Our research focuses on the regulation of gene expression, in particular the mechanisms controlling cellular and viral mRNA expression. 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. The dissection of the mechanisms of posttranscriptional control and nucleocytoplasmic trafficking of macromolecules is relevant to understanding the processes involved in cellular gene expression as well as virus expression. Understanding the basic mechanisms of mRNA expression led to the development of RNA/codon optimization as a key strategy to improve gene expression, which also led to the development of efficient expression vectors for both HIV/SIV antigens and cytokines. 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. The study of Rev also prompted us to derive the concept that all retroviruses and 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 and retroelement export could provide important tools to understand essential and complex steps in cellular gene expression. In fact, this strategy resulted in our past identification and characterization of NXF1, which is the key nuclear receptor for the export of cellular mRNAs. 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. CTE is a highly conserved RNA element located next to the 3LTR in SRV-1, SRV-2, MPMV/SRV-3, and the recently discovered SRV-4 as well as in some murine LTR-retroelements. CTE is essential for the expression and mobility of these retroviruses. We discovered that cellular NXF1 protein acts as the key nuclear receptor for cellular mRNAs and that this function is conserved in metazoa and is essential. We also identified that the expression and mobility of a different subclass of murine LTR-retroelements depends on the presence of a distinct cis-acting RNA transport element, RTE, which acts like CTE but does not share its sequence or structural features. Thus, despite a complex evolutionary history, retroelements and retroviruses share a dependency on posttranscriptional regulation, but the detailed molecular mechanisms are distinct. We identified the mechanism that promotes the export of the RTE RNA export and reported that the RNA binding motif 15 (RBM15) protein acts as the cellular factor that binds and exports RTE-containing mRNAs via the NXF1 export pathway. RBM15, a novel mRNA export factor, belongs to the SPEN family of proteins and is conserved among metazoa. We reported that another SPEN protein, RBM15b/OTT3, acts as an RNA export co-factor like RBM15. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knock-down of RBM15 in mouse is embryonic lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, likely have distinct biological roles. Since RBM15 and OTT3 are cofactors of NXF1, we speculate that they may be part of a developmental or/and tissue-specific switch that controls mRNA export rates or/and specificity. How does NXF1 guide the transcripts through the nuclear pore complex (NPC)? The DEAD-box ATPase DBP5, located at the NPC, is thought to mediate the directional passage of mRNA through the NPC via an unknown mechanism, since it does not bind to NXF1. We reported that RBM15 provides the missing link, since it binds to both DBP5 and NXF1 and thus, it acts as molecular link to the NXF1 export pathway. Our recent findings identified a RNA export element present in another class of retroelements (mus D retroelements). This export element (termed MTE) is distinct in sequence and structure compared to the previously identified export elements. We identified that structure of MTE and identified two classes of tertiary interactions, namely kissing loops and a pseudoknot. We showed that the complex tertiary structure allows for distinct long-range molecular interactions that are essential for function. This work constitutes the first example of an RNA transport element requiring such structural motifs to mediate nuclear export. 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. 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. Interestingly, we found that the RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59 and provide a link to the NXF export pathway. We further noted that RBM15 and OTT3 also interact with the ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69, demonstrating conservation of the interaction of RBM15 and OTT3 with the posttranscriptional regulators of different herpes family members. Thus, despite a complex evolutionary history, 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 are highly regulated at several posttranscriptional as well as posttranslational steps. 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 posttranscriptional and posttranslational regulation strategies. As a result, the formation and secretion of the subunits and heterodimers are highly regulated steps. Using this information, we have generated optimized expression vectors, which allow their efficient use in animal models. These optimized cytokine DNAs provide us with important molecular tools to be tested as molecular adjuvants in vaccine and in cancer immunotherapy, with promising future translational applications.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010334-12
Application #
8348980
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2011
Total Cost
$796,657
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Jayanthi, Srinivas; Koppolu, Bhanu Prasanth; Nguyen, Khue G et al. (2017) Modulation of Interleukin-12 activity in the presence of heparin. Sci Rep 7:5360
Ng, Sinnie Sin Man; Nagy, Bethany A; Jensen, Shawn M et al. (2017) Heterodimeric IL15 Treatment Enhances Tumor Infiltration, Persistence, and Effector Functions of Adoptively Transferred Tumor-specific T Cells in the Absence of Lymphodepletion. Clin Cancer Res 23:2817-2830
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Bergamaschi, C; Kulkarni, V; Rosati, M et al. (2015) Intramuscular delivery of heterodimeric IL-15 DNA in macaques produces systemic levels of bioactive cytokine inducing proliferation of NK and T cells. Gene Ther 22:76-86
Li, Jinyao; Valentin, Antonio; Ng, Sinnie et al. (2015) Differential effects of IL-15 on the generation, maintenance and cytotoxic potential of adaptive cellular responses induced by DNA vaccination. Vaccine 33:1188-96
Purzycka, Katarzyna J; Pilkington, Guy R; Felber, Barbara K et al. (2015) The ""complex"" RNA post-transcriptional element of a ""simple"" retrovirus. Mob Genet Elements 5:1-4
Hanson, Miranda L; Hixon, Julie A; Li, Wenqing et al. (2014) Oral delivery of IL-27 recombinant bacteria attenuates immune colitis in mice. Gastroenterology 146:210-221.e13
Jayanthi, Srinivas; Koppolu, Bhanu prasanth; Smith, Sean G et al. (2014) Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag. Protein Expr Purif 102:76-84
Pilkington, Guy R; Purzycka, Katarzyna J; Bear, Jenifer et al. (2014) Gammaretrovirus mRNA expression is mediated by a novel, bipartite post-transcriptional regulatory element. Nucleic Acids Res 42:11092-106
Byrareddy, Siddappa N; Ayash-Rashkovsky, Mila; Kramer, Victor G et al. (2013) Live attenuated Rev-independent Nef¯SIV enhances acquisition of heterologous SIVsmE660 in acutely vaccinated rhesus macaques. PLoS One 8:e75556

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