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制转录成单个9.2kb的初级转录本。转录受到病毒蛋白TAT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，病毒制定了许多策略来调节其转录本的剪接。剪接和非剪接RNA之间微妙平衡的改变可以极大地影响HIV-1的感染性和致病机制。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，这可能构成新的治疗靶点。我们已经确定了一些调节特定病毒mRNAs剪接的细胞因子和机制。我们已经改变了表达HIV-1的细胞系中几个RNA加工因子的表达水平。细胞蛋白hnRNP A1的过度表达导致病毒复制急剧下降，这是因为编码病毒蛋白Env、Tat和Rev.的mRNAs的加工显著减少。病毒转录因病毒反式激活因子TAT的表达减少而严重减少。HnRNP A1上调导致稳定细胞系中病毒复制减少200倍以上，而不影响细胞活力。我们建议评估在外周血单个核细胞(PBMC)培养中表达hnRNP A1的治疗潜力。我们将利用VSV-G伪型逆转录病毒系统转导原代PBMC培养物，并将hnRNP A1表达盒与荧光蛋白EGFP融合。PBMC将被不同的病毒株感染，并将分析hnRNP A1表达对病毒复制和细胞活力的影响。我们还将开发一种专门针对感染HIV-1的细胞的递送系统，以消除因hnRNP A1在未感染细胞中过度表达而可能产生的副作用。最后，我们将确定病毒抑制所需的最小hnRNP A1序列，创建一个随机N端和C端截断的文库。活性片段的有效性和毒性将与受不同病毒株挑战的PBMCs中的全长蛋白进行比较。 公共卫生相关性：目前用于治疗艾滋病毒-1感染者的药物不能完全消除病毒，而且新的具有多药耐药性的艾滋病毒毒株正在日益频繁地出现。这些药物中的绝大多数针对病毒蛋白。不幸的是，由于HIV-1的高变异率，新的多药耐药株正在以越来越高的频率出现。最近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必要性。由于病毒利用大量宿主细胞蛋白进行有效复制，识别这些因子可能会为新的治疗靶点提供帮助。细胞靶标的优点是不会发生突变，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，它可以在不改变细胞活力的情况下下调病毒复制200倍以上。我们建议创造在白细胞中传递这种基因或其部分，以抑制病毒复制和阻止感染的进展。白细胞是病毒的主要靶点。

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.