Abstract

1. Abstract Assembly of viral proteins and RNA on plasma membranes of host cells is a critical step in the processing of infectious particles. The Major Homology Region (MHR) is a highly conserved sequence throughout all retroviruses, including HIV-1. Its role in assembly is crucial but unknown. The MHR is a 20 amino acid stretch found in the capsid domain of HIV-1 Gag. In order to determine the role of the MHR in viral assembly, membrane and RNA fluorescence based binding studies were conducted. As models, LUVs, composed of POPS, and tRNA, were used. GagWT exhibits approximately 2 fold greater binding to lipid bilayers, but there is not any difference in tRNA binding as compared to the GagMHR mutant. These data indicate that the MHR may be involved in the protein-protein interactions that drive assembly. To begin to characterize these interactions, a set of intrinsic fluorescence and homotransfer studies was used to determine free energy diagrams for membrane and RNA binding. In vitro studies show first that the change in free energy difference between Gag to membrane and Gag to RNA is within RT (600 cal/mol*K). Therefore, initial binding of Gag can be to either substrate, but due to proximity in the cell, it is most plausible that Gag binds to RNA first. Secondly, GagMHR shows a decreased affinity for membrane when initially bound to RNA and for association on tRNA as compared to wild type. This indicates that the MHR mediates protein-protein interactions. Further studies to better understand these protein interactions will be presented. NIH GM53132

2. Figure 1: Structure of Capsid1 C Terminus (near RNA binding domain, NC) MHR N Terminus (near membrane binding domain, MA)

3. Introduction • What? • Determine the role of the Major Homology Region (MHR) of HIV-1 Gag in RNA binding, Gag-Gag interaction, and membrane binding. • Why? • MHR is highly conserved throughout all retroviruses. (Table 1) • The MHR’s function is unknown. • How? • Experiments designed to determine the change in free energy for GagWT and GagMHR. (fig. 2)

4. Table 1: MHR of Various Retroviruses2 A - indicates a non-conserved residue. The Red amino acids are 100% conserved throughout all these viruses and correspond to point mutants that are being utilized in further studies. GagDMHR is a deletion of aa 284-308.

5. Figure 2: Event Sequence Determination for Particle Formation (DG = -RT ln(K) ) G Diagram Methods G Diagram Methods Gag (mono) + RNA Gag (mono) + Membrane Intrinsic Fluorescence Intrinsic Fluorescence Binding DG1 DG4 Gag (mono)* RNA Gag (mono)* Membrane Fluorescein Homotransfer Fluorescein Homotransfer DG2 DG5 Assembly Gag (associated)* RNA Gag (associated)* Membrane Dansyl labeled Lipid DG3 DG6 Intrinsic Fluorescence Binding Membrane *Gag *RNA RNA *Gag * Membrane DG1+DG2+DG3 = DG4+DG5+DG6

6. Figure 3: POPS Binding

7. Figure 4: tRNA Binding

8. Figure 5: Homotransfer

9. Figure 6: Gag/POPS Complexes Binding to tRNA

10. Figure 7: Gag/tRNA Complex Binding to POPS

11. Figure 8: GagWT G Diagram for Particle Formation Gag (mono) + Membrane Gag (mono) + RNA DG = -832 cal/mol*K DG = -1075 cal/mol*K Binding Gag (mono)* RNA Gag (mono)* Membrane DG = -1660 cal/mol*K DG = -2014 cal/mol*K Assembly Gag (associated)* Membrane Gag (associated)* RNA DG = -978 cal/mol*K DG = -734 cal/mol*K Binding Membrane *Gag *RNA RNA *Gag * Membrane -3823 cal/mol*K -3470 cal/mol*K

12. Figure 9: GagMHR G Diagram for Particle Formation Gag (mono) + Membrane Gag (mono) + RNA DG = -1444 cal/mol*K DG = -902 cal/mol*K Binding Gag (mono)* RNA Gag (mono)* Membrane DG = -1907 cal/mol*K DG = N/A Assembly Gag (associated)* Membrane Gag (associated)* RNA DG = -1104 cal/mol*K DG = -1835 cal/mol*K Binding Membrane *Gag *RNA RNA *Gag * Membrane -4455 cal/mol*K -2737 cal/mol*K +N/A

13. Conclusions • The difference in change in free energy between Gag binding to membrane and Gag binding to RNA is within RT(600 cal/mol*K). This suggests that Gag can initially bind either RNA or membrane, but when cellular proximity issues are considered (Goff, poster 1007), Gag/RNA binding would seem most likely to occur first. • The MHR of HIV-1 Gag exhibits its largest effect on protein-protein interactions, as shown by the homotransfer of GagDMHR on tRNA (fig. 4) and by the the binding of the GagDMHR mutant when it is first bound to tRNA and binding to POPS is followed (fig. 6).

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

15. GagDMHR Particle Formation Model GagDMHR binds to RNA GagDMHR does not associate on RNA Legend GagDMHR Trimer MA CA NC GagDMHR/RNA complex binds with less affinity to membrane RNA Membrane

16. Figure Legends • Figures 3,4,6: Intrinsic Fluorescence - The substrate (POPS or tRNA) was titrated into 200nM protein in buffer (0.5M NaCl, 40mM HEPES, 1mM DTT). Intrinsic fluorescence was followed by ex. 280nm, em. 320-400nm. • Figure 4: Homotransfer - Fluorescein labeled protein was titrated into 83mM POPS or 33mM tRNA in buffer. Energy homotransfer was followed by anisotropy ex. 480nm, em. 525nm. • Figure 7: Dansyl fluorescence - Dansyl DHPE was added to POPS in a 1% solution. This was titrated into 200nM protein in buffer. Dansyl fluorescence was followed by ex. 336nm, em. 517nm. • All experiments performed on an ISS PC1.

17. Acknowledgements • We would like to thank Indralatha Jayatilaka, Louisa Dowal, Marjorie Bon Homme and Arthur Goff for technical assistance, and Dr. Stephen Cusack for providing the structure of capsid. • This work was made possible by funding through the National Institutes of Health (NIH 53132) • Berthet-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. • Ebbets-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.