Giardiavirus: an update.

Giardiavirus is the only virus that infects Giardia duodenalis, a highly prevalent parasite worldwide, especially in low-income and developing countries. This virus belongs to the Totiviridae family, being a relative of other viruses that infect fungi and protozoa. It has a simple structure with only two proteins encoded in its genome and it appears that it can leave the cell without lysis. All these characteristics make it an interesting study model; however, its research has unfortunately made little progress in recent years. Thus, in this review, we summarize the currently available data on Giardiavirus, from their structure, genome and main proteins, to the uses that have been given to them and the possible health applications for the future.

Three-dimensional structure of a protozoal double-stranded RNA virus that infects the enteric pathogen Giardia lamblia.

UNLABELLED: Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented double-stranded RNA (dsRNA) virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses, including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 Å. Its outermost diameter is 485 Å, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and transmit through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans. IMPORTANCE: Numerous pathogenic bacteria, including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae, are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa, including Giardia lamblia, Leishmania species, and Trichomonas vaginalis are persistently infected with dsRNA viruses, and growing evidence indicates that at least some of these protozoal viruses can likewise enhance the pathogenicity of their hosts. Understanding of these protozoal viruses, however, lags far behind that of many bacteriophages. Here, we investigated the dsRNA virus that infects the widespread enteric parasite Giardia lamblia. Using electron cryomicroscopy and icosahedral image reconstruction, we determined the virion structure of Giardia lamblia virus, obtaining new information relating to its assembly, stability, functions in cell entry and transcription, and similarities and differences with other dsRNA viruses. The results of our study set the stage for further mechanistic work on the roles of these viruses in protozoal virulence.

Recent advances in molecular biology of parasitic viruses.

The numerous protozoa that can inhabit the human gastro-intestinal tract are known, yet little is understood of the viruses which infect these protozoa. The discovery, morphologic details, purification methods of virus-like particles, genome and proteome of the parasitic viruses, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and the Eimeria sp. are described in this review. The protozoan viruses share many common features: most of them are RNA or double-stranded RNA viruses, ranging between 5 and 8 kilobases, and are spherical or icosahedral in shape with an average diameter of 30-40 nm. These viruses may influence the function and pathogenicity of the protozoa which they infect, and may be important to investigate from a clinical perspective. The viruses may be used as specific genetic transfection vectors for the parasites and may represent a research tool. This review provides an overview on recent advances in the field of protozoan viruses.

A La autoantigen homologue is required for the internal ribosome entry site mediated translation of giardiavirus.

Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5' untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200-348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.

Giardia duodenalis: genetic recombination and its implications for taxonomy and molecular epidemiology.

Traditionally, species within the Giardia genus have been considered as eukaryotic organisms that show an absence of sexual reproduction in their simple life cycles. This apparent lack of sex has been challenged by a number of studies that have demonstrated (i) the presence in the Giardia duodenalis genome of true homologs of genes specifically involved in meiosis in other eukaryotes, and their stage-specific expression; (ii) the exchange of genetic material in different chromosomal regions among human isolates of the parasite; (iii) the fusion between cyst nuclei (karyogamy) and the transfer of genetic material (episomal plasmids) between them. These results are pivotal for the existence of sexual recombination. However, many details of the process remain elusive, and experimental data are still scarce. This review summarizes the experimental approaches and the results obtained, and discusses the implications of recombination from the standpoint of the taxonomy and molecular epidemiology of this widespread pathogen.

Structural elements in the 5'-untranslated region of giardiavirus transcript essential for internal ribosome entry site-mediated translation initiation.

Translation of uncapped giardiavirus (GLV) mRNA in Giardia lamblia requires the presence of a 5'-untranslated region (5'-UTR) and a viral capsid coding region. We used dicistronic viral constructs to show that the downstream 253 nucleotides (nt) of the 5'-UTR plus the initial 264-nt capsid coding region constitute an internal ribosome entry site (IRES). Predicted secondary structures in the 253-nt 5'-UTR include stem-loops U3, U4a, U4b, U4c, and U5. Chemical and enzymatic probing analysis confirmed the presence of all predicted stem-loops except U4a. Disruption of stem-loop structures U3 and U5 by site-directed mutagenesis resulted in a drastic reduction in translation of a monocistronic viral transcript, which could be restored by compensatory sequence changes. Mutations disrupting stem-loops U4b and U4c do not exert an appreciable effect on translation, but certain sequences in the U4a region and in U4b do appear to play important roles in the IRES. Structural analysis also suggests that an 8-nt U3 loop sequence (nt 147 to 154) pairs with an 8-nt downstream sequence (nt 168 to 175) to form a pseudoknot. Disruption of this pseudoknot by mutagenesis resulted in a drastic reduction in translation, which could be restored by compensatory sequence changes. This study has defined the secondary structure in the 5'-UTR of the IRES. Together with the previous results, we have now completed analysis of the entire structure of GLV IRES and fully defined the functionally essential structural elements in it.

