The understanding of basic mechanisms of mRNA expression has been instrumental in the identification of molecular mechanisms mediating posttranscriptional control of gene expression and led to the development of RNA optimization (also referred to as codon optimization) as a key strategy to improve gene expression, a method which is presently a key technology for many gene therapy applications including HIV vaccines. In combination with the methodologies to modify the trafficking of proteins at the posttranslational level, these approaches led us to develop efficient expression plasmids for both HIV/SIV antigens as well as for cytokine genes. Analyses of retroviral regulatory systems, pioneered by research on HIV-1, have shed light into important aspects of nuclear mRNA export and provided critical insights into mechanisms governing cellular mRNA and protein transport. Retroviral model systems, and in particular the regulation of HIV-1, have led to major discoveries in the field of mRNA metabolism. HIV Rev was the first identified viral mRNA export factor, and its discovery was instrumental in the discovery of molecular mechanisms mediating posttranscriptional control of gene expression as well as our development of methods to increase the expression of viral proteins, i.e. development of RNA optimization (also referred to as codon optimization), which is presently a key technology for many gene therapy applications, including HIV vaccines and cytokine vectors. The study of Rev also prompted us to derive the concept that all retroviruses/retroelements use posttranscriptional control mechanisms essential for their replication and indeed we provided the proof for this. These controls require a combination of viral RNA elements and cellular/viral factors able to efficiently link the viral mRNA to the nuclear export pathways and to promote translation. These studies provided important tools to understand essential and complex steps in cellular gene expression and resulted in our past identification and characterization of NXF1, which is the key nuclear receptor for the export of cellular mRNAs and its cofactor the RNA binding motif 15 (RBM15) protein and its related RBM15b/OTT3, both members of the SPEN family. RBM15 provides the missing link for nuclear translocation, since it binds to both DBP5 and NXF1 and thus, it acts as molecular link to the NXF1 export pathway. We identified the mechanisms essential for the expression and mobility of RNAs of retroviruses/retroelements: (1) SRV/D related retrovirus and some murine LTR-retroelements export is mediated by the cis-acting RNA export element (CTE) mediated by the cellular protein NXF1; (2) some murine LTR-retroelement export depends on the presence of a distinct element, RTE or MTE; (3) MuLV export is mediated by a novel, bipartite post-transcriptional regulatory element, PTE. Together, our findings show that these posttranscriptional regulatory elements evolved convergently as high-affinity RNA ligands of certain key components of the NXF1 mRNA export pathway. Thus, despite a complex evolutionary history, retroelements/retroviruses share a dependency on posttranscriptional regulation, but the detailed molecular mechanisms are distinct. Comparative studies of different viral models, spearheaded by our research on HIV and HTLV, provides unique tools to address the complex network of molecular steps controlling viral and cellular mRNA expression. The dissection of these mechanisms are relevant to understand processes involved in cellular gene expression as well as virus expression and HIV pathogenesis. Indeed, we generated HIV and SIV variant viruses with have the Rev/RRE regulatory system replaced by the SRV-1 CTE, which resulted in attenuation of the virus with complete lack of pathogenicity in infected neonatal and juvenile macaques. Thus, a change of the viral regulatory RNA export significantly altered the pathogenicity of SIV. Posttranscriptional regulation is also key to control the production of viruses such as KSHV and is exerted via ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We found that the RBM15 and OTT3 participate in ORF57-enhanced expression of ORF59 and provide a link to the NXF export pathway, a conserved interaction among different herpes family members. Thus, retroelements, retroviruses and viruses like the Herpes virus family, share the same basic concepts of posttranscriptional regulation. Understanding basic mechanisms of gene expression has been instrumental for our understanding on the expression of cytokine genes, which are highly regulated at posttranscriptional and posttranslational level and this has practical applications for cancer immunotherapy and as molecular adjuvant for DNA vaccine regimens. The use of cytokine pDNAs (IL-12&IL-15) as molecular vaccine adjuvants was found to improve the quantity and alter the quality of the immune responses. To optimally use these cytokines, we found that IL-15/IL-15Ra (hetIL-15) as well as the IL-12 cytokine family use similar highly regulated steps posttranscriptional and posttranslational regulation strategies. We reported that coexpression of IL-15 & IL-15Ra from the same cell is critical for the production of this cytokine and analyzed its complex glycosylation pattern. We demonstrated that intramuscular delivery of hetIL-15 DNA in macaques produces systemic levels of the bioactive cytokine inducing proliferation of NK & T cells. To use IL-12 DNA to its full potential, we studied the biology of this glycosylated 70 kDa heterodimeric cytokine to maximize cytokine production. Although the production of each subunit is independently regulated, coexpression of both molecules in the same cell is essential to form biologically active heterodimer. Prompted by our findings on the critical intracellular regulatory step of IL-15/IL15Ra cross-stabilization, we investigated the posttranscriptional regulation and interaction of the p35 and p40 subunits leading to optimal IL-12p70 production. Investigating molecular steps controlling IL-12p70 biosynthesis, we found that the combination of RNA-optimized gene sequences, and importantly, fine-tuning of the relative expression levels of the two subunits within a cell resulted in a 1 log increased production of the IL-12p70 heterodimer. Importantly, we discovered that p40 enhances the p35 stability and promotes its intracellular trafficking, resulting in formation of a stable, efficiently secreted IL-12p70 complex. Dual expression plasmids for IL-12p70 were designed to obtain favorable relative levels of the two subunits and optimal IL-12 expression. Like IL-12, the related IL-23 & IL-27 are similarly regulated and cytokine production is controlled by the p40 and EBI3 chains, respectively. These cytokine pDNAs provide important molecular tools to be tested as molecular adjuvants in vaccine and in cancer immunotherapy, with promising future translational applications. Towards this, we further developed high expresser mammalian cell lines grown under defined serum-free conditions and reported an efficient one-step purification of IL-12 protein without affinity tag. Our understanding of the molecular biology of these cytokines provided the critical information necessary for efficient cytokine production. We are testing applications of the hetIL-15 in immunotherapy against cancer and HIV. hetIL-15, as a lymphocyte growth factor, represents a promising candidate in HIV immunotherapies, since it has the potential that harness the patient's own immune system and to induce CTL to reduce or clear the reservoir. Similarly, cancer immunotherapy aims to harness the patient's anti-tumor CTL. Thus, HIV and cancer immunotherapy share underlying immunologic features and our studies using cytokine therapy approach may shed light towards the cure of both.
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