Targeted Reduction of Nucleoside Triphosphate Hydrolase by Antisense RNA Inhibits Toxoplasma gondii Proliferation*

Valerian Nakaar, Benjamin U. Samuel, Emily O. Ngo, and Keith A. JoinerDagger

From the Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022

    ABSTRACT
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Abstract
Introduction
References

Nucleoside triphosphate hydrolase (NTPase) is a very abundant protein secreted by the obligate intracellular parasite Toxoplasma gondii shortly after invasion of the host cell. When activated by dithiols, NTPase is one of the most potent apyrases known to date, but its physiological function remains unknown. The genes encoding NTPase have been cloned (Bermudes, D., Peck, K. R., Afifi-Afifi, M., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260). We have recently shown that the enzyme is tightly controlled within the vacuolar space and may influence parasite exit from the host cell (Silverman, J. A., Qi, H., Riehl, A., Beckers, C., Nakaar, V., and Joiner, K. A (1998) J. Biol. Chem. 273, 12352-12359). In the present study, we have generated an antisense NTP RNA construct in which the 3'-untranslated region is replaced by a hammerhead ribozyme. The constitutive synthesis of the chimeric antisense RNA-ribozyme construct in parasites that were stably transfected with this construct resulted in a dramatic reduction in the steady-state levels of NTPase. This inhibition was accompanied by a decrease in the capacity of the parasites to replicate. The reduction in parasite proliferation was due to a specific effect of antisense NTP RNA, since a drastic inhibition of hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) expression by a chimeric antisense HXGPRT RNA-ribozyme construct did not alter NTPase expression nor compromise parasite replication. These data implicate NTPase in an essential parasite function and suggest that NTPase may have more than one function in vivo. These results also establish that it is possible to study gene function in apicomplexan parasites using antisense RNA coupled to ribozymes.

    INTRODUCTION
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Abstract
Introduction
References

Apicomplexan parasites (e.g. Toxoplasma, Plasmodium, Cryptosporidium, and Eimeria) are prevalent worldwide and cause significant diseases in humans and animals. In particular, the obligate intracellular parasite Toxoplasma gondii is an opportunistic infection associated with AIDS and congenital neurological birth defects (1). Treatment regimens for this infection are not always effective, and this has mandated the search for novel drug targets.

As reported previously, we and others have identified the gene encoding nucleoside triphosphate (NTPase),1 a very abundant protein secreted by T. gondii shortly after invasion of host cells (2-5). The 63-kDa NTPase isoforms are encoded by two genes, NTP1 and NTP3, which share more than 97% sequence identity (2). When activated in vitro by dithiols, NTPase is one of the most potent apyrases known to date, capable of sequentially degrading ATP to ADP and AMP (4). Because T. gondii is a purine auxotroph, it is thought that NTPase may participate in purine salvage (2, 6, 7). Recently we have shown that the enzyme may facilitate parasite exit from infected host cells (8). However, the biological function of NTPase in vivo is yet to be defined.

It is possible to generate null mutants by gene knock out in the apicomplexan parasites (9-11). This strategy enables the study of gene function that can lead to the identification of potential drug targets. However, the presence in the genome of multiple copies of a gene (such as NTP) combined with low frequency of homologous recombination in T. gondii, hamper gene targeting efforts in these organisms. Moreover, it is not feasible to investigate the biological function of essential genes using this approach because of the lethality of the phenotype. To circumvent these difficulties, we sought to develop an antisense strategy to inhibit gene expression in T. gondii.

Antisense RNA has been shown to regulate gene expression in bacteria (12), Dictyostelium (13), Leishmania (14), Drosophila (15), Xenopus oocytes (16), mammalian cells, (17) and plants (18-20) but has not yet been demonstrated in the apicomplexan parasites. This strategy is at present still by trial and error. This may be partly ascribed to the finding that in eukaryotes, 3'-end formation of mRNA promotes the export of the mature transcript to the cytoplasm (21). Since antisense-induced mRNA degradation in eukaryotes occurs in the nucleus (22), the use of a polyadenylated RNA may reduce the overall efficiency of antisense RNA. Eckner et al. (21) have shown that substituting the normal polyadenylation signal with cis-acting ribozymes leads to the nuclear retention of the product RNAs by generating export-deficient transcripts (21). Ribozymes are catalytic RNA molecules that are active in vitro, both in cell-free systems and in living cells (23-26) and are currently being developed for gene therapy applications as inhibitors of gene expression and viral replication (27-28).

