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Freeze-Fracture Electron Microscopy of HIV-1 p24 capsid protein interaction with the Viral Envelope.
Eric Newmanhttp://www.wired.com/2011/03/virus-therapy-hiv/
Human Immunodeficiency Virus
MOSS, J.A. (2013). HIV/AIDS REVIEW. RADIOL TECHNOL. 84(3):247-267HUANG, M., ORENSTEIN, J., ET. AL. (1995). P6GAG IS REQUIRED FOR PARTICLE PRODUCTION FROM FULL-LENGTH HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 MOLECULAR
CLONES EXPRESSING PROTEASE. JOURNAL OF VIROLOGY. 69(11): 6810-6818
• Enveloped
• Lentivirus: Infects non-dividing cells, Long incubation
• ~1010 Virions per infected cell
• 3 x10-5 per nucleotide base pair per replication cycle mutation rate.
• ssRNA(+), Retroviral integration
Specific Aims
1) Visualize the viral envelope and capsid interaction
2) Arrest viral bud release
3) Obtain multiple fracture planes throughout the Virion
Arresting Bud Release
•p6Gag protein has been show to interact with
lipid membranes
•Site directed mutagenesis of p6
•p6 knockout inhibited virion release 20-fold
•Increase in Virion density
Huang, M., Orenstein, J., Et. al. (1995). p6Gag is Required for Particle Production from Full-Length Human Immunodeficiency Virus Type 1 Molecular Clones Expressing Protease. Journal of Virology. 69(11): 6810-6818
Polyprotein precursor
Freeze-Fracture
http://www.leica-microsystems.com/science-lab/brief-introduction-to-freeze-fracture-and-etching/
Freeze-Fracture
•Freeze under high pressure to form
amorphous ice
•Fracture using super-cooled blade
•Etch-process under high vacuum
•Heavy metal shadowing
•Carbon lattice
•Acid digestion
•1.1 nm resolution
Gross, H. Bas, E. Moor, H. (1978). Freeze-fracturing in ultrahigh vacuum at -196 degrees C. J Cell Biol. 76(3):712-28.
Transmission Electron Microscopy (TEM)
•Electron beam transmitted through Ultra thin
specimen
•Magnified and focused
•0.05 nm resolution
•10,000,000X Magnification
http://www.allposters.com/-sp/Freeze-Fracture-Technique-Used-to-Show-the-Nuclear-Pores-Nuclear-Pores-are-Structures-Posters_i9001685_.htm
FF-TEM of nuclear pores
HIV Statistics
•35 million current infections (WHO 2013)
•2.1 million new infections (WHO 2013)
•1.5 million AIDS deaths (WHO 2013)
•Global funding for HIV/AIDS $31.7 Billion in 2016 (KSS)
•Research spending in the U.S. $2,978 million (NIH 2014)
http://kff.org/global-health-policy/fact-sheet/u-s-federal-funding-for-hivaids-the-presidents-fy-2016-budget-request/http://www.who.int/hiv/data/epi_core_dec2014.png?ua=1
References•http://www.who.int/hiv/data/epi_core_dec2014.png?ua=1
•Barrera, F. Hurtado-Gomez, E. Lidon-Moya, M. Neira, Jose. (2006). Binding of the C-terminal domain of the HIV-1 capsid protein to lipid membranes: a biophysical characterization. Biochem J. 394:345-353
•Huang, M., Orenstein, J., Et. al. (1995). p6Gag is Required for Particle Production from Full-Length Human Immunodeficiency Virus Type 1 Molecular Clones Expressing Protease. Journal of Virology. 69(11): 6810-6818
•Gross, H. Bas, E. Moor, H. (1978). Freeze-fracturing in ultrahigh vacuum at -196 degrees C. J Cell Biol. 76(3):712-28.