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For the next granting period, we plan: <ol>
<li> to encapsulate drug resistance genes into the viral particles to express foreign gene in all infected trophozoites under drug pressures; </li>
<li> 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; </li>
<li> 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;
<li> to complete characterizations of the slippage heptamer and pseudoknot for understanding mechanisms behind the translational frame shift; </li>
<li> to identify, purify and characterize the Giardia cysteine protease involved in GLV maturation and the mechanisms behind its induction by viral infection; </li>
<li> 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. </li></ol></p>
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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.</p>
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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. </p>
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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: <ol>
<li> purified GLV is able to infect, and </li>
<li> GLV mRNA is able to transfect, Giardia trophozoites. </li></ol></p>
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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. </p>