HIV Integrase Structural Biology

描述（由申请方提供）：尚不清楚病毒感染细胞中HIV-1整合酶（IN）与细胞质整合前复合物（PIC）内病毒DNA末端的分子相互作用。了解这些相互作用对于了解针对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鉴定了瞬时突触复合物（SC），其具有与PIC相关的特性。我们将利用这些新的进展来理解协同整合：1）确定使用纯化的IN单体的HIV SC组装的功能机制; 2）探索使用小DNA寡核苷酸（ODN）作为底物，在没有或存在链转移抑制剂（STI）的情况下产生同质HIV IN-DNA复合物的条件; 3）启动HIV IN-DNA复合物的共结晶研究， 最先进的机器人技术 原型泡沫病毒（PFV）整合体（Cherepanov组）的晶体结构极大地增强了我们对IN亚基与同源DNA末端相互作用以促进协同整合的理解。PFV IN主要是允许DNA末端之间的结构域间互补以形成整合体的单体。PFV整合体和HIV SC在协同整合方面功能等同。HIV SC的原子分辨率研究是必要的，因为PFV和HIV IN之间的氨基酸同一性在它们各自的催化中心之外是最小的。 为什么重组HIV IN不能有效地利用ODN进行协同整合，这一谜团存在了20年。我们已经取得了两个最近的进展，以解决这个谜最近发表在去年10月（生物化学50：9788-9796，2011）。首先，我们确定了我们广泛研究的HIV-1 IN作为高度可溶的单体存在。第二，我们发现我们的纯化的HIV IN使用U 5 LTR ODN（18 bp至42 bp，3' OH凹陷或平端）用于高效的协同整合活性，其中IN和ODN都处于微摩尔浓度。最后，我们最近还鉴定了几种STI（>200 nM），其使用钝端或凹端U 5底物选择性地产生新的IN-单DNA（ISD）复合物。 总之，我们假设HIV IN单体促进活性四聚体组装到病毒DNA上以进行协同整合。我们将探索条件，以产生可溶性HIV IN-DNA复合物，没有和STI，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.