Studies into host macrophage transcriptional control by the African Swine Fever Virus protein A238L

Abstract

African swine fever virus (ASFV) is a large double-stranded DNA virus which causes a lethal haemorrhagic fever in domestic pigs. This virus primarily infects cells from the monocyte/macrophage lineage and its ability to manipulate the function of these cells is key to the pathogenesis of this disease. ASFV encodes several proteins involved in immune evasion. One of these proteins, A238L, has been shown to inhibit host macrophage gene transcription. This protein has been shown to interact with several cellular proteins involved in signal transduction: a serine/threonine protein phosphatase, calcinerurin (CaN), the transcription factor NF-кB, and most recently the transcriptional co-activator CREB binding protein (CBP/P300). However its exact mechanism of action is not fully understood. Previous work has been limited to the investigation of individual signaling pathways and/or the expression of individual host genes. The aim of this study was to investigate the global effect of A238L on host macrophage gene transcription and also to carry out further investigation into the mechanism by which this protein functions. To determine the global effect of A238L on host macrophage gene transcription differential gene expression between porcine cells expressing A238L and control cells was examined using a porcine oligonucleotide microarray. These results demonstrated that A238L was a potent inhibitor of host macrophage gene expression. Functional characterisation of the annotated genes showed that a large proportion of A238L down-regulated genes are typically induced in response to cell stress. Significantly, genes regulated by the I kappa B kinase (IKK), mitogen-activated protein kinase (MAPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways were all shown to be down regulated by A238L. Genes associated with the MAPK pathways were particularly enriched. The transcription of A238L-regulated genes is controlled by numerous different transcription factors, including NF-кB. All of the transcription factors identified interact with the transcription co-activator CBP/P300. This provides a common link between these factors, and indicates that A238L may target CBP/P300 to inhibit gene transcription. This observation supports recent work demonstrating that A238L interacts with and inhibits CBP/P300 function. To explore the potential mechanisms involved in the nuclear localisation of A238L, ASFV-infected Vero cells, expressing A238L under the control of its own promoter, were examined under a range of conditions using confocal microscopy. The results demonstrated that A238L was actively imported into the nucleus and exported by a CRM 1 mediated pathway, although a pool of A238L protein remained in the cytoplasm. Sequence analysis of A238L identified the presence of two putative nuclear localisation signals (NLS-1 and NLS-2). NLS-2 was located within A238L’s CaN docking motif. Mutation of these motifs indicated that both NLS-1 and NLS-2 are active and exhibit functional redundancy. Mutation of the CaN docking motif alone, in the presence of intact NLS-2, resulted in a dramatic increase in the nuclear localisation of A238L. These results are consistent with a model in which A238L functions within both the nucleus and the cytoplasm and suggest that binding of CaN to A238L masks NLS-2, contributing to the cytoplasmic retention of A238L.