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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 lwnblia, a parasitic flagellate in human and other mammals, and effclently infects other virus-free G. lamblia. A single-stranded copy of the viral RNA can be electroporated into uninfected G. amblia cells to complete the viral replication cyde. 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-te l region.But the 190-kDa protein makes a -1 switch ofits reading frame beyond the presumed sippage heptamer and is therefore a -1

A linear 5.5-kilobase double-stranded RNA, identified in many strains and isolates of the parasitic protozoan Trichomonas vaginalis in a previous study, is found largely intact in ribonuclease-treated homogenates of the parasite. It can be pelleted with membranes from the homogenate at 12,500 x g and further purified in CsCl buoyant density-gradient centrifugations. The purified sample contains the doublestranded RNA as well as one major protein with an estimated molecular mass of 85 kDa in NaDodSO4/PAGE. Electron microscopic examinations indicated the presence of icosahedral virus-like particles of 33-nm diameter in the purified preparation. The exact location of the virus in T. vaginalis is not clear, except that it is not found in the nuclear fraction and is probably membrane-bound. No free virus can be recovered from the culture medium of T. vaginalis, and no successful infection of virus-free T. vaginalis strains by purified virus has yet been accomplished. There is no viral genomic sequence identifiable in host DNA. So far as we know, it is the first time a double-stranded RNA virus has been identified in a protozoan.Trichomonas vaginalis is a sexually transmitted protozoan parasite found primarily in the human vaginal tract. Recent studies indicated the presence of a double-stranded RNA (ds RNA) in the nucleic acid extract of this organism (1). The ds RNA has a linear structure with an estimated contour length of 1.5 gm (1). It consists of 23.4% guanine, 23.4% cytosine, 23.0% adenine, and 30.3% uracil, and has a transition temperature of 81.7°C with a hyperchromicity of 7-15% in 75 mM NaCl/7.5 mM sodium citrate, pH 7.0 (0.5x SSC; lx SSC = 0.15 M NaCl/0.015 M sodium citrate). It migrates in 0.8% agarose gel electrophoresis, with a mobility equivalent to a DNA size of 5.5 kilobases (kb), and it can be readily stained by ethidium. It is digestable by alkali and sensitive to RNases A and T1 in low-salt solutions. The sensitivity toward RNase T1 is, however, much reduced in high-salt solutions, suggesting a structure of ds RNA.Since this report (1) appears to be the first to identify the presence of a ds RNA in protozoa, the biological significance in this finding is not immediately clear. This ds RNA has since been identified in some 40 different strains or isolates of T.vaginalis at densities ranging from 280 to 1380 copies per cell. There have been, however, four strains of T. vaginalis found not to contain the ds RNA. Three of the four strains turned out to be resistant to the anti-trichomonial agent metronidazole (2). There seems, thus, a connection between the presence of this ds RNA and the sensitivity of T. vaginalis toward metronidazole, since all the 40 ds RNA-containing T. vaginalis samples are also sensitive to metronidazole. However, the one T. vaginalis strain 375, which has no detectable ds RNA but remains susceptible to metronidazole, argues against a possible role of the ds RNA in metronidazole sensitivity. Furthermore, a cattle protozoan parasite Tritrichomonasfoetus, which is closely...

SummaryGiardia lamblia is a primitive eukaryotic microorganism that derives its metabolic energy primarily from anaerobic glycolysis. In trophozoites, pyruvate-ferredoxin oxidoreductase (PFOR) converts pyruvate to acetylCoA 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.

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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 aminoacyltRNA synthetase per amino acid, these organisms employ prolyltRNA 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-(Lprolyl)-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.

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An isolate of Giardia lamblia infected with the double-stranded RNA virus (GLV) has two major species of RNA that are not present in an uninfected isolate. One of these species is the previously characterized double-stranded RNA genome of GLV (1). The second species of RNA appears to be a full length copy of one strand of the double-stranded RNA genome. This full length single-stranded RNA is not present in viral particles isolated from the growth medium. The cellular concentration of the single-stranded RNA changes during exponential and stationary phases of cell growth in a fashion consistent with a viral replicative intermediate or mRNA. The single-stranded species does not appear to be polyadenylated.