In this report we show that the expression of NTPase can be substantially inhibited by stably introducing into the parasite genome an antisense gene in which its 3' end is modified by a hammerhead ribozyme. The attenuation of NTPase activity inhibits parasite proliferation, suggesting a role for NTPase in parasite metabolism. The inhibition of parasite replication was due to specific reduction in NTPase by antisense RNA, since a drastic reduction in hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) expression did not alter NTPase expression nor compromise parasite replication. The chimeric antisense RNA-ribozyme strategy should be useful for the study of gene function in other protozoan parasites.

    MATERIALS AND METHODS

Parasite Cultures, Transfection, and Parasite Replication-- RH-strain of T. gondii parasites were cultivated in Vero cells or in primary human foreskin fibroblasts (HFF), and the tachyzoites were transfected with plasmid constructs palpha NTP-RZ, pASHXRZ, or p5'NTP-RZ (see Fig. 1) subcloned into a vector containing the dihydrofolate reductase-thymidylate-synthase (DHFR-TS) gene, which confers pyrimethamine resistance for selection (29). Stable transformants were selected in 1 µM pyrimethamine, and individual clones were isolated. Confluent cultures of HFF cells in 10-cm plates or 25-cm2 T-flasks were inoculated with 106 freshly lysed-out parasite tachyzoites and incubated at 37 °C for 4 h, and the cultures were replaced with fresh medium so as to remove parasites that had not yet invaded. The doubling of intracellular parasites was followed by counting the number of tachyzoites per parasitophorous vacuole at the various times (at least 50 vacuoles were scored at each time point). The number of parasite divisions since infection was determined using log2 (parasite number) formula (30). Statistical significance was assessed by measures of analysis of variance and two-tailed t test.

Enzyme and Uracil Incorporation Assays-- NTPase enzymatic activity was done as described previously using equivalent amounts of parasite extracts (2). Briefly, 1-10 µg of cell lysate was incubated with [2,8-3H]ATP (Amersham Pharmacia Biotech) in the presence of 100 mM HEPES-KOH, pH 7.2, 30 mM magnesium acetate, 10 mM ATP, 0.1 mg/ml soybean trypsin inhibitor, and 1 mM dithiothreitol at 37 °C for 10 min. The reaction products were separated by TLC, spots corresponding to the nucleobase and 5' products were excised, and the radioactivity was counted under liquid scintillation. HXGPRT activity and parasite replication were assayed by measuring the incorporation of [8-3H]xanthine (Moravek Biochemicals) and [5,6-3H]uracil (Amersham Pharmacia Biotech), respectively. 2-5 × 105 parasites/well were used to infect monolayers of HFF in a 24-well plate at 37 °C. Unattached parasites were removed 4 h later, and after 24 h post-infection, 1 µCi of radiolabel was added to each well. Incubation was continued for 2-6 h before the monolayers were fixed with trichloroacetic acid, rinsed, and counted as described (31, 32).

Northern and Western Blot Analyses-- Total RNAs were isolated from parasites with TRIzol reagent (Life Technologies, Inc.), and Northern blot analyses were performed with 10 µg of total RNA using gel-purified NTP cDNA probe (corresponding to the 1.98-kilobase XhoI-EcoO109IA fragment of NTP1; see Fig. 1) labeled by random priming with [alpha 32P]dCTP (Amersham Pharmacia Biotech). Hybridizations were done at 65 °C in a solution containing 5× saline/sodium phosphate/EDTA, 5× Denhardt and 0.5% SDS, and 100 µg/ml tRNA. The filters were washed at 65 °C with several changes of 1× saline, sodium phosphate, EDTA, 0.1% SDS followed by 0.1× saline, sodium phosphate, EDTA, 0.1% SDS and exposed for autoradiography. Between 106 and 107parasites were separated on SDS-polyacrylamide electrophoresis gels, and the material was transferred onto polyvinylidene difluoride membranes (Millipore). The filters were probed with anti-HXGPRT antiserum (generously given by Buddy Ullman, Portland, Oregon) or with anti-NTPase polyclonal antibody, GRA3, and SAG1 monoclonal antibodies as described previously (2, 33). The primary antibodies were generally used at 1:1000 dilution. Secondary anti-mouse or anti-rabbit were horseradish peroxidase conjugates (Boehringer Mannheim) used at 1:2000 dilution followed by detection with ECL kit (Amersham Pharmacia Biotech).

