The double-stranded RNA in Trichomonas vaginalis may originate from virus-like particles

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 83, pp , October 1986 Microbiology The double-stranded RNA in Trichomonas vaginalis may originate from virus-like particles ALICE L. WANG AND CHING C. WANG Department of Pharmaceutical Chemistry, University of California, School of Pharmacy, San Francisco, CA Communicated by H. A. Barker, June 23, 1986 ABSTRACT 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. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact 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 related to T. vaginalis and also highly susceptible to metronidazole, contains no ds RNA (1). It is thus conceivable that the ds RNA could be eliminated from T. vaginalis under the pressure of metronidazole. In the yeast Saccharomyces cerevisiae (3), two ds RNA species, L ( kb) and M ( kb), were found associated with the virus-like particles. L encodes primarily the capsid protein of the virus-like particles, whereas M encodes a polypeptide toxin of 109 amino acids capable of causing an irreversible change in the plasma membrane of S. cerevisiae and, consequently, the loss of intracellular ions and ATP (4). The ds RNA in T. vaginalis has apparently no killing effect, and no virus-like structures have yet been identified in sectioned T. vaginalis by electron microscopy (1). The purpose of the present investigations is to further test a possible association between the ds RNA and a virus in T. vaginalis similar to that observed in yeast. EXPERIMENTAL PROCEDURES Cultures. T. vaginalis ATCC and two metronidazoleresistant strains IR78 and CDC85 (2) were maintained and cultivated in TYM medium (ph 6.2) supplemented with 10% heat-inactivated horse serum as described (5, 6). All strains of T. vaginalis have been cloned and verified to be free of bacteria, mycoplasma, and plasmids (7). Electron Microscopy. Samples of cell suspension were fixed with 2.5% glutaraldehyde and pelleted by a brief centrifugation (7). The pellets were then fixed with 1% osmium tetroxide, dehydrated, embedded in epon, sectioned, and stained with uranyl acetate in searching for virus-like particles in situ. The purified virus-like particles were fixed and negatively stained by a similar procedure described by Haschmeyer and Meyers (8). Radiolabeling of ds RNA and Hybridization with DNA Digests. The ds RNA was extracted from purified virus-like particles with phenol by a standard procedure (9) and labeled at its 3'-end with [32P]pCp by T4 RNA ligase (10). The remaining free [32P]pCp was removed by Sephadex G-25 chromatography. Restriction fragments of T. vaginalis DNA were fractionated by 0.8% agarose gel electrophoresis (7) and transferred to a nitrocellulose filter (10). The filter was prehybridized in 50% deionized formamide/0.9 M NaCl/50 mm sodium phosphate, ph 7.4/5 mm EDTA/0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin/denatured salmon sperm DNA (100,g/ml) at 42 C for 4 hr. One microgram of the purified 32P-labeled ds RNA (50-60 x 106 cpm) was boiled for 5 min, chilled, and added to the hybridization solution. After 16 hr at 42 C, the filter was washed three times in 2x SSC and 0.1% NaDodSO4 at room temperature followed by incubation with lx SSC and 0.1% NaDodSO4 at 50 C for 1.5 hr. It was then rinsed twice with 1x SSC at room temperature, dried, and exposed to Kodak XAR-5 film between intensifying screens at -70 C. Abbreviations: ds RNA, double-stranded RNA; kb, kilobase(s).

