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summerspowerpoint2013 (2)

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summerspowerpoint2013 (2)

  1. 1. Expression of the Murine Leukemia Virus CANC Domains of the Gag Polyprotein and Transcription of the Core Encapsidation Signal Alexander Emmanuelli1, Ibinabo Feddy-Inimgba1, Meleake Sahlu3, Deborah Girma, Michael F. Summers, Ph.D.2 1Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250. 2Professor,Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250. 3Mount St. Michael Academy, 4300 Murdock Avenue, Bronx, NY 10466. ABSTRACT The Human Immunodeficiency Virus (HIV) is a retrovirus that causes Acquired Immunodeficiency Syndrome (AIDS) in humans. The Murine Leukemia Virus (MLV) is a retrovirus that causes cancer in mice. MLV and HIV share a common life cycle and use the same structural proteins. MLV is simpler than HIV, lacking accessory proteins and possessing a smaller genome, making MLV a great animal model for structural studies of the viral structural proteins. The Gag polyprotein, is essential to the assembly of new virus particles. Gag consists of three domains; Matrix, Capsid, and Nucleocapsid . Nucleocapsid binds to the viral RNA during assembly and CA engages in gag-gag lattice interactions but has been shown to play a role in gag-RNA interaction. Our project focuses on the expression, purification, and concentration of the Capsid and Nucleocapsid domains (CANC). We have successfully expressed CANC in BL21 DE3 E. Coli cells. We have employed a GST fusion tag system to purify CANC. While expression of the protein has been successful we are working towards increasing solubility at higher concentrations. We aim to carry out gel shift assays with the CANC protein which will provide useful information on the protein-RNA interactions during the late phase of the viral life cycle. Dimerization and packaging of the viral genome is directed by a 350 nucleotide packaging signal (Psi) and the nucleocapsid (NC) domain of assembling Gag polyproteins. The core encapsidation signal, a 101 nucleotide sequence located with in the Psi-site, is essential for efficient genome packaging during viral assembly. We are currently optimizing conditions for in vitro transcription of the core encapsidation signal.   Figure 1: Viral Life Cycle EXPERIMENTAL METHODS: PROTEIN 1.  Grow starter culture of BL21 DE3 E. Coli cells overnight, inoculate cells until OD reaches 0.600 2.  Induce CANC expression using IPTG 3.  Lyse Cells in microfluidizer 4.  Nucleic acid precipitation 5.  Isolate and purify protein using GST tags and glutathione resin. 6.  Cation exchange for further purification 7.  Concentrate protein RESULTS   Figure 6: Core with GA start sequence CONCLUSIONS • Successfully expressed and purified the CANC protein • Identified optimal conditions for solubility • 0.05% triton and Argon solution evaporation is the best method to achieve high concentrations of CANC • Core RNA can be transcribed and purified with a GA start sequence. REFERENCES Figure 1: Turner, Brian; Summers, Michael. “Structural Biology of HIV”. Figure 2: Miyazaki, Yasuyuki. “An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus”. JMB. 2010 February 12; 396(1): 141– 152. ACKNOWLEDGEMENTS This research is funded in part by NIH grant R01AI81604. Figure 4: Expression results; lanes from left to right: Ladder, pre- induction, lysate supernatant, lysate pellet, wash, elution 1, E2, and E3. Figure 5: Denaturing gel results; bands from top to bottom: uncleaved sequence, cleaved sequence and hammerhead ribozyme. Observing poor cleavage. INTRODUCTION CANC STUDIES The CANC domains of the gag polyprotein interact with RNA during viral assembly, but genome recognition by CANC is not well understood. It is known that the nucleocapsid domain binds to specific sites (psi sites) on the viral genome with high affinity, and that the capsid domain helps direct this binding. Figure 2: Secondary structure of the MLV 5’ UTR packaging signal. Core encapsidation signal is marked by the dashed lines and NC binding sites are highlighted Expression condition A B Temperature 30oC 37oC ✓ Shaking speed 225 rpm ✓ 250rpm Induction time Six hours ✓ Overnight Concentration Centrifugation Argon evaporation ✓ Buffer Chaotropes Triton ✓ None Cleavage Buffer pH 7.5 8.4✓ INTRODUCTION RNA STUDIES The 5’ UnTranslated Region (5’ UTR) of the MLV genome interacts with the nucleocapsid domain of the group specific antigen (Gag) polyprotein in a process of the retroviral life cycle known as Genome Recognition but this process is not yet fully understood. After initial work with hammerhead ribozyme we are currently optimizing conditions for in vitro transcription of the core encapsidation signal using a construct which has GA start sequence. Figure 3: Transcriptions run concurrently on small scale denaturing gels to determine the optimal conditions for a large scale. All volumes in microliters. EXPERIMENTAL METHODS: RNA 1. Mega prep 5. Trial Transcription 2. Plasmid Digestion 6. Large Scale 3.Phenol Extraction 7. Elutrap 4. Ethanol Precipitation 8. Wash