    RESULTS AND DISCUSSION

We targeted the gene encoding the abundant NTPase in T. gondii for inhibition by antisense RNA. Previous experiments by others suggest that a high ratio of transfected antisense RNA to the endogenous mRNA is required for effective reduction of expression of the target gene (34, 35). To increase the effectiveness of the antisense expressing vector (Fig. 1), we designed a construct that incorporated potentially enhancing features: a potent NTP promoter (36), the inclusion of an autocatalytic ribozyme structure and RNA structural elements that may act to stabilize the antisense RNA, as well as the possible enrichment for antisense RNA within the nuclear compartment (21, 37). Substituting the polyadenylation signal with a cis-acting ribozyme that cleaves in a site-specified manner leads to nuclear retention of the product RNAs by generating export-deficient transcripts (21, 37). We inserted a hammerhead ribozyme (23) immediately downstream of the antisense DNA, thus replacing the 3'-untranslated region of the gene. This operation potentially creates a pool of antisense RNA in the nucleus comparable with the endogenous mRNA. Since 3'-end formation promotes the export of mature polyadenylated RNA from the nuclear compartment where antisense effects most likely occur in eukaryotes, we did not include in these experiments a plasmid containing NTP antisense sequences with a polyadenylation signal (21, 22, 34, 35, 37).


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Fig. 1.   Strategy for design of antisense-ribozyme expression constructs. A, step 1. A 1.7-kilobase fragment of the NTP 5'-flanking region was isolated from pBSNTP1 with SpeI and BglII digests and subcloned at SpeI-BamHI sites in the polylinker of pBS-RZ (kindly provided by G. Carmichael). The resultant construct has the entire 3'-untranslated region replaced by a hammerhead ribozyme and a histone stem-loop structure, which has been included as a possible stabilizing element against 3'-to-5' exonuclease degradation of ribozyme-cleaved RNAs (23, 37). The histone-ribozyme sequence is presented as a folded structure with the site of self-cleavage indicated by a small arrow. A, step 2. A 1.98-kilobase XhoI-EcoO109IA fragment was excised from the pBSNTP1, and the ends were filled in with the Klenow DNA polymerase. The fragment was cloned in the antisense orientation into the ribozyme cassette cleaved with EcoRI and filled in. B, a 690-base pair polymerase chain reaction fragment encompassing the coding region of HXGPRT derived from pminCAT vector was blunt-ended and cloned into p5'NTPRZ in the antisense orientation as described in A. Orientation of the coding region is indicated by the arrowheads. Figs. are not drawn to scale. Sp, SpeI; Xh, XhoI; Bg, BglII, Av, AvrII, RI, EcoRI, and EcoO, EcoO109IA. Av and Bg were generated by site-directed mutagenesis.

Clones expressing antisense NTP RNA (and hereafter referred to as AS-NTP) displayed a dramatic reduction in NTPase enzymatic activity, ranging from ~4 to >19-fold (Fig. 2A). The variation in the degree of inhibition as revealed by the enzymatic assay is considered a general feature of antisense inhibition (20, 38). This variation may be related to the difference in the steady-state levels of antisense RNA generated due to chromosomal location of the gene or in copy number of the integrated gene (38, 39). To ascertain that reduction in enzyme activity was due to reduced synthesis of the protein, the amount of NTPase protein was assessed by immunoblot. NTPase levels were reduced between 86 to 88% when compared with WT in selected clones expressing antisense RNA and correlated with the decrease in enzymatic activity (Fig. 2B). To control for the possible nonspecific effects of antisense RNA, we measured the levels of another dense granule protein, GRA3, and of the surface antigen, SAG1. The level of each of these proteins was comparable in all the samples analyzed. By immunofluorescence, NTPase signal was reduced in the transformants, whereas SAG1 expression was not affected (data not shown). The controls in these experiments indicated that the expression of antisense NTP RNA did not globally impair parasite metabolism.


