CHAIN TRANSLOCATION IN A BIOLOGICAL CONTEXT INJECTING VIRAL GENOMES INTO HOST CELLS Roya Zandi

1. CHAIN TRANSLOCATION IN A BIOLOGICAL CONTEXT INJECTING VIRAL GENOMES INTO HOST CELLS Roya Zandi Mandar Inamdar David Reguera Rob Phillips Joe Rudnick [CHUCK KNOBLER]

2. ANIMAL CELL ENTRY *Virus gets in by binding to receptor in cell membrane *Whole viral particle enters cell *Virus forms and exits via budding

3. Plasmodesmata (shared-wall channels) *Cells are each surrounded by a rigid (cellulose) wall, which must be “broken” (e.g., by abrasion) in order for viral particles to enter *Consequently, a large number of viral particles enter the cell simultaneously, where they are disassembled and replicated *New virions leave cell through existing shared-wall channels

4. BACTERIAL CELL INFECTION BY VIRUS * Virus binds to receptor and ejects genome *Viral particlestays outside cell! Only its genome enters*Virion leaves via lysis of cell

5. Bacteriophage l Its dsDNA genome, 17000 nm long, is highly stressed in its capsid (30 nm radius), due to: Electrostatic Repulsion DNA is packed at crystalline density and is highly crowded Bending Energy Persistence length, 50 nm, implies DNA is strongly bent 30 nm Can calculate energy (U) of DNA as a function of length (L-x) inside U 104 kBT -(dU/dx)=f 10 pN 0 0 0 0.5 1.0 0 0.5 1.0 x/L x/L

6. This internal force drives the genome out along its length. But, it falls sharply as ejection proceeds, and… There is a an opposing force, resisting entry of the chain into the cell, equal to the work per unit length that must be done against the osmotic pressure (P) in the cell 50 Internal Force, pN 20 Osmotic Force: fosmotica P 0 0 30 60 Percentage of genome ejected

7. feject = fresist EXPERIMENT: COUNTERBALANCE EJECTION FORCE BY ESTABLISHING AN EXTERNAL OSMOTIC PRESSURE Capsid permeable to H2O and to ions, but not to PEG Measure DNA concentration by 260-nm absorption -- but mustdistinguish DNA ejected from that remaining in capsid

8. Experimental Design PEG8000 Phages And nuclease (not shown explicitly) Ejected/digested DNA nucleotides Add receptor v Spin down phage by centrifugation Phages (sedimenting material) v Ejected/digested DNA + PEG

9. Evilevitch, Lavelle, Raspaud, Knobler and Gelbart Proc. Nat. Acad. Sci. (USA) 100, 9292 (2003). UV absorbance of DNA ejected from phage as a function of PEG8000 concentration. PEG

10. Extent of Ejected DNA vs Osmotic Pressure in Solution

11. EFFECT OF GENOME LENGTH ON EJECTION FORCE A. Evilevitch C. M. Knobler W. M. Gelbart P. Grayson M. Inamdar P. Purohit R. Phillips 3-4 atms ONLY PART OF GENOME IS DELIVERED TO HOST CELL?!

12. TRANSLOCATION (DIFFUSION) INVOLVING PARTICLE BINDING, AND…RATCHETING Stiff chain of length L is “threaded” into a solution of particles that can bind to it at sites separated by distance s; chain diffusion constant is Drod s viral capsid L Binding particles interact with sites on chain via e-s LJ potential (s=s, in this case) mimic of bacterial cytoplasm

13. SUPPPOSE BINDING PARTICLES STICK IRREVERSIBLY AT EACH ENTERING SITE… [G. Oster et al.]

14. BUT, OTHERWISE…

15. MORE GENERAL TREATMENT OF TRANSLOCATION… Rigid rod (with black monomers) of length L moves distance x into cell (radius Rs) containing N binding particles Brownian Molecular Dynamics (MBD)

16. f(kBT/s) x(s) The filled squares show the force calculated directly in the MBD simulation, for 2Rs=24, L=16, N=100, e/kBT=5, and Drod=Do/16; the open circles show the same for Drod 60 times smaller. Solid curve is computed from the full, coupled, equations for chain diffusion in the presence of binding particles; dashed curve is obtained from assumption of fast equilibration of particle binding.

17. Fast equilibration of particle binding

18. f(kBT/s) x(s) Dashed curve is obtained from solution to the quasi-equilibrium equation for r(x,t); solid curve is computed by solving the full, coupled, diffusion equation for r(x,n,t). The filled squares show the force calculated directly in the MBD simulation, for 2Rs=24, L=16, N=100, e/kBT=5, and Drod=Do/16; the open circles show the same for Drod 60 times smaller.

19. TRANSLOCATION, INCLUDING PUSHING AND PULLING FORCES

20. RECALL THAT DNA is packed at crystalline density and is highly crowded, hence involving a large energy of self-repulsion AND because its persistence length is larger than the capsid size, a significant bending energy is also involved ENERGY ‘COST” (U) IS RELIEVED AS EJECTED LENGTH (x) INCREASES U 104 kBT -(dU/dx)=f 10 pN 0 0 0 0.5 1.0 0 0.5 1.0 x/L x/L

21. EFFECT OF RATCHET ON U(x) 0.5 x 10-4 1 (L2/D) 4 6 Internal force + Langmuir Langmuir

22. EFFECT OF LANGMUIR FORCE ONU(x) --ADDe/sTOfi’s:

23. EFFECT OF OSMOTIC FORCE -- SUBTRACT fosmoticFROMfi’s: driving force drops below 1pN when fraction ejected reaches 50%

24. BINDING/UNBINDING (ON/OFF) EQUILIBRIUM COMPETING TIME SCALES FOR TRANSLOCATION

25. WHERE IS tdiff=s2/Drod ON THE TIME SCALE OF BINDING/UNBINDING? toffton diffusion ratcheting pulling

26. FUTURE WORK: GENOME EJECTION -- PHAGE Build mimics of the bacterial cell, i.e., reconstituted vesicles -- either lipid bilayers or A-B-A block copolymers Investigate effects on injection, of: internal osmotic pressure (all) DNA-binding proteins (e.g., T5) RNA polymerase (e.g., T7) Complement with single-cell, in vivo, studies, monitoring -- in real time -- the entry of the viral genome into bacterial cytoplasm [P. Grayson, R. Phillips]

27. L. T. Fang, C. M. Knobler, W. M. Gelbart + DNA-binding proteins…