HIV-1 Reverse Transcriptase Expression, Purification, Crystallization And Functional Study

Human immunodeficiency virus type l(HIV-1) is the iducement for the Acquired Immunodeficiency Syndrome (AIDS) HIV-1 is a retrovirus which encodes a reverse transcriptase (RT) that is required for viral replication. RT is responsible for the conversion of the single-stranded RNA viral genome into double-stranded DNA that subsequently integrased into the host DNA by integrase.The rapid replication of HIV-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. In the absence of an effective vaccine, drugs are the only useful treatment. Anti-HIV drugs work, so far drug therapy has saved more than three million years of life. Unfortunately, HIV-1 develops resistance to all of the available drugs. Although a number of useful anti-HIV drugs have been approved for use in patients, the problems associated with drug toxicity and the development of resistance means that the search for new drugs is an ongoing process. The three viral enzymes, reverse transcriptase (RT), integrase (IN), and protease (PR) are all good drug targets. Two distinct types of RT inhibitors, both of which block the polymerase activity of RT, have been approved to treat HIV-1 infections, nucleoside analogs (NRTIs) and nonnucleosides (NNRTIs), and there are promising leads for compounds that either block the RNase H activity or block the polymerase in other ways.In order to develop more effective inhibitors of HIV-1 reverse transcritase, we designed various of small molecular inhibitors and synthesized by solid-phase reaction method. At the same time, by utilizing Methods of Molecular Biology, the sequences of the subunit P66 and P51 segment of HTV-1 RT was amplified by PCR and recombinant plasmid contain the subunit P66 and P51 segment was successfully constructed. Large scale protein expression in E.coli, and three step purification was performed with AKTA FPLC, then we obtained abundent RT of high purity for further biochemical analysis and crystallography experiment. We verified the interaction between RT and ds-DNA, RT and ss-DNA, RT and integrase(IN), and P66 subunit of RT is the substrate of the HIV-1 Protease(PR), and P66 can be partially hydrolyzed by the PR to generate RT. At last, we get the crystal of RT, after optimized by various ways, we solved the three-dimensional structure of RT at 2.1 A resolution. Unfortunately, we failed to get the X-ray structure of the complex of RT and small molecular inhibitor. However, our research can provide a important platform for further research and development of more effective HIV-1 RT inhibitors.