HIV Integrase Structural Biology

描述（由申请人提供）：在病毒感染的细胞中，HIV-1整合酶（IN）与细胞质预整合复合体（PIC）内的病毒DNA末端的分子相互作用尚不清楚。了解这些相互作用对于理解针对IN的链转移抑制剂（STI）、对这些抑制剂的耐药性以及第二代抑制剂的开发至关重要。使用纯化的HIV IN和病毒DNA底物，我们在天然琼脂糖凝胶上发现了瞬时突触复合物（SC），它们具有与PIC相关的特性。我们将利用这些新进展来理解协同整合：1)利用纯化的IN单体确定HIV SC组装的功能机制；2)探索在没有或存在链转移抑制剂（STI）的情况下，利用小DNA寡核苷酸（ODN）作为底物生产同质HIV in -DNA复合物的条件；3)启动HIV IN-DNA复合物的共结晶研究

DESCRIPTION (provided by applicant): Molecular interactions of HIV-1 integrase (IN) with the viral DNA ends within the cytoplasmic preintegration complex (PIC) in virus infected cells are not clearly understood. Knowledge of these interactions is critical to understand strand transfer inhibitors (STI) directed against IN, drug-resistance to these inhibitors, and development of second generation inhibitors. Using purified HIV IN and viral DNA substrates, we have identified the transient synaptic complex (SC) on native agarose gels that shares properties associated with the PIC. We will use these new advancements for understanding concerted integration to: 1) determine the functional mechanisms of HIV SC assembly using purified IN monomers; 2) explore conditions to produce homogenous HIV IN-DNA complexes using small DNA oligonucleotides (ODN) as substrates, without or in the presence of strand transfer inhibitors (STI); and 3) initiate co-crystallization studies of HIV IN-DNA complexes using state-of-the-art robotic technologies. The crystal structure of the prototype foamy virus (PFV) intasome (Cherepanov group) has greatly enhanced our understanding of IN subunit interactions with the cognate DNA ends to promote concerted integration. PFV IN is mainly a monomer that allows interdomain complementation between DNA ends to form the intasome. The PFV intasome and the HIV SC are functionally equivalent for concerted integration. Atomic resolution studies of the HIV SC are necessary because the amino acid identity between PFV and HIV IN is minimal outside their respective catalytic centers. A mystery existed for 20 years why recombinant HIV IN was not capable of efficiently using ODN for concerted integration. We have made two recent advances towards solving this mystery recently published this last October (Biochem. 50:9788-9796, 2011). One, we determined that our extensively studied HIV-1 IN exists as a highly soluble monomer. Two, we discovered that our purified HIV IN uses U5 LTR ODN (18 bp to 42 bp, 3' OH recessed or blunt) for highly efficient concerted integration activity where both IN and ODN are in the micromolar concentrations. Finally, we also recently identified several STI (>200 nM) that selectively produce a new IN-single DNA (ISD) complex using either blunt or recessed ended U5 substrates. In summary, we hypothesize that HIV IN monomers facilitate the assembly of the active tetramer onto viral DNA for concerted integration. We will explore conditions to produce soluble HIV IN-DNA complexes, without and with STI, for co-crystallization studies.