Feline Immunodeficiency Virus Orf-A a Model for HIV Vpr

描述(由申请人提供)：猫免疫缺陷病毒(FIV)动物模型为评估针对人类艾滋病毒/艾滋病的新型抗病毒策略提供了一个独特而重要的机会。然而，对与HIV编码蛋白具有保守功能的FIV辅助基因的鉴定将是开发这种动物模型的关键，该动物模型将表征用于治疗人类免疫缺陷病毒-1(HIV-1)感染的靶向干预策略。 FIV基因OH-A，又名orf-2，编码77个氨基酸的辅助蛋白。我们最近的研究表明，Orf-A在FIV生命周期中参与病毒粒子形成的后期步骤和参与病毒感染性的早期步骤中都是重要的，并绘制了这些复制步骤所需的关键Orf-A结构域。在另一项单独的研究中，我们报道了以前未被认识到的新的Orf-A功能。GFP-Orf-A融合蛋白在哺乳动物细胞中的表达证实了这种FIV辅助蛋白的核定位，并促进了对FIV Orf-A核输入至关重要的核定位信号(NLS)的定位。最后，对瞬时表达GFP-Orf-A的细胞的细胞周期曲线的评估表明，Orf-A导致细胞周期的第二个间隙(G2)停滞。基于这些新的发现，我们假设FIV oh-A编码一种病毒蛋白，其表达的特定特性类似于HIV-1 VPR所表达的特性。此外，我们推测OH.A蛋白-蛋白的特异性相互作用可能是由非灵长类慢病毒(FIV和山羊关节炎脑炎病毒)或灵长类慢病毒(HIV和猿猴免疫缺陷病毒)编码的辅助基因的共同特性。我们建议通过研究Orf-A在病毒复制中的功能，对复制至关重要的Orf-A结构域，以及Off-A影响病毒复制的机制来进一步表征FIV Orf-A。重要的是，我们将比较轮状病毒Orf-A和HIV VPR的表达和亚细胞定位对宿主细胞功能的影响，包括细胞周期和细胞活力。最后，我们将通过实验性的猫接种研究来检测编码Orf-A突变的FIV分子克隆，以检验Orf-A功能在病毒发病中的作用。FIV动物模型将用于分析特定病毒基因活动(G2期停滞、细胞凋亡等)的影响。FIV Off-A和HIV-1 VPR在体内病毒致病方面的共同研究，并相应地检测特定VPR结构域作为基于HIV-1 VPR的抗病毒治疗的靶点的价值。

DESCRIPTION (provided by applicant): The feline immunodeficiency virus (FIV) animal model presents a unique and significant opportunity for assessing novel antiviral strategies for HIV/AIDS in humans. However, characterization of FIV accessory genes that share conserved functions with HIV-encoded proteins will be crucial to development of this animal model for characterizing targeted intervention strategies useful for treatment of human immunodeficiency virus-1 (HIV-1) infection of humans. FIV gene OH-A, also designated orf-2, encodes a 77 amino acid accessory protein. Our recent studies have shown Orf-A to be important in late steps of the FIV life cycle involved in virion formation and in early steps involved in virus infectivity and have mapped critical Orf-A domains needed for these steps in replication. In a separate study we reported novel previously unrecognized Orf-A functions. Expression of GFP-Orf-A fusion proteins in mammalian cells demonstrated nuclear localization for this FIV accessory protein and facilitated mapping of a nuclear localization signal (NLS) critical for nuclear import of FIV Orf-A. Lastly, assessment of cell cycle profiles of cells transiently expressing GFP-Orf-A demonstrated that Orf-A causes an arrest at the second gap (G2) of the cell cycle. Based on these novel findings, we hypothesize that FIV oH-A encodes a viral protein that expresses specific properties similar to those expressed by HIV-1 Vpr. Furthermore, we hypothesize that specific oH.A protein-protein interactions may be common properties of accessory genes encoded by either non-primate lentiviruses (FIV and caprine arthritis encephalitis virus) or primate lentiviruses (HIV and simian immunodeficiency virus). We propose to further characterize FIV Orf-A by investigating Orf-A functions in virus replication, Orf-A domains critical for replication, and mechanisms by which Off-A affects virus replication. Importantly, we will compare effects of RV Orf-A and HIV Vpr expression and subcellular localization on host cell functions including cell cycle and cell viability. Lastly, we will examine effects of Orf-A function on viral pathogenesis through experimental cat inoculation studies testing FIV molecular clones encoding Orf-A mutations. The FIV animal model will be used to analyze the impact of specific viral gene activities (G2 arrest, apoptosis, etc.) shared by FIV Off-A and HIV-1 Vpr on viral pathogenesis in vivo and to accordingly examine the value of specific Vpr domains as targets for antiviral therapeutics based on HIV-1 Vpr.