Virus-Mediated Expression of Firefly Luciferase in the Parasitic Protozoan Giardia lamblia

Abstract

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.

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

“…The in vitro transcripts of various GLV-luciferase chimeric cDNA mutants were each introduced into GLV-infected WB strain G. lamblia trophozoites (WBI) by electroporation as described (Yu et al+, 1995;Garlapati et al+, 2001)+ Approximately 4 ϫ 10 6 trophozoites were transfected with 100 mg of the in vitro-synthesized transcript+ Mutant and wild-type transcripts were each used in triplicate for transfection study in every duplicated experiment+…”

“…The transfected G. lamblia trophozoites were lysed and assayed for luciferase activity 20 h posteletroporation as described (Yu et al+, 1995)+ Each mutant transfectant in triplicate from two independent transfection experiments was examined, with the wild-type pC631-luc transfectant as a positive control+ Luciferase activity was calculated in relative light units (RLU) per microgram of crude lysate protein determined by the Bradford method (Bradford, 1976)+ Enzymatic probing of RNA structure Enzymatic probing was carried out essentially as described (Moazed et al+, 1986;Stern et al+, 1988)+ cDNA encoding the 264-nt RNA fragment was amplified by polymerase chain reactions (PCR) using pC631-luc as template, and the amplified product was cloned into a pGEM-T-easy vector (Promega)+ The recombinant plasmid was linearized by HindIII and transcribed in vitro to produce the 264-nt transcript using the T7 Megascript transcription kit (Ambion)+ The RNA was initially denatured at 65 8C for 15 min in a probing buffer (160 mM HEPES, pH 7+5, 50 mM KCl, 10 mM MgCl 2 ) followed by a slow cooling to ambient temperature for an hour+ Approximately 10 mg of the renatured RNA was used for each enzymatic digestion+ RNaseV (Pharmacia), RNaseT1 (Life Technologies, Inc+), RNaseT2 (Life Technologies), and RNaseA (Ambion) were each diluted in the probing buffer and the enzymatic activities were titrated (in units or nanograms) to identify the optimal probing condition for each enzyme+ The following optimal amount of each enzyme was determined and used in subsequent experiments: 0+35 U of RNaseV, 0+1 U of RNaseT1, 2+5 U of RNaseT2, and 12 ng of RNaseA+ All the enzymatic digestions were performed in a final volume of 100 mL at 37 8C for a period of 10 to 20 min+ The reactions were stopped by adding phenol-chloroform to the digestion mixture, and the RNA substrates were extracted and recovered by ethanol precipitation in the presence of 0+3 M NaOAc and 10 mg yeast tRNA+ RNA pellets were dissolved in DEPC-treated water and subjected to primer extension analysis to determine the enzyme-cleaved sites in the RNA molecule+ Chemical probing of RNA structure Chemical probing was carried out essentially as previously described (Moazed et al+, 1986;Stern et al+, 1988;Garlapati et al+, 2001) except that the RNA substrate was renatured to assume its native structure prior to chemical probing as in the enzymatic probing+ Approximately 10 mg of the 264-nt transcript were used for each type of modification+ For DMS and KE probing, 10 mg of the RNA sample were suspended in 300 mL and 270 mL of HMK buffer (160 mM HEPES, pH 7+2, 50 mM KCl, 10 mM MgCl 2 ), respectively+ For CMCT probing, the same amount of RNA sample was suspended in 150 mL of BMK buffer (70 mM potassium-borate, pH 8+0, 50 mM KCl, 10 mM MgCl 2 )+ The RNA samples were denatured in their respective buffers at 65 8C for 15 min and slowly renatured to ambient temperature for an hour+ DMS modification of RNA was carried out by adding 12 mL of DMS (diluted 1:12 in ethanol) to the renatured RNA in 300 mL of HMK buffer, and incubated at 37 8C for varying lengths of time (0, 5, 10, and 20 min)+ The reaction was stopped by adding 75 mL of the DMS stop buffer (1 M Trisacetate, pH 7+5, 1 M b-mercaptoethanol, 1+5 M sodium acetate, 0+1 mM EDTA)+ For KE treatment, 30 mL of KE at 37 mg/mL in 20% (v/v) ethanol was added to the renatured RNA in 270 mL of HMK buffer+ The reaction was carried out as described and stopped by stabilizing the modified RNA with 40 mM potassium borate, pH 7+0...…”

