HIV Entry into the Nucleus and Organization of the Genome

1. HIV Entry into the Nucleus and Organization of the Genome Over the Next 3 lectures we will analyze the mechanism of virus entry into the nucleus and its propagation in the nucleus. Principles to be covered Directed Diffusion Osmolarity Motor and Topoisomerase Function

2. Structure of HIV Reverse Transcriptase

3. Nucleosome Structure

4. Nuclear Localization Signals Cause Transport to Nucleus

5. Nuclear Transport is Diffusion-Dependent In Smaller Cells Transport from the Cytoplasm to the Nucleus is Dependent upon thermal energy through random diffusion. One-dimensional random walk with a flip of a coin and a step in + or - direction gives a Gaussian distribution with many particles and long time.

6. Diffusive Processes Can be Described by Diffusion Equation Einstein-Smoluchowski equation relates the friction coefficient of a particle moving through a medium to the diffusion coefficient of the particle in that medium. Dsphere = kT/d = kT/6r Frictional Coefficient d = 6r Where  = viscosity and r = radius

7. Diffusive Movements Depend Upon the Square of Distance For one-dimensional diffusion 2D1t = <X2> For two-dimensional diffusion 4 D2t = <X2+Y2> Example: 1 mm sphere Dsphere = 4.4 x 10-9 cm2/sec After 10s <X2> = 8.8 x 10-8 cm2 X = 3 x 10-4 cm = 3 mm

8. Large Particles (~26 nm) Can be Transported (Diffusive Pores <9 nm)

9. Nuclear Pore Complex Structure Princ of Virology. Flint et al. Fig. 5.19

10. Model of Nuclear Import Princ of Virology. Flint et al. Fig. 5.20

11. Ran-GTP-GDP Exchange Catalyzes the Transport

12. Another Way to Enter the Nucleus? Some viruses don’t have a nuclear localization signal. The other way to enter the nucleus is to wait for nucleus breakdown in mitosis. At that time the nuclear membrane moves into the ER and vesiculates.

13. Nucleus Moves into ER-like Vesicles During Mitosis

14. Nuclear Lamins Provide a Structural Support for Nucleus

15. DNA Packaging Problem There are 2 physical problems that must be overcome in packaging the 2 meters of double stranded DNA in a nucleus of about 6 mm in diameter. Packing Problem: How to fold the DNA to fit into the nucleus. Osmolarity: Each base has a negative charge and that charge needs to be balanced by a plus charge, commonly potassium is cytoplasm.

16. Osmolarity in the Nucleus Osmolarity in cytoplasm: typically 310 milli osmolar, which is the sum of the concentration of potassium, sodium, chloride, phosphate, and other charged small molecules. Neutrality is always preserved (concentration of minus equals conc of minus ions) Osmolarity in nucleus: cytoplasmic ions and small molecules plus counterions for DNA? (2 X 6 x 109 Phosphate anions- or 2 x 10-14 moles of P- in 4/3r3 = 4-10 X 10-14 l or 0.2-0.5 M of negative charge that needs counterions)

17. Structure of DNA in Nucleus

18. Nucleosome Structure

19. DNA Superhelicity and Knots Unwinding of DNA for replication or transcription introduces superhelicity in the DNA. Movement of DNA can create knots or tangles (as in fishing line) that can only be straightened out by breaking the line.

20. Topoisomerase I

21. Topoisomerase II

22. Reading for Next Class Alberts et al., MBOC (vol.4) Chapters 5 and 6 on DNA replication and transcription. Alternatively, read the equivalent chapters in one of the other Cell Biology Texts.