Our research focuses on the regulation of gene expression, in particular the mechanisms controlling cellular and viral mRNA expression, and on the development of DNA vectors for vaccine and immunotherapy. 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-1 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. 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. The study of retrovirus/retroelement export has provided important tools to understand essential and complex steps in cellular gene expression. This strategy 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. Both NXF1 and RBM15 are essential cellular proteins. We identified the mRNA export requirement of the simian type D retroviral transcript, which is mediated by the cis-acting RNA export element (CTE) present in SRV/D related retroviruses and in some murine LTR-retroelements, and its binding partner, the cellular protein NXF1, and is essential for the expression and mobility of these retroviruses. We also identified that the expression and mobility of a different subclasses of murine LTR-retroelements depends on the presence of a distinct cis-acting RNA transport element, RTE or MTE, which act like CTE but does not share its sequence or structural features. Our findings suggest that the posttranscriptional regulatory elements in modern retroelements 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. Posttranscriptional regulation is also key to control the production of viruses such as the Kaposi's sarcoma-associated herpesvirus (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 KSHV ORF59 and provide a link to the NXF export pathway, a conserved interaction among different herpes family members. We recently demonstrated that ORF57, although very capable of promoting expression of many KSHV genes, is not able to provide RNA export function. Although not a bona fide export factor, ORF57, like the related proteins of other herpes viruses, plays a critical role in the posttranscriptional regulation of many viral genes and remains essential for the virus production. Thus, retroelements, retroviruses and virus like the Herpes virus family, share the same basic concepts of posttranscriptional regulation. Together, comparative studies of different viral models provide unique tools to address the complex network of molecular steps controlling viral and cellular mRNA expression and this has led to major discoveries on the factors regulating cellular nuclear export mechanisms. We also studied regulation of expression of some cytokine genes, which have practical applications for cancer immunotherapy and as molecular adjuvant for DNA vaccine regimens. The use of cytokine DNAs (IL-12 and 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 are studying their regulation and have found that IL-15/IL-15Ra as well as the IL-12 cytokine family use similar highly regulated steps posttranscriptional and posttranslational regulation strategies. 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 and 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 the p40 subunit plays a critical role in enhancing the p35 stability and promoting its intracellular trafficking, through the trans-Golgi network, resulting in formation of a stable, efficiently secreted IL-12p70 complex. Based on these observations, dual expression plasmids for IL-12p70 were designed to obtain favorable relative levels of the two subunits and optimal IL-12 expression. Our expression-optimized human IL-12 DNA showed higher cytokine production compared to DNAs expressing the native sequences that are currently employed in clinical trials. These optimized cytokine DNAs provide important molecular tools to be tested as molecular adjuvants in vaccine and in cancer immunotherapy, with promising future translational applications. Development of novel HIV DNA vaccine plasmids. An ideal HIV vaccine should provide protection against all HIV-1 variants. HIV sequence diversity and the presence of potential immunodominant "decoy" epitopes are hurdles in the development of an effective AIDS vaccine. To address these problems, we are exploring approaches to maximize immunological strength and breadth focusing on highly conserved regions of HIV to induce immune responses to nearly invariable proteome segments, essential for the function of the virus, while excluding responses to variable and potentially immunodominant "decoy" epitopes. We developed a prototype vaccine targeting regions within the p24gag, benefitting from our experience in RNA and protein expression and trafficking. Optimized plasmids were tested in mice and successful candidates were further tested in macaques. In proof-of-concept studies in mice and macaques, we demonstrated that immunization with this DNA elicits robust cellular and humoral immune responses against CE, which cannot be achieved by p55gag DNA vaccination. The translation of this novel concept is currently being pursued in an HVTN/DAIDS-supported clinical trial.
|Jalah, Rashmi; Rosati, Margherita; Ganneru, Brunda et al. (2013) The p40 subunit of interleukin (IL)-12 promotes stabilization and export of the p35 subunit: implications for improved IL-12 cytokine production. J Biol Chem 288:6763-76|
|Chertova, Elena; Bergamaschi, Cristina; Chertov, Oleg et al. (2013) Characterization and favorable in vivo properties of heterodimeric soluble IL-15·IL-15R? cytokine compared to IL-15 monomer. J Biol Chem 288:18093-103|
|Bergamaschi, Cristina; Jalah, Rashmi; Kulkarni, Viraj et al. (2009) Secretion and biological activity of short signal peptide IL-15 is chaperoned by IL-15 receptor alpha in vivo. J Immunol 183:3064-72|
|Uranishi, Hiroaki; Zolotukhin, Andrei S; Lindtner, Susan et al. (2009) The RNA-binding motif protein 15B (RBM15B/OTT3) acts as cofactor of the nuclear export receptor NXF1. J Biol Chem 284:26106-16|
|Bergamaschi, Cristina; Rosati, Margherita; Jalah, Rashmi et al. (2008) Intracellular interaction of interleukin-15 with its receptor alpha during production leads to mutual stabilization and increased bioactivity. J Biol Chem 283:4189-99|
|Zolotukhin, Andrei S; Schneider, Ralf; Uranishi, Hiroaki et al. (2008) The RNA transport element RTE is essential for IAP LTR-retrotransposon mobility. Virology 377:88-99|