2 Protein Analysis. Samples were boiled for 5 min in a buffer containing 62.5 mm Tris HCl, 2% NaDodSO4, 10% (vol/vol) glycerol, and 5% 2-mercaptoethanol (ph 6.8). NaDodSO4/ PAGE was carried out in 11% gels according to the method of Laemmli (12). The protein bands were identified by silver staining (13). Materials. Pancreatic RNase A and proteinase K were purchased from Sigma. Restriction enzymes and X DNA were from Bethesda Research Laboratories. T4 RNA ligase was obtained from New England Biolabs, and [32P]pCp was from Amersham. All other chemicals were of the highest purity commercially available. RESULTS Localization of the ds RNA. T. vaginalis grown to the late logarithmic phase were harvested, washed in phosphatebuffered saline (PBS), and resuspended in 10 vol of PBS. The cell suspension was homogenized at 0C-40C in a Potter-Elvehejm tissue grinder driven by a lab stirring motor (H. K. Heller, Bellerose, NY) for 20 min. The crude homogenate was then subjected to differential centrifugations at 3000 x g for 15 min and at 12,500 x g for 90 min, with the pellets collected after each spin designated P1 and P2 (Fig. 1). The P2 fraction was resuspended in 10 vol of PBS, homogenized again for 10 min to free the virus-like particles from membrane fragments, and centrifuged at 12,500 x g for 90 min (S3 and P3). Small samples of the original crude homogenate and the separated fractions were extracted with phenol, precipitated with ethanol, and analyzed in 0.8% agarose gel electrophoresis. The results (Fig. 2) indicate that the ds RNA remained largely intact in the crude homogenate. It was not in the nuclear fraction P1, but it was found in S1, P2, S2, P3, and S3 (Fig. 2). Ribosomal RNA was found in crude homogenate and S2 only. The ds RNA was originally brought down at 12,500 x g (P2) but released back to the supernatant fraction (S3) upon further grindings, which may suggest an original association of the ds RNA with particles or membrane vesicles sedimenting at 12,500 x g (P2). Electron micrographs of the P2 fraction indeed indicate the presence of aggregated virus-like particles inside membrane vesicles (Fig. 3) not readily penetrated by negative stain. Identification of the ds RNA with Virus-Like Particles. Pancreatic RNase A was added to the crude homogenate of T. vaginalis to a final concentration of 10 ug/ml and incu- CELL SUSPENSION I CRUDE HOMOGENATE PELLET (Pi) PELLET (P3) Microbiology: Wang and Wang Homogenization, 20 min Centrifugation, 3,000 xg, 15 min PELLET (P2) SUPERNATANT (S.) Centrifugation, 12,500 xg, 90 min SUPERNATANT (S2) Homogenization, 10 min Centrifugation, 12,500 xg, 90 min SUPERNATANT (S3) FIG. 1. The protocol of purifying virus-like particles from T. vaginalis. Proc. Natl. Acad. Sci. USA 83 (1986) 7957 H Pi P2 S FIG. 2. Agarose gel electrophoretic analysis of samples from the fractionation of T. vaginalis crude homogenates. Gels of 0.8% agarose were cast in 89 mm Tris borate, ph 8.3/2.5 mm EDTA. Bands of nucleic acids were identified by staining with ethidium bromide (0.5,ug/ml). The size markers (in kb) are from the X DNA HindIII fragments. H, crude homogenate; P1, 3000 x g pellet, this lane was purposely overloaded to show the absence of ds RNA; P2, 12,500 x g pellet; S2, 12,500 x g supernatant. bated at 37 C for 30 min. The treated homogenate showed the presence of intact ds RNA after phenol extraction, suggesting that the ds RNA remained largely unaffected by the RNase pretreatment. However, when 0.1% NaDodSO4 or proteinase K (100,g/ml) was added to the crude homogenate with or without the RNase treatment, the ds RNA became no longer detectable in gel electrophoresis (data not shown). Apparently, the ds RNA in the crude homogenate was protected from both exogenous and endogenous RNase, but the protection could be removed by NaDodSO4 or proteinase K. The S2 and S3 fractions were pooled and CsCl was added to a final density of 1.39 g/ml. The mixture was centrifuged in an SW41 rotor at 34,000 x g and 20 C for 16 hr. Fractions of the CsCl buoyant-density gradient were collected in a 0.5-ml vol, and the absorbance at 260 nm of each fraction was monitored with a Beckman DU7 spectrophotometer (Fig. 4). Samples of individual fractions were extracted with phenol and analyzed in gel electrophoresis. Fig. 4 indicates that the peak fractions of absorbance at 260 nm identified at the center of the gradient (p = g/ml) are also the fractions containing the ds RNA. Ribosomal RNA is found exclusively at the bottom of the gradient together with a substantial amount of the ds RNA (Fig. 4). Electron microscopic examinations of this bottom fraction again indicated the virus-like particles entrapped in membrane vesicles as shown in Fig. 3. Fractions 9-11 were pooled, diluted with 15 vol of PBS, and centrifuged in an SW41 rotor at 109,000 x g and 4 C for 2 hr. The pellets were negatively stained and examined by electron microscopy (8). Fig. 5 shows that the pellets consist ofuniformly virus-like particles. They are icosahedral with an estimated diameter of 33 nm. No such virus-like particles

