1. Abstract The major homology region (MHR) is a highly conserved sequence in the Gag gene of all retroviruses, including HIV-1. Its role in assembly is unknown, but deletion of the motif significantly impairs membrane binding and viral particle formation. Preliminary studies in transfected cells indicated that GagΔMHR was defective in formation of an RNA-containing,membrane-bound replication intermediate. To determine the role of the MHR in formation of this complex, fluorescence-based binding studies were conducted in vitro, using tRNA and large unilamellar vesicles of 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS-LUVs) as model membranes. As previously reported, GagΔMHR bound to lipid bilayers with slightly reduced affinity compared to wild-type Gag (GagWT) and no differences in tRNA binding between GagWT and GagΔMHR were detected. From our studies, we were able to determine the change in free energy (ΔG) for all of these associations and find that, the energies for membrane binding and for tRNA binding are within room temperature indicating that in vitro Gag will not preferenitally bind to either substrate. However, we speculate that, due to proximity, in vivo RNA binding would be preferred. Interestingly, GagWT showed a strong tendency to self-associate on both tRNA and membranes whereas self- association of GagΔMHR on tRNA was not detectable. These results suggest that, during infection, the MHR plays a key role in promoting productive protein-protein interactions on RNA.

2. What? –Determine if the interaction between HIV-1 Gag and RNA is a prerequisite for membrane binding. –Determine the assembly event sequence of HIV-1 viral particles. –Gain an insight into the role of the MHR in this assembly. Why? –The steps that precede the formation of viral budding structures at the plasma membrane are not well defined. –MHR is highly conserved throughout all retroviruses. –The MHR’s function is unknown. How? –Experiments designed to determine the change in free energy for Gag WT and Gag  MHR. Introduction

3. Figure 1: Sucrose Gradient Method and Rationale Gag proteins were expressed in mammalian cells 20% 60% 20% 60% Cell lysates were subjected to sucrose density gradient centrifugation Fractions were separated by gel electrophoresis

4. Figure 2: Event Sequence Determination for Particle Formation (  G = -RT ln(K) )  G 1 +  G 2 +  G 3 =  G 4 +  G 5 +  G 6 Gag (mono) + Membrane Gag (mono) + RNA Gag (mono)* Membrane Gag (mono)* RNA Gag (associated)* Membrane Gag (associated)* RNA RNA *Gag * Membrane Membrane *Gag *RNA Intrinsic Fluorescence Fluorescein Homotransfer Intrinsic Fluorescence Fluorescein Homotransfer Intrinsic Fluorescence  G Diagram Methods G1G1 G1G1 G2G2 G2G2 G3G3 G3G3 G4G4 G4G4 G5G5 G5G5 G6G6 G6G6 Binding Assembly Binding

5. Figure 6: Binding of Gag to POPS and tRNA Intrinsic Fluorescence - The substrate (POPS or tRNA) was titrated into 200nM protein in buffer (0.5M NaCl, 40mM HEPES, 1mM DTT), 83  M POPS or 33  M tRNA. Intrinsic fluorescence was followed by ex. 280nm, em. 320-400nm.

6. Figure 7: Monitoring Gag Assembly on POPS and tRNA by Homotransfer Homotransfer - Fluorescein labeled protein was titrated into 83  M POPS, 33  M tRNA, or buffer. Energy homotransfer was followed by anisotropy ex. 490nm, em. 516nm.

7. Figure 8: Gag Complex Binding to Second Substrate Intrinsic Fluorescence - The substrate (POPS or tRNA) was titrated into 200nM protein in buffer (0.5M NaCl, 40mM HEPES, 1mM DTT), 83  M POPS or 33  M tRNA. Intrinsic fluorescence was followed by ex. 280nm, em. 320-400nm.

8. Figure 9: Gag WT  G Diagram for Particle Formation -3763 cal / mol*K  -3469 cal / mol*K Gag (mono) + MembraneGag (mono) + RNA Binding Gag (mono)* Membrane Gag (mono)* RNA Assembly Gag (associated)* MembraneGag (associated)* RNA RNA *Gag * Membrane Membrane *Gag *RNA  G = -1075 cal / mol*K  G = -1960 cal / mol*K  G = -728 cal / mol*K  G = -830 cal / mol*K  G = -1658 cal / mol*K  G = -981 cal / mol*K Binding

9. Figure 10: Gag  MHR  G Diagram for Particle Formation -5014 cal / mol*K  -2739 cal / mol*K +N/A  G = -1439 cal / mol*K  G = -2473 cal / mol*K  G = -1102 cal / mol*K  G = -904 cal / mol*K  G = N/A  G = -1835 cal / mol*K Gag (mono) + MembraneGag (mono) + RNA Gag (mono)* Membrane Gag (mono)* RNA Gag (associated)* MembraneGag (associated)* RNA RNA *Gag * Membrane Membrane *Gag *RNA Binding Assembly Binding

10. Conclusions Gag binds initially to RNA, and this complex facilitates efficient membrane binding. The MHR plays a critical role in Gag-Gag interactions which enables the RNA/Gag complex to present a membrane binding face.

11. Gag WT Particle Formation Model Gag WT binds to RNA Gag WT /RNA complex binds with higher affinity to membrane Gag WT associates on RNA Legend Gag WT Trimer MA CA NC RNA Membrane

12. Gag  MHR Particle Formation Model Gag  MHR binds to RNA Gag  MHR /RNA complex binds with less affinity to membrane Gag  MHR does not associate on RNA Legend Gag  MHR Trimer MA CA NC RNA Membrane

13. Acknowledgements We would like to thank Indralatha Jayatilaka, Fadila Bouamr, Lynn VerPlank, Louisa Dowal and Marjorie Bon Homme for technical assistance. This work was made possible by funding through the National Institutes of Health (NIH 53132) 1Berthet-Colominas, C., et al., Head-to-tail dimers and interdomain flexibility revealed by the crystal structure of HIV-1 capsid protein (p24) complexed with a monoclonal antibody Fab. Embo J, 1999. 18(5): p. 1124-36. 2Ebbets-Reed, D., S. Scarlata, and C.A. Carter, The major homology region of the HIV-1 gag precursor influences membrane affinity. Biochemistry, 1996. 35(45): p. 14268-75. 3Lee, Y. M., B. Liu, and X. F. U. 1999. Formation of virus assembly intermediate complexes in the cytoplasm by wild- type and assembly defective mutant human immunodeficiency virus type 1 and their association with membranes. J. Virol. 73:5654-5662.

14. Figure 4: Intermediate Complexes Contain RNA and are Membrane Bound Cytoplasmic extracts were prepared from cells expressing Pr55 Gag and treated with Panel A: untreated Panel B: RNase Panel C: EDTA Panel D: 1% nonionic detergent Gradient fractions were subjected to density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis and immunoblotting using an anti p6 antibody (IGEPAL)

15. Figure 5: Formation of Assembly Intermediates Require the MHR Cytoplasmic extracts were prepared from transfected cells containing Panel A: Pr55 Gag Panel B: Pr55 Gag-  MHR Panel C: Pr55 Gag-Myr Supernatants were subjected to density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis and immunoblotting using an anti CA antibody. The graph above each gradient indicates the density in g/ml of each fraction.