Abstract
African swine fever virus is a large double-stranded DNA virus which causes an acute haemorrhagic fever in domestic swine, but is apathogenic in its natural hosts, the warthog and the bushpig, in which the virus persists for long periods. The virus can also persist in, and be transmitted by, soft ticks of the genus Ornithodoros which can incubate the virus for several years and transmit the virus transovarially. Previous studies on the virus have identified a number of viral genes encoding proteins involved in host-cell interactions and immune evasion. One of these proteins, A238L, has been shown to inhibit the NF-B and calcineurin pathways, two key pathways involved in regulating the immune response. These studies have shown that A238L protein inhibits NF-kappaB-dependent reporter gene expression, is able to prevent the p65 subunit of NF-kB binding to DNA and that A238L protein co-immunoprecipitates with the p65 protein. The aims of this study were to further investigate the mechanism by which A238L inhibits the NF-kappaB pathway. This study shows that p65 is present in the nucleus following ASFV infection, and that the 32 kDa form of A238L accumulates in the nucleus at late times post infection. A238L is unable to move to the nucleus in p65-/- cell lines, suggesting that A238L binds to NF-kappaB in the cytoplasm and shuttles to the nucleus in complex with NF-kappaB. A proportion of A238L remains in the cytoplasm and this is mainly the 28 kDa form of the protein; the cytoplasmic pool of A238L remains when nuclear export is inhibited by leptomycin B suggesting that this cytoplasmic pool is not exported from the nucleus. A negative effect of A238L protein on the rate of cell proliferation was also observed. This effect was similar in cells transfected with a mutant form of A238L which is unable to bind to calcineurin. This suggests that the cause for this effect is via NF-kappaB inhibition. In summary, this study provides evidence for a model in which the A238L protein acts to inhibit NF-kappaB in the nucleus, rather than by sequestering the complex in the cytoplasm.