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Fig. 2.   Antisense NTP RNA inhibits NTPase expression. A, equal numbers of parasites were assayed for NTPase activity as described under "Materials and Methods." Values are expressed as % ATP hydrolyzed ±S.E. relative to control (WT), which is taken as 100% (n = 6-10). Values between the control (WT) and antisense-expressing clones (AS-NTP) are significantly different (p < 0.005) as determined by the two-tailed t test. B, analysis of the steady-state levels of protein of AS-NTP clones was done by immunoblot with equal numbers of parasites using a polyclonal anti-NTPase antibody. Quantitation was performed by densitometric scanning of autoradiographs, and values obtained were expressed relative to WT, which is arbitrarily set as 1. AS-NTP 17, 12, 13, and 14 had values of 0.16, 0.12, 0.12, and 0.16 respectively. GRA3 and SAG1 were used as internal controls. C, Northern blot analysis of steady-state levels of RNA was performed with equal amounts of total RNA (10 µg/lane). As judged by staining intensity on agarose gel (indicated by 18 S and 28 S rRNA), the WT lane is underloaded; nevertheless, the band corresponding to NTP is more intense than in lanes containing the antisense clones. Values for clones AS-NTP 12, 17, and 14 as assessed had values of 0.16, 0.41, and 0.29, respectively, relative to WT, which is set at 1. Note that NTP1 and NTP3 genes (97% sequence similarity) encode two isoforms, indistinguishable from each other by immunoblot (molecular mass = 63 kDa) and by Northern blot analysis (2.7 kilobases).

We determined the steady-state levels of NTP transcripts by Northern blot analysis (Fig. 2C). NTP RNA levels were depressed at least by 2- to 6-fold in clones expressing antisense RNA. There appeared to be a good correlation between the steady-state levels of NTP RNA and the synthesis of the protein as assessed by Northern blot and immunoblot, respectively. Using a double-stranded DNA probe in Northern hybridization, we did not detect antisense RNA, although dot blot analysis revealed the presence of the gene in the genomic DNA of stably transfected clones (data not shown). One possible reason for this is that the antisense transcripts or their cleavage products are highly unstable and are degraded extremely rapidly in the parasite. However, the reduced steady-state levels of NTP mRNA coupled with the lack of detection of antisense RNA in the transfected clones is consistent with observations in other systems (38-40). This is generally assumed to be caused by duplex formation between antisense RNA and the target mRNA in the nucleus, thereby interfering with normal nuclear processing, cytoplasmic export, and translation of mRNA (34, 35). A direct inhibition of the production of NTPase through disruption of mRNA processing and transport seems plausible since ribozyme-processed RNA has been shown to be retained in the nuclear compartment (21, 37). These results indicate that antisense RNA can block mRNA and protein expression in T. gondii.

Parasites expressing antisense RNA were impaired in intracellular replication but not invasion. At 8 h after infection, equal numbers of parasitophorous vacuoles were established by both control (108 ± 4.9) and antisense-expressing clones (114 ± 7.2, 111 ± 14.3; Table I), indicating that transgenic parasites that had a normal morphology invaded host cells normally. However, once inside the cell, the transgenic parasites divided more slowly than wild type, as evidenced by two different assays for parasite replication: intracellular doubling (Table I; Fig. 3, A, B, and C) and nucleobase uptake (Fig. 3D). For example, at 24 h after inoculation, the average doubling for the control was 2.89, which differed from 2.17 and 1.67 (p < 0.01), displayed by the antisense-expressing parasites (Table I). For the control parasites, this doubling corresponds to a replication time of about 8 h, similar to that previously reported (30). The reduced replication of the antisense parasites is coincident with reduced [3H]uracil incorporated into parasite nucleic acid pools as shown by the lower values (37 and 48%) in transformants relative to wild type (Fig. 3D). The lag in replication of the transformants in the first 24-32 h after infection may reflect the deficit in initial NTPase levels and represents the time needed to overcome this deficit by synthesis, packaging into dense granules, and exocytosis of a threshold amount of NTPase into the vacuolar space.

