HIV-1 viral protein R (Vpr) is a multifaceted protein that modulates host cells through a number unique ways to facilitate HIV-1 infection and viral pathogenesis. Vpr is required for HIV-1 infection in non-dividing cells such as monocytes, macrophages and incomplete activated T-lymphocytes because of its ability to facilitate nuclear transport of the viral pre-integration complex (PIC) into the nucleus. Vpr induces cell cycle G2/M arrest that is shown to optimize viral replication and escape of host immune responses. In addition, Vpr causes apoptosis that is believed to contribute to depletion of CD4 T-cell depletion. Activation of viral replication and depletion of CD4 T-cell depletion are considered as hallmark of causing AIDS. The findings that rhesus monkeys, chimpanzees or human subjects infected with Vpr defective viruses are associated with slow disease progression indicate that anti-Vpr agents could be useful in anti-retroviral therapies against HIV-1 infection. Because of the unique and efficient ability of Vpr to interrupt host cell cycle G2-M transition and to induce apoptosis, identification of new molecular probes against these Vpr activities could also have potential utility in new drug designs against cancerous cells. The goal of this proposal is to adapt multiple fission yeast cell-based assays, established in our laboratory, for the use in automated high throughput molecular screening (HTS) for new molecular probes against three specific Vpr activities, i.e., apoptosis, cell cycle G2/M regulation and nuclear transport. Each Vpr activity will be first measured by a primary assay and confirmed by a secondary assay. Corresponding mammalian assays will also be used to validate the findings from yeast screenings. Each of these assays will be adapted and validated for 96-well formats, further miniaturized if possible. Finally all of these assays will be configured into HTS format and pilot testing will be carried out through test runs with a small ChemDiv40000 compound library. Significance of the """"""""hits"""""""" identified in the primary HTS will be further verified in mammalian cell systems either with Vpr alone or in the context of HIV-1 infection.
Two specific aims are proposed to adapt and configure the fission yeast cell-based assays into the HTS format to screen for small molecule probes of Vpr-mediated activities. In the first aim, we will adapt three sets of the fission yeast-based assays, which were developed in our laboratory over the last 11 years, into 96-well microtiter plate format by optimizing them for easy handling and automation. Corresponding mammalian methods will also be used to verify the small molecules identified in the yeast screenings. Completion of this specific aim would allow us to demonstrate the robust nature and reproducibility of each of the described assays, furthermore, to simplify the readouts and to make them more amenable to automated analyses; the second Specific Aim will be carried out at the HTS Core Facility of the University of Maryland at Baltimore (UMB) to further configure our assays for HTS and to conduct a pilot run by using a small ChemDiv40000 compound library. After adaptation and validation, these fission yeast cell-based assays should be ready for HTS and to participate in the Molecular Libraries Screening Centers Network (MLSCN) to identify additional molecular probes against each of the specific Vpr activities. All together, successful completion of the proposed studies will enable us to adapt the fission yeast-based assays into the formats for HTS, to conduct primary drug screening against Vpr for the first time and to provide opportunities for us to participate in the NIH Molecular Libraries Roadmap Initiative and the Molecular Libraries Screening Centers Network (MLSCN). The identified small molecules should provide functionally significant lead compounds to help us in the future design of additional drugs against HIV and cancers. ? ? Public Health Relevance Statement HIV/AIDS is one of the most devastating diseases in the world with approx. 33 million people living with HIV in 2007 and approx. 2.5 million new infections in the same year. Activities of HIV-1 viral protein R (Vpr) has been linked to activation of viral replication and depletion of CD4 T-cell depletion, both of which are hallmark of AIDS. In addition, rhesus monkeys, chimpanzees or human subjects infected with HIV carrying defective Vpr typically show slow disease progressions. Thus finding drugs that could limit the Vpr's activities could potentially alleviate the detrimental effects of Vpr on HIV-infected patients. At present, there is no antiviral drug or HTS system that is designed to screening drugs against Vpr. In this proposal, multiple fission yeast cell-based assays, which have been developed in our laboratory over the last 11 years to measure specific activities of Vpr such as nuclear transport, cell cycle arrest and apoptosis, will be adapted to the high throughput platform to screen potential inhibitors of Vpr. The proposed assays could additionally identify compounds that affect cell proliferation and apoptosis, two cellular processes that are involved in the development of cancers. ? ? ?
| Benko, Zsigmond; Elder, Robert T; Li, Ge et al. (2016) HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe. PLoS One 11:e0151286 |
| Nkeze, Joseph; Li, Lin; Benko, Zsigmond et al. (2015) Molecular characterization of HIV-1 genome in fission yeast Schizosaccharomyces pombe. Cell Biosci 5:47 |
| Zhao, Richard Y; Bukrinsky, Michael I (2014) HIV-1 accessory proteins: VpR. Methods Mol Biol 1087:125-34 |
| Benko, Zsigmond; Zhao, Richard Y (2011) Zeocin for selection of bleMX6 resistance in fission yeast. Biotechniques 51:57-60 |
| Zhao, Richard Y; Li, Ge; Bukrinsky, Michael I (2011) Vpr-host interactions during HIV-1 viral life cycle. J Neuroimmune Pharmacol 6:216-29 |
