This project aims to understand the role and mechanism of function of HIV regulatory and accessory proteins in the virus life cycle and disease development; to dissect the mechanisms of nucleocytoplasmic trafficking of macromolecules; and to examine the mechanism of Vpr function as transcription activator and as cell cycle modulator. The conclusions of this work are applied towards the design of better vaccine approaches against AIDS. We study the function of several HIV-1 proteins, including Tat, Rev, and Vpr. We showed that the defect of Rev in rodent cells is caused by delayed import of Rev in the nucleus. The factors responsible for this effect are under investigation and may lead to new types of interventions targeted against the essential Rev factor. A new inhibitor of CRM1-directed nuclear export has been identified and characterized. This inhibitor will be useful for studies of Rev and many cellular proteins exported through the CRM1 pathway. We identified a new RNA element (named RTE) from the mouse genome that is able to replace the Rev and RRE of HIV-1, using a mutated HIV-1 DNA proviral clone as a new type of molecular trap. The factors binding to this element are under investigation. This result showed the presence of additional RNA elements in the genome that are able to direct nuclear export and may define a distinct pathway for nucleocytoplasmic transport. The role of Vpr on virus life cycle is complex and needs to be examined in more detail. Although several cellular proteins were shown to interact with Vpr, the mechanisms of its function are not fully elucidated. We have detected direct binding of Vpr to additional cellular proteins and have studied the effect of this binding on the cell cycle and on transactivator effects of Vpr. We have shown that Vpr interacts directly with the glucocorticoid and other nuclear receptors, and is found in complexes with these factors during active transcription. We also found that Vpr binds directly to p300, an important coactivator of many cellular promoters. This binding leads to increased activation of such promoters, and explains some of the transactivation effects of Vpr. The understanding of the regulatory mechanisms of HIV gene expression has been applied for the development of improved DNA vaccination approaches. We have developed a general method to increase the expression of unstable mRNAs by introducing multiple point mutations in their coding regions. This results in efficient transport, and increased stability and translation of several mRNAs. Based on our results with gag and env sequences of HIV, we developed efficient expression vectors for DNA-based immunization. We showed that better antigen expression results in increased immunogenicity. This led to DNA vaccines that are efficiently expressed and achieve the production of antibodies, CTL, and helper responses in mouse. In contrast, primates do not achieve an equally effective immune response, indicating differences among the species. We developed additional DNA vaccine vectors producing modified antigens in order to elicit more potent immune responses in primates. We tested several such vectors expressing either secreted or intracellularly degraded antigens and showed that some combinations appear to boost immunogenicity. Results in mice were used to design monkey vaccination experiments. We work on further optimization of the vaccine vectors to improve the methodology of DNA vaccination, aiming at efficient vaccination using combinations of vectors against multiple HIV antigens.

Agency
National Institute of Health (NIH)
Institute
Division of Clinical Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01SC010387-03
Application #
6948366
Study Section
Vector Biology Study Section (VB)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Clinical Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Egan, Michael A; Chong, Siew Yen; Megati, Shakuntala et al. (2005) Priming with plasmid DNAs expressing interleukin-12 and simian immunodeficiency virus gag enhances the immunogenicity and efficacy of an experimental AIDS vaccine based on recombinant vesicular stomatitis virus. AIDS Res Hum Retroviruses 21:629-43
Daelemans, Dirk; Costes, Sylvain V; Lockett, Stephen et al. (2005) Kinetic and molecular analysis of nuclear export factor CRM1 association with its cargo in vivo. Mol Cell Biol 25:728-39
Boyer, Jean D; Robinson, Tara M; Maciag, Paulo C et al. (2005) DNA prime Listeria boost induces a cellular immune response to SIV antigens in the rhesus macaque model that is capable of limited suppression of SIV239 viral replication. Virology 333:88-101
Daelemans, Dirk; Pannecouque, Christophe; Pavlakis, George N et al. (2005) A novel and efficient approach to discriminate between pre- and post-transcription HIV inhibitors. Mol Pharmacol 67:1574-80
Kutzler, Michele A; Robinson, Tara M; Chattergoon, Michael A et al. (2005) Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help. J Immunol 175:112-23
Rosati, Margherita; von Gegerfelt, Agneta; Roth, Patricia et al. (2005) DNA vaccines expressing different forms of simian immunodeficiency virus antigens decrease viremia upon SIVmac251 challenge. J Virol 79:8480-92
Smulevitch, Sergey; Michalowski, Daniel; Zolotukhin, Andrei S et al. (2005) Structural and functional analysis of the RNA transport element, a member of an extensive family present in the mouse genome. J Virol 79:2356-65
Daelemans, Dirk; Costes, Sylvain V; Cho, Edward H et al. (2004) In vivo HIV-1 Rev multimerization in the nucleolus and cytoplasm identified by fluorescence resonance energy transfer. J Biol Chem 279:50167-75
Lueders, Kira K; De Rosa, Stephen C; Valentin, Antonio et al. (2004) A potent anti-HIV immunotoxin blocks spreading infection by primary HIV type 1 isolates in multiple cell types. AIDS Res Hum Retroviruses 20:145-50
Otero, Miguel; Calarota, Sandra A; Felber, Barbara et al. (2004) Resiquimod is a modest adjuvant for HIV-1 gag-based genetic immunization in a mouse model. Vaccine 22:1782-90

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