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首页> 外文期刊>Molecular urology >Virion-targeted viral inactivation: new therapy against viral infection.
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Virion-targeted viral inactivation: new therapy against viral infection.

机译：针对病毒颗粒的病毒灭活：针对病毒感染的新疗法。

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BACKGROUND: Acquired immune deficiency syndrome (AIDS) is resistant to all current therapy. Gene therapy is an attractive alternative or additive to current, unsatisfactory AIDS therapy. MATERIALS AND METHODS: To develop an antiviral molecule targeting viral integrase (HIV IN), we generated a single-chain antibody, termed scAb, which interacted with human immunodeficiency virus type 1 (HIV-1) IN and inhibited virus replication at the integration step when expressed intracellularly. To reduce infectivity from within the virus particles, we made expression plasmids (pC-scAbE-Vpr, pC-scAbE-CA, and pC-scAbE-WXXF), which expressed the anti-HIV IN scAb fused to the N-terminus of HIV-1-associated accessory protein R (Vpr), capsid protein (CA), and specific binding motif to Vpr (WXXF), respectively. All fusion proteins were tagged with a nine-amino acid peptide derived from influenza virus hemagglutinin (HA) at the C terminus. RESULTS: The fusion molecules, termed scAbE-Vpr, scAbE-CA, and scAbE-WXXF, interacted specifically with HIV IN immobilized on a nitrocellulose membrane. Immunoblot analysis showed that scAbE-Vpr, scAbE-CA, and scAbE-WXXF were incorporated into the virions produced by cotransfection of 293T cells with HIV-1 infectious clone DNA (pLAI) and pC-scAbE-Vpr, pC-scAbE-WXXF. A multinuclear activation galactosidase indicator (MAGI) assay revealed that the virions released from 293T cells cotransfected with pLAI and pC-scAbE-Vpr, pC-scAbE-WXXF had as little 1000-fold of the infectivity of the control wild-type virions, which were produced from the 293T cells transfected with pLAI alone. Furthermore, the virions produced from the 293T cells cotransfected with pLAI and an scAb expression vector (pC-scAb) showed only 1% of the infectivity of the control HIV-1 in a MAGI assay, although scAb was not incorporated into the virions. In either instance, the total quantity of the progeny virions released from the transfected 293T cells and the patterns of the virion proteins were hardly affected by the presence of scAb, scAbE-Vpr, or scAbE-WXXF, as determined by virion-associated reverse transcriptase assay and by immunoblot analysis, respectively. Because G418-selected HeLa clones carrying the expression plasmid for scAbE-WXXF were obtained much more frequently than those for scAbE-Vpr, scAbE-WXXF was inferred to be less toxic to cells than scAbE-Vpr. The result that scAbE-WXXF with viral incorporation achieved more than a 10-fold reduction in infectivity of the progeny virions than scAb without incorporation suggests that scAbE-WXXF is a potential antiviral molecule, inhibiting replication by neutralization of HIV IN activity both within cells and within virions. Moreover, it is nontoxic to human cells. We termed this gene therapy virion- antiviral therapeutics." CONCLUSION: This new gene therapy has the potential for wide application in many viral infectious diseases.
机译：背景：获得性免疫缺陷综合症（AIDS）对目前所有的治疗方法均具有抵抗力。基因疗法是目前不令人满意的艾滋病疗法的一种有吸引力的替代方法或添加剂。材料和方法：为了开发靶向病毒整合酶（HIV IN）的抗病毒分子，我们产生了一种单链抗体，称为scAb，它与人免疫缺陷病毒1型（HIV-1）IN相互作用并在整合步骤中抑制了病毒复制当在细胞内表达时。为了减少病毒颗粒内部的感染力，我们制备了表达质粒（pC-scAbE-Vpr，pC-scAbE-CA和pC-scAbE-WXXF），该质粒表达与HIV N端融合的抗HIV IN scAb -1相关的辅助蛋白R（Vpr），衣壳蛋白（CA）和与Vpr的特异性结合基序（WXXF）。所有融合蛋白均在C端标记有源自流感病毒血凝素（HA）的九氨基酸肽。结果：融合分子，分别称为scAbE-Vpr，scAbE-CA和scAbE-WXXF，与固定在硝酸纤维素膜上的HIV IN特异性相互作用。免疫印迹分析显示，将scAbE-Vpr，scAbE-CA和scAbE-WXXF掺入了293T细胞与HI​​V-1感染性克隆DNA（pLAI）和pC-scAbE-Vpr，pC-scAbE-WXXF共转染的病毒粒子中。多核激活半乳糖苷酶指示剂（MAGI）分析显示，从以pLAI和pC-scAbE-Vpr，pC-scAbE-WXXF共转染的293T细胞中释放的病毒粒子，其感染性仅为对照野生型病毒粒子的1000倍。从单独用pLAI转染的293T细胞中产生了p53。此外，尽管未将scAb掺入病毒颗粒，但在MAGI分析中，用pLAI和scAb表达载体（pC-scAb）共转染的293T细胞产生的病毒颗粒仅显示了对照HIV-1的1％感染力。在任一种情况下，通过病毒体相关逆转录酶测定，scAb，scAbE-Vpr或scAbE-WXXF的存在几乎不会影响转染的293T细胞释放的子代病毒体的总量和病毒体蛋白的模式。检测和免疫印迹分析。因为带有scAbE-WXXF的表达质粒的G418选择的HeLa克隆比scAbE-Vpr的HeLa克隆获得的频率要高得多，所以推断scAbE-WXXF对细胞的毒性比scAbE-Vpr低。与未掺入scAb相比，掺入病毒的scAbE-WXXF的后代病毒体感染率降低了10倍以上，这表明scAbE-WXXF是一种潜在的抗病毒分子，通过中和细胞内和细胞内的HIV IN活性抑制复制。在病毒粒子内。而且，它对人体细胞无毒。我们将这种基因疗法称为病毒体-抗病毒疗法。”结论：这种新的基因疗法具有在许多病毒感染性疾病中广泛应用的潜力。

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