DESCRIPTION: (provided by the applicant): Giardia lamblia has been proposed as a primitive eukaryotic microorganism. Double-stranded (ds) RNA viruses, using RNA-dependent RNA polymerases for replication, have been regarded as the most ancient form of viruses. Giardiavirus (GLV) is such a virus that infects Giardia. The virus was recently developed into a transfecting vector capable of either over expressing a foreign gene or knocking out the expression of a specific gene in Giardia. These technical advantages enabled us to identify a downstream 264-nucleotide sequence in the viral transcript that acted as the internal ribosome entry site (IRES) for translation initiation. The finding is particularly meaningful in Giardia, because its mRNAs have 5'-untranslated regions of only 0 to 14 nucleotides, which may have to depend on an IRES element inside the open reading frame for translation initiation. A knockout of the expression of pyruvate: ferredoxin oxidoreductase (PFOR) from Giardia by the viral vector enabled the organism to grow aerobically with enhanced metronidazole resistance. Furthermore, recombinant GLV RDRP has been successfully expressed. A putative GLV receptor was identified on the membrane surface of GLV-susceptible Giardia. The receptor was isolated and cloned recently. A cysteine protease CP2 in Giardia was found responsible for the maturation of GLV. These experimental outcomes prompted us to enlist four specific aims in our future research plan. We intend to perform a thorough structure-function analysis of the viral IRES and use the information for a search of similar IRESs among the cellular mRNAs in Giardia. A double knockout of PFOR and alcohol dehydrogenase E (ADHE) coupled with an over expressed Fe-superoxide dismutase will be attempted to convert Giardia into an obligated aerobe. Conversely, an overexpression of ADHE or lactate dehydrogenase may turn Giardia into a strict anaerobe. Analyses of the interactions between GLV RDRP and the viral replication and viral transcription initiation sites and the viral packaging site will be pursued. Two distinct forms of the same enzyme protein performing the functions of replicase and transcriptase, respectively, will be determined. The GLV-receptor will be further characterized, and the encoding gene will be expressed in Giardia originally missing the receptor for verification. CP2 will be expressed arid characterized and its Specific modification of GLV capsid protein for viral maturation will be further verified. Valuable knowledge will be gained on the primitive organism and the ancient virus from these future studies.