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