Structural and biophysical studies of HIV-1 Rev protein




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Rev is a key HIV-1 virus regulatory protein, which exports the unspliced and partially\r\nspliced viral mRNA out of the nucleus, thereby inducing the switch from the early phase\r\nto the late phase of the viral replication cycle. Because it is prone to form filaments at\r\nvery low concentration, the X-ray crystallography or NMR approaches to solving Rev\r\nstructure are very difficult. In my dissertation studies, I have designed several structural\r\nmutants of Rev. Under physiological conditions, I found RevC mutant behaves as a\r\nnatively unfolded monomer in solution. The segmentally disordered character of RevC\r\nwas supported by CD and HSQC studies. Another structural Rev mutant is the Rev loop\r\ndeletion. I used a Gly-Ser-Gly-Ala linker to take the place of the Rev loop and most of\r\nthe Arg-rich domain. This mutant can stay in solution as oligomers in sub-millimolar\r\nconcentration for NMR studies. The HSQC spectra of the Rev loop deletion and the\r\nRevC can be well overlaid except the HSQC of Rev loop deletion which has less peaks\r\nunder the same threshold. It brings a filament model with a thin wall formed by Rev Nterminal\r\ntwo helixes and a flexible Rev C-terminal freely moving inside. The flexible\r\ncharacter of Rev C-terminal is also proved to be true for the Rev wild type in comparing\r\nthe HSQC of Rev wild type and RevC. The HSQC of Rev wild type was obtained from\r\n15N labeled Rev and 132B23 complex. B23 is a major cellular component which binds\r\nRev. I designed an N-terminal core of B23, 132B23, and found that it binds with Rev and\r\nforms a decamer with Rev and 132B23 heterodimer.