title: DH82 cells: a macrophage cell line for the replication and study of equine infectious anemia virus.
abstract: In vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (EIAV). Laboratory investigations of EIAV/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. In addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. This report describes an adherent canine macrophage-like cell line, DH82, that supports the replication of EIAV. This cell line was easily transfectable and supported EIAV Tat transactivation of the LTR. Electrophoretic mobility shift assays were carried out to determine which transcription factor binding sites within the LTR enhancer region were bound by DH82 nuclear extracts. It was found that five different motifs were occupied. The ets motifs that are bound by PU.1 in primary macrophage nuclear extracts specifically interacted with DH82 nuclear extracts. In addition, the PEA-2, Lvb and Oct motifs that are occupied by fibroblast nuclear extracts were also bound by DH82 nuclear extracts. Finally, the methylation-dependent binding protein (MDBP) site that is bound by all nuclear extracts investigated to date demonstrated specific interactions with DH82 nuclear extracts. The observation that both macrophage-specific and fibroblast-specific motifs were utilized by DH82 nuclear extracts suggested that both macrophage-adapted and fibroblast-adapted EIAV could replicate in DH82 cells. Indeed, infectivity studies demonstrated that strains of virus that exclusively replicate in macrophages can replicate in DH82 cells and fibroblast-adapted strains of virus can also replicate in these cells. Finally, these cells could be transfected readily with the EIAV molecular clone, pSPeiav19-2, and virus spread was detected within the culture. In conclusion, this study has identified a useful cell line that should facilitate the study of EIAV expression and replication.
authors.affiliation: University of South Dakota, Lee Medical Building, 414 E Clark St., Vermillion, SD 57069, USA.
authors.forename: R
authors.initials: R
authors.lastname: Hines
authors.forename: W
authors.initials: W
authors.lastname: Maury
mesh_terms: D000818:Animals; D002457:Cell Extracts; D002460:Cell Line; D002467:Cell Nucleus; D004285:Dogs; D005347:Fibroblasts; D015696:Gene Products, tat; D004860:Infectious Anemia Virus, Equine / Q000235:genetics / Q000502:physiology*; D008264:Macrophages / Q000166:cytology / Q000821:virology*; D020079:Terminal Repeat Sequences; D014162:Transfection; D014779:Virus Replication
chemical_list: D002457:Cell Extracts; D015696:Gene Products, tat