Our research focuses on the regulation of gene expression, in particular the mechanisms controlling cellular and viral mRNA expression. A critical step in the mRNA metabolism is the transport of the mRNA from the nucleus to the cytoplasm. Analysis of retroviral systems, pioneered by research on HIV-1, have shed light into some important aspects of nuclear mRNA export and have provided critical insights into mechanisms governing cellular mRNA and protein transport. We are utilizing retroviral systems to identify and study mechanisms of mRNA metabolism using a combination of biochemistry, functional genomics, and proteomics. The dissection of the mechanisms of posttranscriptional control and nucleocytoplasmic trafficking of macromolecules are relevant to understand processes involved in cellular gene expression as well as virus expression. We identified the mRNA export requirement of the simian type D retroviral transcript which is mediated by the cis acting RNA export element (CTE) and its binding partner, the cellular protein NXF1. We further found that the cellular NXF1 protein acts as the key nuclear receptor for cellular mRNAs, and that this function is conserved in metazoa. We identified that the mobility of the murine LTR-retroelements (Intracisternal A particle retroelements) depends on the presence of the cis-acting RNA transport element RTE. The export and expression of the retroelement transcript depends on RTE. Thus, RTE acts like the CTE and is a potent signal for a cellular RNA export factor. This finding reveals that, despite a complex evolutionary history, retroelements and retroviruses share the dependency on posttranscriptional regulation. We identified the RNA binding motif 15 (RBM15) protein as the cellular factor that binds and exports RTE-containing mRNAs. RBM15, a novel mRNA export factor, belongs to the SPEN family of proteins and is conserved among metazoa. Importantly, we found that RBM15 acts as molecular link and tethers the RTE-mRNAs to the NXF1 export pathway. Thus, these experiments have identified another important factor of the mRNA export route. Since HIV gag and env mRNAs are poorly expressed in the absence of potent posttranscriptional Rev-RRE regulation, these mRNAs serve as excellent reporters to study improvement of mRNA expression at the posttranscriptional level. We demonstrated that the combination of RNA export elements CTE and RTE synergistically improves gag and env expression. Thus, this discovery provided us with a simple novel technology to improve gene expression for DNA mediated gene transfer applications. We also studied an example of regulated cellular mRNA expression, namely the expression of cytokine genes, in particular interleukin 15 (IL-15). IL-15 is a multifunctional cytokine expressed in many tissues. Its use as molecular adjuvant in vaccine and in cancer immunotherapy is promising. In addition to regulation at the transcriptional level, IL-15 expression is controlled at several posttranscriptional and posttranslational steps such as mRNA stability, translation, intracellular trafficking and secretion. As a direct extension of our research of retroviral mRNAs, we studied expression of IL-15. Expression from the native mRNA is poor and upon RNA-optimization we found great improvement for murine, rhesus macaque and human IL-15. Thus, the methodology utilized to optimize HIV gene expression also let to improved IL-15 expression from simple DNA vectors. The combination of posttranscriptional and posttranslational modification led to the generation of efficient expression plasmids producing several hundred fold higher levels of bioactive IL-15. Such expression plasmids are part of our cocktail of DNA plasmids used in preventive and immunotherapeutic vaccination protocols in SIV. These optimized expression vectors have potential applications in vaccine and immunotherapy approaches against AIDS and cancer.