Giardia lamblia: stable expression of green fluorescent protein mediated by giardiavirus.

Giardia lamblia, an early diverging eukaryote that infects several species including humans and a major agent of water-borne diarrhea throughout the world, can be infected with a double-stranded RNA virus, giardiavirus (GLV). A chimeric GLV cDNA and green fluorescent protein (GFP) according to the cis-acting signals of the GLV genome required for expression of foreign gene was constructed and its in vitro transcript was electroporated into GLV-infected G. lamblia trophozoites, GFP was expressed transiently. pGDH5/NEO/GLV was constructed by combining the neomycin resistance cassette in which the neomycin phosphotransferase gene was flanked by Giardia glutamate dehydrogenase (GDH) uncoding regions and the transcription cassette in which the chimera of GLV cDNA and GFP was located downstream from GDH gene promoter on a single plasmid. This plasmid was electroporated into G. lamblia and the transfectants persistently expressed GFP under G418 selection. This stable transfection system should provide a valuable tool for genetic study of G. lamblia.

Identification of an essential pseudoknot in the putative downstream internal ribosome entry site in giardiavirus transcript.

Enhanced translation of giardiavirus-luciferase chimeric mRNA in Giardia lamblia requires the initial 264-nt viral capsid coding region as a putative internal ribosomal entry site (IRES). Essential structural elements in this site include (1) a downstream box (DB) complementary to the anti-DB at the 3' end of 16S-like rRNA, (2) stem-loops I, II, III, and IVA, and (3) a pentanucleotide 5'-UCUCC-3' immediately downstream from stem loop IVA. A search for the structural role of the pentanucleotide suggested that it may form a pseudoknot with another pentanucleotide 5'-GGAGA-3' in loop II. Alteration of the two pentanucleotides by site-directed mutagenesis resulted in a drastic reduction in translation of the transcript. But the loss was recovered by compensatory changes in the two sequences, suggesting Watson-Crick base pairings between them. Results from in vitro enzymatic and chemical structural probing supported the presence of such a pseudoknot 143 nt downstream from the initiation codon. Minor repositioning of this codon led invariably to a complete loss of translation, suggesting that the initiation site is confined within a rigid position defined by all the structural elements in the IRES including the pseudoknot. This is the first pseudoknot of its kind shown to play an important role in a downstream IRES of a viral transcript. The finding is particularly interesting because it could reflect a unique feature of translation initiation in Giardia, which is known to have exceedingly short (1-6 nt) 5' untranslated regions in its mRNAs.

Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia.

Enhanced translation of giardiavirus (GLV)-luciferase chimeric mRNA in Giardia lamblia requires the presence of the initial 264 nucleotides of the viral capsid-coding region. A 13 nt downstream box (DB) sequence within this region, complementary to a 15 nt sequence near the 3' end of G. lamblia 16 S-like ribosomal RNA (rRNA), was found to be essential for the enhanced translation. However, DB is located 64-78 nt downstream of the initiation codon, whereas an exponential increase of translation efficiency depends on a further increment of the coding region from nucleotides 111 to 264. Thus, there could be additional structural requirements for translation enhancement in the region downstream from DB. Four major stem-loop structures, designated I to IV, were identified in the MFOLD-predicted secondary structure of the 264 nt capsid-coding region with an estimated minimum free energy (DeltaG degrees ) of -77.16 kcal x mol(-1). Our chemical probing analysis of the free 264 nt RNA molecule in solution supports the predicted presence of stem-loops I, II and III, but casts doubts on stem-loop IV. It suggests, instead, the presence of a stem-loop IVA at a nearby location in the molecule. Site-directed mutagenesis designed to disrupt stem-loop structures I, II, III or IVA resulted in drastic reduction of translation efficiency, which was restored by compensatory sequence changes to regenerate individual stem-loop structures. Mutations disrupting the originally designated stem-loop IV did not exert any detectable effect on translation. However, alterations of the sequence UCUCC between nucleotides 216 and 220 in the flexible loop region of the revised secondary structure led to a precipitous drop in translation. Another stem-loop predicted by MFOLD that consists of a major portion of the DB sequence was examined by chemical probing but found little experimental support. Changes of the DB sequence without affecting the postulated stem structure led to drastic losses of translation efficiency. Thus, a simple structural basis for the enhanced translation could be that stem-loops I, II, III and IVA and the UCUCC sequence may facilitate the interaction between DB and the anti-DB in 16 S-like rRNA in initiating translation of GLV mRNA in G. lamblia.

