DOUBLE STRANDED RNA VIRUS IN GIARDIA

贾第虫病毒（GLV）是第一个被发现的原生动物病毒 在全病毒科中。 36 nm二十面体病毒体含有 6，277个碱基对的dsRNA基因组和蛋白质衣壳。 其有义链 RNA显示两个开放阅读框（ORF）。 100个ORF 1代码 千道尔顿（kDa）衣壳蛋白，而ORF 2与ORF 1重叠 220个核苷酸（nt），并通过a-1移码分离，具有所有的 病毒RNA依赖性RNA聚合酶（RDRP）的共有基序。 一 -1核糖体移码所需的七聚体/假结结构， 在220 nt重叠中发现，这导致190 nt的合成， 在5%的GLV蛋白中存在kDa gag-pol样融合蛋白。 特别是蓝氏贾第鞭毛虫的滋养体， 人类贾第虫病和最早的真核生物之一。 它会倍增 在感染贾第虫滋养体的细胞核内， 感染细胞或改变其生长速度或致病性。 两 GLV区别于其他全病毒的特征：1）纯化的GLV能够 2）GLV mRNA能感染贾第鞭毛虫滋养体。 这些特点导致了近年来稳定转染的成功 GLV感染的滋养体与萤火虫荧光素酶的转录本 基因 在病毒中发现外源基因以dsRNA形式被包裹， 与GLV一起感染滋养体的颗粒。 GLV受体初步确定的表面上的第一组 贾第虫滋养体。 病毒蛋白的蛋白水解修饰 在GLV成熟过程中，观察到并发现由宿主半胱氨酸催化 由病毒感染诱导的蛋白酶。在下一个授予期， 我们计划：（1）将耐药基因封装到病毒中， 在药物作用下在所有感染的滋养体中表达外源基因的颗粒 （2）将腺苷脱氨酶基因包封到 病毒颗粒，预期酶的表达 活性将破坏G. Lamblia，并测试 该重组病毒作为潜在的抗贾第虫剂;（3） 鉴定GLV mRNA和（-）RNA中用于包装的结合位点， 复制和转录，以及RDRP中用于结合 GLV RNA的;（4）完成滑移的表征 七聚体和假结的理解背后的机制， 翻译移码;（5）鉴定、纯化和表征 贾第虫半胱氨酸蛋白酶参与GLV成熟及其机制 病毒感染诱导GLV的背后;（6）分离GLV受体 通过四种不同的方法;转染受体缺陷型贾第虫 用野生型DNA文库，然后用耐药病毒感染; 通过光敏化非极性叠氮化物的受体标记， 荧光素标记的GLV;表面等离子体共振和酵母双- 混合系统 这些研究的结果应该提供有效的 探讨G. Lamblia，并为控制贾第鞭毛虫病提供新的线索。

Giardiavirus (GLV) was the very first protozoan virus to be recognized in the Totiviridae family. The 36 nm icosahedral virion contains the 6,277 base pair dsRNA genome and the protein capsid. Its sense strand RNA reveals two open reading frames (ORFs). ORF1 codes for the 100 kilodalton (kDa) capsid protein, whereas ORF2, overlapping with ORF1 by 220 nucleotides (nts) and separated by a-1 frameshift, has all the consensus motifs of viral RNA-dependent RNA polymerases (RDRPs). A heptamer/pseudoknot structure required for -1 ribosomal frameshift is found within the 220 nts overlap, which causes the synthesis of the 190 kDa gag-pol like fusion protein in 5% of GLV proteins.GLV infects specifically the trophozoites of Giardia lamblia, the causative agent of human giardiasis and one of the earliest eukaryotes. It multiplies inside the nuclei of infected Giardia trophozoite without either lysing the infected cells or altering their growth rate or pathogenicity. Two features distinguish GLV from other totiviruses: 1) purified GLV is able to infect, and 2) GLV mRNA is able to transfect, Giardia trophozoites. These feasibilities have led to the recent success in stable transfection of GLV-infected trophozoites with transcripts of the firefly luciferase gene. The foreign gene was found to be encapsulated as dsRNA in viral particles that were infectious to the trophozoites together with GLV. GLV-receptors were tentatively identified on the surfaces of Group I Giardia trophozoites. Proteolytic modification of the viral protein during GLV maturation was observed and found catalyzed by a host cysteine protease(s) induced by the viral infection. For the next granting period, we plan: (1) to encapsulate drug-resistance genes into the viral particles to express foreign gene in all infected trophozoites under drug pressures; (2) to encapsulate the adenosine deaminase gene into the viral particles, with the anticipation that expression of the enzyme activity will disrupt the purine metabolism in G. lamblia, and to test this recombinant virus as potential antigiardiasis agent; (3) to identify the binding site(s) in GLV mRNA and (-) RNA for packaging, replication, and transcription, as well as domains in RDRP for binding to the GLV RNA's; (4) to complete characterizations of the slippage heptamer and pseudoknot for understanding mechanisms behind the translational frame shift; (5) to identify, purify and characterize the Giardia cysteine protease involved in GLV maturation and the mechanisms behind its induction by viral infection; (6) to isolate the GLV receptor by four different approaches; transfection of receptor-deficient Giardia with a wild-type DNA library followed by drug-resistant viral infection; receptor labeling via photosensitization of an apolar azide by fluorescein-labeled GLV; surface plasmon resonance and the yeast two- hybrid system. The outcome from these studies should provide effective means of looking into the mechanisms regulating gene expressions in G. lamblia, as well as offering new leads in controlling giardiasis.