hnRNP A1 inhibition of HIV-1 replication

描述（由申请人提供）：整合的HIV-1前病毒基因组由宿主转录机构转录成单个9.2 kb初级转录物。转录受病毒蛋白达特与几种细胞因子相互作用的严格调控。为了表达病毒复制所需的九种不同的基因产物，病毒已经开发了许多策略来调节其转录物的剪接。剪接和未剪接RNA之间微妙平衡的改变可以显著影响HIV-1的感染性和发病机制。病毒基因组的转录和加工涉及病毒RNA序列、病毒蛋白和宿主细胞因子之间的相互作用，这些相互作用可能构成新的治疗靶点。我们已经确定了一些细胞因子和机制，调节特定的病毒mRNA的剪接。我们已经改变了表达HIV-1的细胞系中几种RNA加工因子的表达水平。细胞蛋白hnRNP A1的过表达导致病毒复制的急剧减少，这是由于编码病毒蛋白Env、达特和Rev的mRNA的加工显著减少。病毒转录通过表达的病毒反式激活因子达特的量的减少而严重减少。hnRNP A1上调诱导稳定细胞系中病毒复制减少超过200倍，而不影响细胞活力。我们建议评估hnRNP A1在外周血单核细胞（PBMC）培养物中表达的治疗潜力。我们将利用VSV-G假型逆转录病毒系统，以hnRNP A1表达盒融合到荧光蛋白eGFP的原代PBMC培养物。将用不同的病毒株感染PBMC，并分析hnRNP A1表达对病毒复制和细胞活力的影响。我们还将开发一种特异性靶向HIV-1感染细胞的递送系统，以消除由于hnRNP A1在未感染细胞中过度表达而产生的潜在继发性影响。最后，我们将确定病毒抑制所需的最小hnRNPA1序列，建立随机N-和C-末端截短文库。将活性片段的功效和毒性与用不同病毒株攻击的PBMC中的全长蛋白质进行比较。 公共卫生相关性：目前用于治疗HIV-1感染者的药物不能完全消除病毒，此外，新的多药耐药HIV毒株出现的频率越来越高。这些药物中的绝大多数靶向病毒蛋白。不幸的是，由于HIV-1的高突变率，新的多药耐药菌株越来越频繁地出现。最近在疫苗开发方面的挫折突出了开发具有新作用机制的药物的需要。由于病毒利用大量的宿主细胞蛋白质来有效复制，因此这些因子的鉴定可能提供新的治疗靶点。细胞靶点具有不突变的优势，因此不太可能出现耐药病毒株。我们已经确定了一种细胞因子（hnRNP A1），如果在人类细胞中高水平表达，它可以下调病毒复制超过200倍，而不会改变细胞的活力。我们建议创造一种方法，将这种基因或其一部分传递到白细胞中，白细胞是病毒的主要靶点，以抑制病毒复制并阻断感染的进展。

DESCRIPTION (provided by applicant): The integrated HIV-1 proviral genome is transcribed by the host transcription machinery into a single 9.2 kb primary transcript. Transcription is tightly regulated by the interaction of the viral protein Tat with several cellular factors. To express the nine different gene products required for viral replication the virus has developed a number of strategies to regulate splicing of its transcript. Alteration of the delicate balance between spliced and unspliced RNAs can dramatically affect HIV-1 infectivity and pathogenesis. Transcription and processing of the viral genome involves interactions between viral RNA sequences, viral proteins and host cell factors, which may constitute novel therapeutic targets. We have identified a number of cellular factors and mechanisms regulating the splicing of specific viral mRNAs. We have altered the expression levels of several RNA processing factors in a cell line expressing HIV-1. Over-expression of the cellular protein hnRNP A1 led to a dramatic decrease in viral replication due to a marked reduction in the processing of mRNAs coding for the viral proteins Env, Tat and Rev. Viral transcription was severely reduced by the decrease in the amount of the viral transactivator Tat expressed. hnRNP A1 up-regulation induced a decrease in viral replication of over 200 fold in a stable cell line without affecting cell viability. We propose to evaluate the therapeutic potential of hnRNP A1 expression in cultures of Peripheral Blood Mononuclear Cells (PBMCs). We will utilize a VSV-G pseudotyped retroviral system to transduce primary PBMC cultures with an hnRNP A1 expression cassette fused to the fluorescent protein eGFP. PBMCs will be infected with different viral strains and the effects of hnRNP A1 expression on viral replication and cell viability will be analyzed. We will also develop a delivery system that specifically targets cells infected by HIV-1 to eliminate potential secondary effects due to hnRNP A1 over-expression in uninfected cells. Finally we will identify the minimal hnRNP A1 sequence required for viral inhibition creating a library of random N- and C- terminus truncation. The efficacy and toxicity of the active fragment will be compared with the full-length proteins in PBMCs challenged with divergent viral strains. PUBLIC HEALTH RELEVANCE: Current drugs utilized in the treatment of HIV-1 infected individuals cannot completely eliminate the virus, furthermore new multidrug resistant HIV strains are appearing with growing frequency. The great majority of these drugs target viral proteins. Unfortunately, because of the HIV-1 high mutation rate new multidrug resistant strains are appearing with growing frequency. Recent setbacks in the development of vaccines have accentuated the need for the development of drugs with novel mechanisms of action. Since the virus utilizes a multitude of host cellular proteins to replicate efficiently the identification of such factors might provide for novel therapeutic targets. Cellular targets have the advantage of not mutating, thus making unlikely the emergence of resistant viral strains. We have identified a cellular factor (hnRNP A1), which, if expressed at high levels in human cells, down-regulates viral replication over 200 folds without altering the viability of the cells. We propose to create to deliver this gene, or portions of it, in leukocytes, the primary target of the virus, to inhibit viral replication and block the progression of the infection.