Role of alcohol dehydrogenase E (ADHE) in the energy metabolism of Giardia lamblia.

Two hammerhead ribozymes flanked by Giardia lamblia alcohol dehydrogenase E (ADHE) antisense RNA fragments, ARzS and ARzL, were designed, synthesized and found capable of cleaving an ADHE mRNA fragment at the anticipated position in vitro. The ribozymes were then electroporated into Giardia trophozoites and expressed via the giardiavirus-mediated RNA expression system. Expression of the ribozyme with two short antisense arms, ARzS, was stabilized under puromycin selection and demonstrated a 33% reduction in ADHE mRNA and 25% decrease in NAD+-dependent ADH activity in the transfectants. Expression of ARzL, the ribozyme with two long antisense arms, cannot be enriched under puromycin without killing the transfected cells, probably due to excessive depletion of ADHE. Without the drug selection, however, transient expression of ARzL 20-40 h after electroporation resulted in an 83.7% loss of ADHE mRNA and an 84.5% reduction in ADH activity in the transfected cells. When the ribozyme moiety was removed from ARzL, the latter retained some of its in vivo activity of lowering ADHE mRNA and ADH activity, suggesting that inhibition of ADHE gene expression in Giardia can be accomplished by the antisense RNA alone, albeit less efficiently. The ADHE deficient transfectant demonstrated relatively poorer anaerobic growth but grew more vigorously than the wild type under aerobic conditions, suggesting that the role of ADHE in providing NAD+ through anaerobic reduction of acetyl-CoA to ethanol could be replaced by a yet unidentified aerobic enzyme(s) in Giardia. The close association consistently observed between the levels of ADHE mRNA and ADH activity in transfected Giardia cells suggests that ADHE could be the only functional alcohol dehydrogenase in Giaradia. One other Giardia gene encoding a putative Class III ADH, GIADH3, was identified and cloned, but no Class III ADH activity could be detected in Giardia by the conventional enzyme assays. This gene is thus probably unexpressed in Giardia trophozoite.

Identification of virus-specific vesicles in Giardiavirus-infected Giardia lamblia.

Giardiavirus (GLV), which infects the parasitic protozoan Giardia lamblia, is a nonsegmented double-stranded (ds) ribonucleic acid (RNA) virus. We previously purified two distinct types of related GLV from infected G. lamblia, and showed differential export of one of the viruses from infected cells. In the present study, fractionation of cell lysate was performed, revealing the presence of viruses in the membranous fraction. Distribution of viral antigens in the infected cells was examined by immunocytochemistry. The signal was enriched in certain regions of the cytoplasm, suggesting that a portion of GLV is confined to certain cellular compartments. A significantly reduced signal was also detected in the nuclei. We directly observed the viruses in the infected cells by electron microscopy. Consistent with previous observations, virus-like particles were clearly observed in some membranous vesicles in the cytoplasm at 48 h postinfection, and virus-like particles were again seen in the cytoplasm and then in the nuclei toward the late phase of virus infection. The virus-associated vesicles and some electron-dense nuclear structures were only observed in virus-infected cells, suggesting that virus infection may induce ultrastructural alteration of G. lamblia.

Inhibition of pyruvate-ferredoxin oxidoreductase gene expression in Giardia lamblia by a virus-mediated hammerhead ribozyme.

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.

Site-specific binding of polymerase-containing particles of the Giardia lamblia double-stranded RNA virus to the viral plus-strand RNA.

The non-segmented, double-stranded RNA genome of the Giardia lamblia virus (GLV) contains two genes encoding the major capsid protein (gag) and a fusion of gag with the viral RNA-dependent RNA polymerase (pol). Computer analysis of the viral RNA genome revealed three putative stem-loop structures that were predicted to mediate replication, transcription and packaging of the GLV genomic RNA by binding to the pol domain of the virus-encoded fusion protein. To provide evidence of these postulated RNA/protein interactions, gel retardation assays were employed to examine the potential binding capacity of various viral RNA genome-related sequences to native GLV protein(s). Viral proteins were obtained by disrupting purified GLV particles under low-ionic-strength conditions. The resulting viral protein particles maintained their RNA polymerase activity in the presence of GLV genomic RNA and thus appeared to be suitable tools for the analyses of GLV-protein-mediated binding reactions. A 72-nt short single-stranded in vitro transcript containing a putative stem-loop structure predicted to participate in the packaging of GLV (+)-strand RNA bound specifically to the disrupted virus particles. RNAs containing modified motifs of this stem-loop structure failed to bind to the GLV capsid.

Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia.

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.

Identification of cis-acting signals in the giardiavirus (GLV) genome required for expression of firefly luciferase in Giardia lamblia.

Giardiavirus (GLV) is a 6,277-bp double-stranded RNA virus of Giardia lamblia, one of the earliest eukaryotic divergents from the prokaryotes. Our previous success in GLV-mediated transfection of G. lamblia has provided an effective way of monitoring the mechanisms underlining GLV gene replication and mRNA translation in this organism. Here we have investigated the cis-acting signals in the GLV genome that regulate replication, transcription, and translation of an inserted firefly luciferase gene in GLV-infected G. lamblia. By modifying the two terminal regions of a full-length GLV cDNA clone used to flank a luciferase gene, various in vitro chimeric transcripts were generated and introduced into GLV-infected G. lamblia via electroporation. Expression of luciferase (+) strand and (-) strand RNAs in the transfected cells was monitored and the luciferase activity assayed. The results indicated that the 5'-untranslated region (UTR) of 366 nt and the 3'-terminal 2,022 nt of the viral transcript are both needed for optimal expression of the two RNA strands. Although the entire 5'-UTR is needed for the chimeric mRNA synthesis, both the primary sequence and the secondary structure at the 3' end of GLV transcript are essential for the synthesis of (-) strand RNA. When the 5' end of GLV transcript was extended 265 nt into the capsid protein open reading frame and fused with that of luciferase, there was no change in the level of luciferase chimeric RNA, but a 5,000-fold increase of luciferase activity was observed that may be attributed to an enhanced translational efficiency of the chimeric mRNA in G. lamblia.

Separation and characterization of two related giardiaviruses in the parasitic protozoan Giardia lamblia.

Giardiavirus encapsidates a 6.2-kb double-stranded (ds) RNA within a capsid that consists of a major 100-kDa capsid protein (p100) and a minor 190-kDa protein (p190). In this study, two nonhomologous 6.2-kb ds RNAs cohabiting in Giardia lamblia trophozoites were found to be separately encapsidated into two distinct virions, one (designated GLV[p100]) whose capsid consists of p100 and p190, and the other (designated GLV[p95]) whose capsid consists of a 95-kDa protein (p95) and a minor p190-equivalent protein. Both types of virions were enriched in the membranous fraction of a lysate from virus-infected G. lamblia cells. Separation of these virions was achieved by CsCl gradient centrifugation following osmotic rupture of the viral particles. By these treatments, the 6.2-kb ds RNA was removed from GLV[p100] whereas that in GLV[p95] remained unchanged, and the two 6.2-kb ds RNAs that had been purified by this protocol displayed differential hybridization properties to viral cDNA probes. Western blotting and peptide mapping experiments show that p100 and p95 were closely related proteins, but each had distinct amino acid sequences. Virus purification and pulse-chase experiments show that GLV[p100] was selectively secreted into the medium whereas GLV[p95] remained within the trophozoites of G. lamblia toward the late phase of cell growth. Secretion of GLV[p100] was not inhibited by Brefeldin A. These findings demonstrate the cohabitation of multiple Giardiavirus species in G. lamblia.

Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia.

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.

Maturation of giardiavirus capsid protein involves posttranslational proteolytic processing by a cysteine protease.

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.

Small extrachromosomal nucleic acid segments in protozoan parasites.

Viruses have been described in the following protozoa: Babesia spp., Trichomonas vaginalis, Giardia lamblia, Leishmania braziliensis and Eimeria spp. In order to study the Babesia bovis virus, merozoites have been prepared from the blood of infected cattle. Agarose gel electrophoresis of nucleic extracts from the bovine protozoa B. bovis and Babesia bigemina were separated into genomic DNA and at least two additional nucleic acids. One molecule with a relative mobility of 5.5 kilobase pairs (kbp) was identified as a double-stranded RNA virus-like particle. Another 6.2 kbp DNA molecule had sequences related to mitochondrial genome.