Cloning and expression of equine NF-kB2

Equine infectious anemia virus (EIAV) is a macrophage-tropic retrovirus that causes persistent disease in horses and ponies. In addition to its structural proteins, EIAV encodes four regulatory/accessory genes, tat, rev, ttm, and S2. It has been documented EIAV S2 gene expression is essential for disease expression of EIAV. Using a yeast two-hybrid assay, it was shown that S2 protein interacts with human NF-KB2. NF-KB2 plays a key role in the alternative or non-canonical NF-KB pathway. In order to determine if the interaction of S2 with NF-KB2 might be relevant to equine disease, a cDNA representing full length equine NF-KB2 was generated in our laboratory using PCR and rapid amplification of cDNA ends. To our knowledge this is the first time that equine NF-KB2 cDNAs have been recovered and characterized. The sequence of equine NF-KB2 was 95% homologous to human overall, however a major difference was found in the ankyrin repeat region where protein-protein interactions occur. Two splice variants of equine NF-KB2 were found that correspond to splice variants of human NF- KB2. We tested the interaction of EIAV S2 and equine NF-KB2 using the yeast two hybrid system (Y2H) and co-immunoprecipitation. Unfortunately we were not able to detect an interaction between EIAV S2 and equine NF-KB2 in either system. Despite this result, NF-KB2 is an important component in the immune response so we examined its expression in equine macrophages. Moreover we were interested to know if EIAV might affect expression levels of equine NF-KB2, as NF-KB2 is a target of other viruses. Hence, the expression level of equine NF-KB2 was measured in uninfected and infected primary equine monocyte- derived macrophage (eMDM). Using quantitative PCR we determined that equine NF-KB2 gene expression is decreased in eMDM after 3 days post plating, about the time that monocytes start to differentiate into mature macrophages. However EIAV infection of eMDM upregulated the expression level of NF-KB2.