                              
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Table I
Distribution of parasitophorous vacuole sizes in antisense-expressing and control parasites
Stably transfected transgenic clones and control RH parasites were inoculated into fresh HFF cells. After 4 h, parasites that did not infect were washed off, and the number of parasites per vacuole was scored at the indicated times. The average number of vacuoles is expressed as % and plotted in Fig. 3. Parasite doublings were determined by log2(parasite number).


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Fig. 3.   Inhibition of parasite proliferation by antisense RNA. The % distribution of vacuole sizes (number of parasites/vacuole) was determined at 24 h (A) and 32 h (B) after infection with control and antisense clones (also see Table I). Since all parasites within a single vacuole replicate synchronously, the number of parasite doublings since infection can be determined by log2(parasite number). At time 24 h after infection, the median vacuole in the control contained 8 parasites (corresponding to 3 parasite doublings), whereas in the antisense clones, this was between 2 and 4 parasites (1-2 doublings), reflecting a delay in replication. Similarly, at 32 h, the control had a median vacuole containing >8 parasites, whereas the transgenic parasites still had 2-4 parasites. Data represent the averages for at least 50 randomly selected vacuoles in four different experiments. black-square, control or WT; triangle , AS-NTP 12; open circle , AS-NTP 14. C, after infection, intracellular parasites were harvested at the time points indicated and counted. AS-NTP 6, 12, 14, 18 over a 24-h period post-infection reproducibly replicated more slowly than the control (p < 0.005). There were approximately half as many parasites with AS-NTP clones as in the WT control at 24 h. The doubling time as calculated from the data for the control was 8.2 h, whereas the AS-NTP clones had doubling times of 13, 14.5, 16, and 18 h, respectively. black-square, control; bullet , AS-NTP 17; , AS-NT 12; open circle , AS-NTP 6; diamond , AS-NTP 14. D, uracil incorporation was assayed using HFF cells infected for 24 h as under "Materials and Methods." Data are expressed relative to control (which is taken as 100%) ±S.E. from three independent experiments done in duplicate. Differences between values of the control and experimental groups were statistically significant (p < 0.005). Note that inhibition of parasite replication is not a consequence of drug selection, because pyrimethamine does not interfere with parasite replication (also see RZ; Fig. 4D and Ref. 29).

To further control for nonspecific effects of antisense RNA, we employed two additional constructs; the histone-ribozyme vector, p5'NTPRZ (Fig. 1A) and pAS-HXRZ, containing the HXGPRT gene in the antisense orientation (Fig. 1B). Stable, pyrimethamine-resistant lines expressing these constructs showed no significant reduction in NTPase levels (Fig. 4A) nor in NTPase enzymatic activity (clones AS-HXRZ-26, 32, and RZ; Fig. 4C). In contrast, parasites harboring pAS-HXRZ displayed a profound reduction in HXGPRT levels (Fig. 4A), which was accompanied by a significant reduction in HXGPRT activity (Fig. 4B). These transgenic parasites did not reveal any morphological changes nor any growth retardation (Fig. 4D) and were comparable with the control HXGPRT knock out strain of RH (KO; Fig. 4, C and D). Similarly, parasite replication was not significantly reduced in parasites harboring the p5'NTPRZ construct (RZ; Fig. 4D). The reduction in HXGPRT activity with no adverse effects on parasite replication is consistent with the finding that HXGPRT is not an essential gene (32). These results also suggest that promoter interference or squelching is not responsible for the inhibition of endogenous NTP expression. Although we have not directly tested these constructs (Fig. 1) in host cells, it is unlikely that they affect host cell growth or viability because they are driven by the developmentally regulated NTP promoter (32). Therefore, we conclude that the inhibition of parasite replication is a consequence of a deficit in NTPase production brought about by antisense NTP RNA.