“…Giardiavirus (GLV) is a double-stranded (ds)RNA virus of the Totiviridiae family, which specifically infects trophozoites of the most primitive protozoan parasite Giardia lambila (Wang & Wang, 1986)+ Its dsRNA genome of 6277 bp encodes two polypeptides; a major 100-kDa capsid protein (Gag) and a minor 190-kDa fusion protein (Gag-Pol) via a Ϫ1 ribosomal frameshift (Wang et al+, 1993;Li et al+, 2001)+ The coding region in GLV (ϩ)-strand RNA is flanked by a 367-nt 59 untranslated region (UTR) and a 301-nt 39 UTR + The ability of purified GLV to infect, and the capability of its (ϩ)-stranded RNA to transfect G. lamblia trophozoites, resulting in intracellular proliferation of infectious GLV particles, are the major distinguishing features of this virus among the totiviruses (Wang & Wang, 1986)+ It has turned the GLV (ϩ)-strand RNA into an effective transfection vector for high-level expression of foreign mRNAs in GLVinfected G. lamblia (Yu et al+, 1995;Yu & Wang, 1996)+ In vitro-transcribed chimeric mRNA containing a fulllength firefly luciferase transcript flanked by the 367-nt GLV 59 UTR and a 2022-nt 39 terminus of GLV (ϩ)-strand RNA can be introduced into GLV-infected G. lamblia trophozoites via electroporation (Yu et al+, 1995)+ The chimeric mRNA thus introduced undergoes vigorous replication and transcription but only a basal level of translation, resulting in an expressed luciferase activity barely above the background level (Yu et al+, 1995;Yu & Wang, 1996)+ This relatively poor translation efficiency was, however, enhanced by 5,000-fold when the initial 264 nt of the capsid-coding region in GLV mRNA were fused in frame with, and upstream from, the luciferase mRNA (Yu & Wang, 1996)+ In our earlier studies, we have identified a 13-nt downstream box (DB) sequence within the initial 264-nt capsid coding region of GLV mRNA at position 66-78 that complements a 15-nt sequence (with two gaps) between nt 1382 and 1396 in the V9 region near the 39 end of the 16S-like ribosomal RNA (rRNA) of G. lamblia (Yu et al+, 1998)+ Deletion or scrambling of this DB sequence led to a significant loss of translation efficiency+ However, although DB is located 66-78 nt downstream of the start codon, inclusion of the first 98 nt of the downstream region encompassing the entire DB exerted little enhancement of translation of the chimeric transcript (Yu & Wang, 1996)+ It was the increment of coding region from nt 111 to 264 that resulted in an exponential increase of translation efficiency up to 5,000-fold (Yu & Wang, 1996)+ The entire 264-nt segment of GLV mRNA has since been thoroughly analyzed for additional structural and sequence-specific elements that may contribute to the enhanced translation+ Results from chemical probing and mutational analysis of the MFOLD-predicted stemloops I (nt 11-35), verified their presence in the RNA molecule and indicated their essential involvement in enhanced translation (Garlapati et al+, 2001)+ Similar experiments also demonstrated the presence and ...…”

Capped mRNA with a Single Nucleotide Leader Is Optimally Translated in a Primitive Eukaryote, Giardia lamblia

“…Serial passages of the in vitro culture were performed at an inoculation ratio of 1:13 once every 3 days into fresh culture medium to maintain a continuous logarithmic cell growth. Transfection of the trophozoites with RNA was performed by electroporation as described previously (25,26) with minor modifications. Logarithmically growing cells were pelleted at 2500 rpm for 10 min and washed twice with the Ca 2ϩ -, Mg 2ϩ -free phosphate-buffered saline.…”

Stage-Specific Expression and Targeting of Cyst Wall Protein–Green Fluorescent Protein Chimeras inGiardia

“…Stable transformation and expression of reporter proteins have been demonstrated only recently in trophozoites, using a viral system Yu et al, 1995Yu et al, , 1996a and subsequently also by electroporation of plasmid-based vectors Singer et al, 1998;Sun et al, 1998). As an initial step toward the complete molecular dissection of regulated protein trafficking in this ancient eukaryotic protozoan, we describe for the first time in an anaerobic organism a model for the study of stage-specific expression, protein targeting, and transport using green fluorescent protein (GFP) as a reporter.…”