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Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5'-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.

Giardia lamblia is a primitive eukaryotic microorganism that derives its metabolic energy primarily from anaerobic glycolysis. In trophozoites, pyruvate-ferredoxin oxidoreductase (PFOR) converts pyruvate to acetyl-CoA with the transfer of a pair of electrons to ferredoxin, which can then reduce metronidazole and activate it into a potent antigiardiasis agent. It is unclear, however, whether this anaerobic disposal of electrons is essential for the energy metabolism in Giardia. In the present study, cDNAs encoding hammerhead ribozyme flanked with various lengths of antisense PFOR RNA were cloned into a viral vector pC631pac derived from the genome of giardiavirus (GLV). RNA transcripts of the plasmids showed high cleavage activities on PFOR mRNA in vitro. They were introduced into GLV-infected G. lamblia trophozoites by electroporation and stablized in the transfected cells via serial passages under puromycin selection. PFOR mRNA and enzyme activity in the transfected cells were decreased by 46-60% with the ribozyme PRzS flanked with 20 nt PFOR antisense RNA on each arm and by 69-80% with the ribozyme PRzL flanked with 600 and 1500 nt PFOR antisense RNA. PRzS without the inserted ribozyme or ribozyme flanked with alcohol dehydrogenase E antisense RNA showed no effect on PFOR mRNA and activity. The ribozyme-transfected cells demonstrated significantly enhanced resistance to metronidazole and grew equally well under anaerobic and aerobic conditions. In contrast, the wild-type cells grew slightly better anaerobically than the transfectants but did not grow at all in aerobic conditions. Thus, the reduced PFOR expression enables Giardia to grow under molecular oxygen and the presence of PFOR enhances the anaerobic growth of Giardia with an increased susceptibility towards metronidazole. In addition, this study demonstrated for the first time the feasibility of using a viral RNA vector to express a ribozyme targeted at a specific mRNA in G. lamblia to reduce the expression of a specific gene.

Giardia lamblia, a parasitic protozoan, has been regarded as one of the most conserved eukaryotes evolved from the prokaryotes. One of its unique features appears to be the unusually short 5'-untranslated regions (UTR) (1-6 nucleotides (nts)) and the apparent absence of 5'-cap structures from its mRNAs. Transfection of the Giardia trophozoites with luciferase-encoding chimeric transcripts, flanked by the 5'- and 3'-ends of giardiavirus (GLV) (+)-strand RNA, indicated that the translational efficiency was enhanced by 5000-fold when the 5'-viral sequence extended 264 nts into the capsid coding region and fused with the luciferase open reading frame (ORF). A 13-nt downstream box (DB) was identified within this region which complements a 15-nt sequence between nts # 1382 and 1396 near the 3'-end of the Giardia 16S-like ribosomal RNA (the anti-DB). Deletion or scrambling of this DB in the mRNA leads to a significant loss of the translational efficiency in Giardia. A Shine-Dalgarno (SD)-like element was also identified at 9-14 nts upstream from the initiation codon in the viral (+)-strand RNA, but alteration of its sequence led to no change in translation. Using the sequence complementary to ribosomal anti-DB to probe the Giardia mRNAs available in the databases, each mRNA was found to contain a putative DB with an average length from 8 to 13 nts. It is thus possible that initiation of translation in Giardia may involve a DB in the coding region of mRNA that may bind to a putative anti-DB in the small ribosomal RNA through base pairing. This mechanism of ribosome recruitment, which finds a potential parallel in Escherichia coli, could illustrate a relatively close distance between Giardia and prokaryotes in terms of translation initiation, and may provide a model for studying the evolution of translation machinery.

Manipulation of gene expression in Giardia lamblia, one of the most ancient eukaryotes, may provide insights into the evolutionary transition from prokaryotes to eukaryotes. Two recent successes in transient expression of the firefly luciferase (luc) gene in G. lamblia were mediated by a 5'-untranslated region (UTR) of the Giardia glutamate dehydrogenase (gdh) gene and a giardiavirus (GLV) genomic transcript, respectively. We now report a stable coexpression of luc gene with a neomycin phosphotransferase (neo(r)) gene in G. lamblia. An in vitro transcript of the construct pC670-Neo; containing the neo(r) encoding region flanked with the 5'670 nucleotides (nt) and the 3'2022 nt portion of GLV positive strand RNA, was electroporated into G. lamblia trophozoites that were infected with GLV. G418-resistant Giardia trophozoites were cloned, and the neo(r) mRNA in these clones was found to increase with increasing G418 pressure. This drug resistance remained stable upon continuous in vitro cultivation in the absence of G418 for over 15 days. Another plasmid pNeo/GDH/Luc, was constructed by inserting luc gene downstream from the neo(r) gene and the 193 nt 5' portion of gdh gene in pC670-Neo, and its bicistronic in vitro transcript was introduced into GLV-infected G. lamblia by electroporation. The transfectants demonstrated G418-resistance and persistent luciferase activity at levels parallel to the amount of G418 used for selection, peaking at a level of several thousand-fold above the background. Taken together, these data indicate that the neo(r) gene provides an effective selection marker for transformation of Giardia trophozoites, and the bicistronic RNA transfection vector may open the way for functional analysis of other genes in Giardia.