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Fig. 4.   Inhibition of HXGPRT expression by antisense RNA does not affect NTPase activity and parasite replication. A, equal numbers of parasites were assayed by immunoblot with a polyclonal antibody that recognizes bands corresponding to two isoforms of HXGPRT (HXGPRT I = 26 kDa and HXGPRT II = 31 kDa) as in Donald and Roos (29). Note that NTPase levels are comparable in all samples. B, HXGPRT activity was expressed by % [3H]xanthine incorporation ±S.E. in two independent experiments done in triplicate; activity from WT was taken as 100%. C, NTPase activity was expressed as % ATP hydrolysis ±S.E. from three independent experiments; activity from WT was taken as 100%. D, rate of [3H]uracil incorporation was expressed relative to control (which is taken as 100%) ±S.E. (n = 3-6). RZ, parasites harboring p5'NTPRZ; KO, HXGPRT knock out strain (kindly provided by D. Roos (29)).

We propose that NTPase is functionally required for proliferation of T. gondii. The molecular mechanism by which the reduction in NTPase levels leads to inhibition of parasite replication is unknown. Nevertheless, this study implicates NTPase for the first time in an essential parasite function. Data from other studies support the notion that NTPase may have a regulatory function in T. gondii growth and replication. First, NTPase activity is tightly regulated in vivo, as unfettered activation of the enzyme leads to rapid exit of parasites from the host cell (8). Second, it has been shown that quercetin (P-type ATPase inhibitor) effectively prevents growth of T. gondii in culture at concentrations that inhibit NTPase by >90% in vitro (41). Third, the secretion of NTPase into the parasitophorous vacuoles occurs shortly before replication of Toxoplasma sporozoites (infective forms within oocysts generated after the sexual cycle in the definitive feline host) (42). Finally, NTP promoter activity is developmentally down-regulated in bradyzoites, the slowly dividing form of T. gondii that persists in the host as a chronic infection (36). Of interest, in gene knock out experiments we have never recovered any clones with a disrupted NTP1 or NTP3 gene locus, although we have employed targeting vectors with large contiguous genomic sequences. This suggests indirectly that NTPase is an essential gene and may thus provide a potential target for chemotherapy in T. gondii.

The experiments were undertaken in this study to demonstrate the biological role of NTPase, one of the most potent ATP degrading enzymes, in the metabolism of the parasite. To this end, we employed antisense RNA coupled to a hammerhead ribozyme to reveal the role of NTPase in parasite proliferation. Although it remains to be formally proven, a complete abrogation of NTPase expression may be incompatible with parasite growth and survival. Since NTPase can also influence parasite exit from the host cell (8), these findings suggest that the physiological role of NTPase may be multifunctional. In this study, we have also shown that even a very abundant protein such as NTPase or HXGPRT is titratable by antisense RNA. Because it is effective and simple, antisense RNA strategy should find widespread application among apicomplexan parasites for the rapid analysis of gene function. For example, genes that are refractory to targeted gene disruption or essential genes that generate a lethal phenotype when they are disrupted, are especially amenable to this strategy.

    ACKNOWLEDGEMENTS

We thank G. Carmichael, Storrs, CT for generously donating pBS-RZ and Buddy Ullman, Oregon Health Sciences University for HXGPRT antibody. We also thank Heinreich Hoppe, Huân Ngô, Christian Tschudi, and Elisabetta Ullu for critically reading the manuscript.

    FOOTNOTES

* This work was supported by a National Institutes of Health Public Health Service Grant from the (to K. A. J.), a National Institutes of Health Minority Supplement Award (to V. N.), and the Markey Physician Scientist Training program (to B. U. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence and reprint requests should be addressed: Infectious Diseases Section, Dept. of Internal Medicine, Yale University School of Medicine, 808 LCI, 333 Cedar St., New Haven, CT 06520-8022. Tel.: 203-785-4140; Fax: 203-785-3864; E-mail: keith.joiner{at}yale.edu.

    ABBREVIATIONS

The abbreviations used are: NTPase, nucleoside triphosphate hydrolase; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyl transferase; GRA3, dense granule antigen 3; SAG1, surface antigen 1; HFF, human foreskin fibroblasts; AS-, antisense; WT, wild type.

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