Structural And Functional Research Of HIV-1CRF07-BC Gp41and EIAV Gp45

AIDS(acquired immunodeficiency syndrome, AIDS) is an infectious disease caused by HIV (human immunodeficiency virus). CRF07-BC is a China-specific recombinant HIV-1strains, first detected in Sichuan and Xinjiang’s IDUs (intravenous drug users) and become China’s major epidemic strain, accounting for over50%of all infected people. From discovery to date, CRF07-BC in China for more than ten years was gradually expanded its popular trend.The same as other HIV-1viruses, CRF07-BC virus infection life cycle from entering the target cell. At the surface of the virus particles, HIV envelope glycoprotein (ENV) is in the form of gp120/gp41trimer within high-energy states. When virus infection of target cells, the HIV envelope glycoprotein gp120binding to the target cell receptor CD4molecule, then the gp120conformation change exposing the binding site of the co-receptor, CXCR4or CCR5. Co-receptor binding causes the release of gp120and then gp41conformation changes, the fusion peptide insert into the cell membrane of target cells, followed by gp41forming a six-helix bundle. HIV gp41play a key role in the fusion process, gp41has three major functional domains:an extracellular domain, a transmembrane domain and an intracellular domain. Its main function is mediated by the extracellular domain.In this research, we solved crystal structure of CRF07-BC gp41ectodomain core, through the analysis; we found that the overall structure of CRF07-BC gp41is very similar to the structure of B-subtype gp41. But they are different in the surface charge and hydrophilicity significantly. By sequence alignment, we found that there is a hyper-mutant ant the middle of gp41HR2, site-directed mutagenesis experiments and cell fusion experiments showed that this region is important to maintain the cell fusion. And during fusion, the hyper-mutant site s conformation may change, form loop to helix. Therefore, we propose the natural state of gp41and revised the gp41-mediated fusion model.Through the structure analysis of the hyper-mutant site, we know that CRF07-BC gp41s function is affected not only by the single amino acid, but also by the neighboring amino acid, so for the design of drugs should also consider the role of the microenvironment of drugs. That would be helpful to design drugs and vaccines target multiple amino acids that have synergistic effect in function or target the microenvironment that important to the function.Equine infectious anemia virus (EIAV) belong to the retrovirus family lentivirus, can infect equine, its distribution is extremely broad. In1970s, scientist form Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, successfully developed EIAV live attenuated vaccine, which is the only large scale application lentivirus virus vaccine in the world. EIAV and HIV belong to the lentivirus; they are similar in the viral structural, viral genomes, the life cycle and the infection symptoms. Therefore, EIAV live attenuated vaccine is a good model for HIV vaccine research and development.In our research, we solved the EIAV gp45structure. And we know that Gp45have similar crystal structure of HIV gp41and SIV gp41, HR1-HR2form stable six-helix bundle structure. EIAV gp45structure is the same as the longest HIV gp41(PDB:2X7R) with FPPR and MPER, as presented in the HR1N terminal of an open conformation. However, gp45is much more open and form a pocket. In the "pocket", some water molecules arranged orderly. The solvent molecules (water molecules) of gp45that aligned along the central axis of are very unique. In HIV gp41there is only three water molecules. Also gp45is much looser in stack than gp41, this may related to the central axis water molecular.Meanwhile, we solved the gp45crystal structure of EIAV vaccine strain. EIAV vaccine strain has the similar crystal structure of wild-type gp45, only have a mutation in position505, valine/isoleucine (wild type strain) to threonine (vaccine strain). Important cause mutations in the d-bit HR2of gp45mutations result in reduced stability occurs, which may also have immune effect of the vaccine strain. This mutation is in HR2d position, and the mutation break the stability of gp45, this may be an important mechanism and explain of the vaccine strain.Moreover, we want to use what we found and do something to HIV vaccine development. Use the vaccine gp45, which thermostability reduced but the fusion stability kept, as standard, we made a mutagenesis screening of HIV gp41HR1and HR2a and d position. We did not find compliant mutant candidate in the single point mutation. As for the functional complementarity in HR2hyper-mutant region, we will do a screen again in the amino acid that have functional complementary. In our research, we not only describe a possible immune mechanism of EIAV vaccine strain, but also we want to use this mechanism in HIV, and we hope this could be helpful in HIV vaccine development. In this thesis, there are72figures,20tables and160references.