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

Mature HIV-1 Virion

http://www.scistyle.com/

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

Questions?

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.