Giardia lamblia is an intestinal protozoan parasite and one of the earliest eukaryotic divergents. The trophozoite multiplies via asexual binary fission and lacks all natural means of lateral gene transfer. A system is developed here for long-term expression of a foreign gene in this organism by exploiting recombinant virions derived from the giardiavirus (GLV), a double-stranded RNA virus that infects many Giardia isolates. An in vitro transcript of the cloned GLV cDNA, comprising the firefly luciferase-encoding region flanked by 5' and 3' fragments of GLV positive-strand RNA, was electroporated into GLV-infected trophozoites. Luciferase activity in electroporated cells peaked on day 2 at levels 6 orders of magnitude above background. Expression of this foreign gene remained at 80% of its peak level after 30 days in the absence of selective pressure. The chimeric RNA was replicated as double-stranded RNA and packaged into virus-like particles. The recombinant virions were partially purified from the wild-type helper virus by CsCl equilibrium density-gradient centrifugation and used to superinfect Giardia trophozoites. At multiplicities of infection of 100 or higher, these chimeric virions were able to initiate new rounds of expression of luciferase activity in the superinfected cells. Thus, the engineered virion can be successfully used to introduce and efficiently express a heterologous gene in this eukaryotic microorganism.

Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.

The double-stranded RNA genome of giardiavirus (GLV) has only two large open reading frame (ORFs). The 100-kDa capsid polypeptide (p100) is encoded by ORF1, whereas the only other viral polypeptide, the 190-kDa GLV RNA-dependent RNA polymerase (p190), is synthesized as an ORF1-ORF2 fusion protein by a (-1) ribosomal frameshifting. Edman degradation revealed that p100 was N-terminally blocked except for 2 to 5% of it that showed free N terminus starting from amino acid residue 33 of ORF1. Studies using antiserum targeted against amino acid residues 6 to 27 indicated that this region (NT) is absent from viral p100 and p190, while pulse-labelling experiments showed that NT is present in nascent p100 synthesized in GLV-infected Giardia lamblia but removed subsequently. In contrast, this region was retained in the two viral proteins synthesized in vitro, and it was not removed upon prolonged incubation or inclusion of microsomal fraction in the in vitro translation reaction mixtures. These results suggest that endoplasmic reticulum is not involved in the protein processing and that the precursors of p100 and p190 are incapable of cleaving themselves or each other. This specific cleavage was reproduced when lysates from GLV-infected G. lamblia were added, but not those from uninfected cells. The cleavage activity was relatively insensitive to phenylmethylsulfonyl fluoride, but it was inhibitable by leupeptin or E-64, two known specific inhibitors of cysteine protease. The possible origin of this processing activity is discussed.

Giardia lamblia virus (GLV) is a small nonenveloped double-stranded RNA virus that infects specifically the parasitic protozoan G. lamblia. Among the many collected strains of G. lamblia, a few turn out to be highly resistant to the virus infection. Two of these strains, Ac and JH, were subjected to electroporation with the RNA from GLV-infected G. lamblia WB strain. Subsequent studies indicated the presence of GLV double-stranded RNA and GLV protein in the electroporated and propagated cells. Virus particles, released by the transfected cells into the culture medium, were capable of infecting the virus-sensitive G. lamblia WB strain. When the WB cells were incubated with GLV at 4 degrees C and treated with the bifunctional cross-linking reagent disuccinimidyl suberate, little GLV protein was detectable inside the cells by immunofluorescent staining. However, patches of fluorescent granules were found on the membrane surface of the cells, suggesting cross-linking of the viruses with a certain membrane component(s). Similar treatment of the resistant strains Ac and JH showed no fluorescence either inside or outside of the cells. Two other closely related parasitic protozoa, Tritrichomonas foetus and Trichomonas vaginalis, cannot be infected by GLV via either viral infection or RNA transfection. The [35S]cysteine-labeled protein profiles in Triton X-114 extracts of G. lamblia WB, Ac, and JH were compared. The profile of the WB strain differs clearly from that of Ac and JH. It remains to be seen, however, whether this difference is related at all to the different susceptibilities to GLV infection.

Giardiavirus is a small, nonenveloped virus comprising a monopartite double-stranded RNA genome, a major protein of 100 kDa, and a less abundant polypeptide of 190 kDa. It can be isolated from the culture supernatant of Giardia lamblia, a parasitic flagellate in human and other mammals, and efficiently infects other virus-free G. lamblia. A single-stranded copy of the viral RNA can be electroporated into uninfected G. lamblia cells to complete the viral replication cycle. Giardiavirus genomic cDNA of 6100 nt was constructed and its sequence revealed the presence of two large open reading frames that are separated by a -1 frameshift and share an overlap of 220 nt. The 3' open reading frame contains all consensus RNA-dependent RNA polymerase sequence motifs. A heptamer-pseudoknot structure similar to those found at ribosomal slippage sites in retroviruses and yeast killer virus was identified within this overlap. Immunostudies using antisera against synthesized peptides from four regions in the two open reading frames indicated that the 100- and 190-kDa viral proteins share a common domain in the amino-terminal region. But the 190-kDa protein makes a -1 switch of its reading frame beyond the presumed slippage heptamer and is therefore a -1 frameshift fusion protein similar to the gag-pol fusion protein found in retroviruses.

The subcellular distribution of Giardia lamblia virus RNA in infected G. lamblia trophozoites was examined by in situ hybridization using biotinylated DNA probe and riboprobe. In G. lamblia Portland I strain, which is chronically infected by G. lamblia viruses, the viral RNA was detected in the cytoplasm as well as in the twin nuclei. When riboprobe was used to examine the course of virus infection in WB strain, accumulation of viral RNA was detected only in the cytoplasm prior to the first 72 hr of infection. Using DNA probe, further accumulation of viral RNA in increasing number of cells occurred after the 72nd hr of infection, with the RNA found in both the cytoplasm and nuclei. Eventually, the cell nuclei showed damaged morphology that deteriorated rapidly toward the final stage of infection. These observations indicate that early phase of viral RNA replication may take place in the cytoplasm of infected G. lamblia, but the nuclei are also involved during the late phase of viral replication.