyte maturation in regulating EIAV expression. Freshly isolated, nonadherent equine peripheral blood monocytes were infected with a macrophage-tropic strain of EIAV, and expression of EIAV was monitored in cells held as nonadherent monocytes and cells allowed to adhere and differentiate into macrophages. A 2- to 3-day delay in viral antigen expression was observed in the nonadherent cells. This restriction of viral expression in monocytes was supported by nuclear run-on studies demonstrating that on day 5 postinfection, the level of actively transcribed viral messages was 4.7-fold lower in monocyte cultures than in macrophage cultures. Electrophoretic mobility shift assays identified three regions of the U3 enhancer that interacted with nuclear extracts from normal equine macrophages. Each region contained the core binding motif of a family of transcription factors that includes the product of the proto-oncogene ets. Antibodies to the Ets family member PU.1 caused a supershifting of retarded bands in an electrophoretic mobility shift assay. Transfection studies of ets motif mutants demonstrated that the U3 ets sites were important in the regulation of EIAV transcription in macrophages. Interactions between the ets motif and nuclear extracts from freshly isolated, nonadherent monocytes, macrophages adherent for 1 or 2 days, or macrophages adherent for 5 days gave different patterns of retarded bands, although the binding specificities were similar with all three extracts. The different complexes formed by monocyte and macrophage nuclear extracts may explain the enhanced ability of mature macrophages to support EIAV expression.
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