Abstract
The double-stranded (ds) RNA-binding protein RAX was discovered as a stress-induced cellular activator of the dsRNA-dependent protein kinase (PKR), a key regulator of protein synthesis in response to viral infection and cellular stress. We now report a novel function of RAX, independent of PKR, to enhance SV40 promoter (origin)/enhancer-dependent gene expression. Several mammalian cell lines including COS-7, CV-1, and HeLa cells were tested. Results reveal that the SV40 large T antigen is required for RAX-mediated, synergistic enhancement of gene expression. RAX augments SV40 regulatory element-dependent DNA replication and transcription. The mechanism requires the SV40 enhancer, a viral transcriptional element that is necessary for efficient SV40 DNA replication in vivo. Mutational analysis reveals that the dsRNA-binding domains of RAX are required for the gene expression enhancing function. Thus, in addition to stimulating PKR activity, RAX can positively regulate both SV40 large T antigen-dependent DNA replication and transcription in a mechanism that may alter the interaction of the cellular factor(s) with the SV40 enhancer via the dsRNA-binding domains of RAX. This novel function of RAX may have implications for regulation of mammalian DNA replication and transcription because of the many similarities between the viral and cellular processes.
Highlights
SV40 is a small DNA virus that has served as a powerful model system for dissecting fundamental biological processes including, DNA replication, transcription, and neoplastic transformation [1,2,3,4]
PKR shuts down protein synthesis in uninfected cells following cellular stress responses that lead to inhibition of cell growth and apoptosis, including interleukin-3 growth factor withdrawal, serum deprivation, and treatment of cells with tumor necrosis factor-␣ and lipopolysaccharide [11, 13, 14]
RAX-mediated augmentation of exogenous gene expression was unexpected because RAX was initially identified and characterized as a cellular activator of PKR that inhibits protein synthesis [15, 16]
Summary
SV40 is a small DNA virus that has served as a powerful model system for dissecting fundamental biological processes including, DNA replication, transcription, and neoplastic transformation [1,2,3,4]. The T-Ag has been found to interact with a number of cellular proteins that regulate DNA replication and transcription (e.g. DNA polymerase ␣, human TFIIBrelated factor, pRb, and p53) and to modify cellular regulatory processes that may efficiently promote viral gene expression and replication in permissive cells [1,2,3,4]. PKR shuts down protein synthesis in uninfected cells following cellular stress responses that lead to inhibition of cell growth and apoptosis, including interleukin-3 growth factor withdrawal, serum deprivation, and treatment of cells with tumor necrosis factor-␣ and lipopolysaccharide [11, 13, 14]. RAX/PACT is a dsRNA-binding protein that directly binds and activates PKR following cellular stresses (such as interleukin-3 withdrawal, sodium arsenite, hydrogen peroxide, serum deprivation, or ceramide treatment) that lead to cell death [15,16,17,18]. Findings reported here indicate that RAX has a novel, unexpected function to augment SV40 T-Ag-dependent DNA replication and transcription that is dependent on its dsRNA-binding domains but independent of PKR
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