Structural Investigation Of SAM68 Interaction With Hiv-1 RRE And REV

Gene expression of retroviruses, such as those of HIV-1, requires transcription, alternative splicing, RNA export and translation steps. All these steps are highly regulated by interactions between viral proteins and cellular proteins. HIV-1 spliced RNAs are exported to the cytoplasm using the classical mRNA export pathway. The export of unspliced RNAs or partially spliced RNAs, however, needs the viral protein Rev (Regulator of Virion) and other cellular proteins including Sam68. This step is crucial for the viral life cycle because these unspliced RNAs are necessary for the expression of the viral structural proteins and for the viral replication. Rev exports unspliced RNA by binding an RNA motif of the HIV-1 RNA composed of approximately 351 nucleotides, which adopts specific secondary structures called an RRE (Rev Response Element). It has been demonstrated that Sam68 plays a crucial role in the export of unspliced RNAs by binding both Rev and the RRE. It is therefore, important to understand the interaction of Sam68 with a viral protein and RNA at molecular level to develop drugs that inhibit HIV-1 replication by disrupting this interaction. Structural and biophysical techniques used to study the interaction of Sam68 with RRE and Rev are described in this thesis. A novel construct of Sam68 designed to bind RRE and Rev simultaneously shows interaction with both binding partners and forms a ternary complex. The EMSA and ITC data provides insight into the binding affinity of Sam68 to RRE, which correlates with the binding affinity of Sam68 with (U/A)AA-N>15-(U/A)AA previously published by our lab. Structural investigation of the binary and ternary complex was carried out using X-ray crystallography. SEC-MALS data provides evidence of a complex formed by interaction of a Sam68 dimer, Rev dimer and one RRE molecule ~161kDa. The region of the RRE recognized by Sam68 is unknown. However, it is known that Sam68 specifically recognizes RNA sequences rich in adenines and uracils. We have analysed the RRE sequence and observed 5 A/U rich regions in the stem-loop structure of the RRE that could be a potential binding site for Sam68. Mutants of RRE have been designed to map the exact binding site of Sam68 on the RRE.