3 7958 Microbiology: Wang and Wang Proc. Natl. Acad. Sci. USA 83 (1986) 0 I' okma FIG. 3.Eeto irgaho egtvl tie 2fato (sefg ) Br 10n were detectable in those fractions without detectable A260 or ds RNA in gel electrophoresis (Fig. 4). Similar experiments were performed on the metronidazole-resistant T. vaginalis strains IR78 and CDC85, which are known to contain no detectable ds RNA (1). No virus-like particles were found in these two strains. Characterization of the Virus-Like Particles. The purified virus-like particles were named T. vaginalis virus. An average yield equivalent to 3-4 ng of T. vaginalis virus protein per 106 T. vaginalis has been established. There is, however, no free T. vaginalis virus detectable in the culture medium up to the stationary phase. Many sections of a T. vaginalis cell have been examined under transmission electron microscope, but no evidence of the presence of mature T. vaginalis virus has yet been found. The purified T. vaginalis virus preparation (-1 gg of ds RNA) was extracted with phenol and the extracted nucleic acid was precipitated with ethanol. Electrophoretic analysis (not shown) indicated the presence of a single ethidiumstainable band with a migration in the gel corresponding to a size of 5.5 kb of DNA. It is thus concluded once again that the ds RNA is the nucleic acid component of T. vaginalis virus. The same purified T. vaginalis virus (=4 ;kg of protein) was then analyzed in NaDodSO4/PAGE for its protein composition. The data presented in Fig. 6 show one major band with an estimated molecular mass of 85 kda. This band is detectable by both silver staining and Coomassie blue staining and is thus most likely a protein. The ds RNA extracted from purified T. vaginalis virus was labeled with [32P]pCp at the 3' end by T4 RNA ligase and was used as a probe to hybridize with DNA fragments of T. vaginalis on Southern blots (10). EcoRI and HindIII digests of T. vaginalis DNA derived from both the T. vaginalis virus-containing strain ATCC and the virus-free strain IR78 were used in these experiments. The results indicated no sign of hybridization between the DNA fragments and the ds RNA, thus suggesting the absence of homology between ds RNA and host DNA. Control experiments with denatured T. vaginalis virus ds RNA on dot blots found highly efficient hybridization with the radiolabeled probe. Finally, an attempt was made to use the purified T. vaginalis virus to infect the two strains of T. vaginalis IR78 and CDC85, known to contain no such virus-like particles (1). Purified T. vaginalis virus was added to the in vitro cultures of the two T. vaginalis strains to a final concentration of 10 Ag of ds RNA per ml. The two strains were found growing at normal rates under such conditions. The late logarithmic phase cells were harvested, homogenized, and fractionated by differential and CsCl gradient centrifugations and checked for virus-like particles and ds RNA. No such particles or ds RNA were detected in the cell homogenates, suggesting the failure of viral infection of T. vaginalis. DISCUSSION The present investigation has provided unequivocal evidence that virus-like particles (T. vaginalis virus) containing the 1.5-gm linear ds RNA (1) and a major protein of 85 kda are present in T. vaginalis. Since the ds RNA has been detected in some 40 independent isolates and strains of T. vaginalis (1), the virus must have a prevalent presence in the parasite. The absence of T. vaginalis virus in the culture medium of T. vaginalis may reflect instability of the virus in an external environment, which may partly explain the failure of purified T. vaginalis virus to infect virus-free T. vaginalis. The purified T. vaginalis virus, however, has apparently retained its nucleic acid component in spite of the relatively intense negative stain of the virus-like particles (Fig. 5), because the ds RNA was successfully extracted from the purified T. vaginalis virus. The finding of one major 85-kDa protein suggests that the latter could be the capsid protein of T. vaginalis virus, but the presence of other viral proteins at lower levels such as the RNA-dependent RNA replicase cannot be ruled out. T. vaginalis virus is undoubtedly multiplied in the growing T. vaginalis. But there is no

4 ~~~~4t Microbiology: Wang and Wang A Fractions S I \' 0~~~~~ ~~~~i\ \I x 0, \% \ A Fractions FIG. 4. CsCl buoyant-density gradient centrifugation analysis of the S2 and S3 fractions of T. vaginalis crude homogenate. (A) Agarose gel electrophoresis profile of individual fractions. Size markers are in kb. (B) Absorbance at 260 nm profile of individual fractions. Fraction 1 is the bottom of the gradient. Y Proc. Natl. Acad. Sci. USA 83 (1986) 7959 evidence of symbiosis between the virus and the protozoan, since the latter can grow normally without the virus. It is not known whether certain aspects of the pathogenicity in T. vaginalis infections could be attributed to the presence of T. vaginalis virus. It is also unclear whether T. vaginalis virus has a specific location inside the infected T. vaginalis, but it may have a close association with the membrane fraction (Fig. 3). The difficulty in identifying T. vaginalis virus in sectioned T. vaginalis under the electron microscope could be due to the relatively small number of T. vaginalis virus ( ; see ref. 1) in a T. vaginalis cell, which has a rather large diameter of 15,um. More intensive in situ searches for the virus are currently under way. This is the only ds RNA virus we know of that has been identified in a protozoan. Mattern et al. (14) observed the presence of virus-like particles in Entamoeba histolytica. The particles have two morphological types-a filamentous form and a polyhedral (icosahedral) form, mostly nm in diameter (14, 15). The latter apparently consists of DNA and is lytic to certain strains of E. histolytica (16). Later studies revealed the presence of a third type of beaded particles in the nuclei of a few amebal strains (17). None of the three virus-like particles bears much resemblance to T. vaginalis virus. There is, however, considerable resemblance between T. vaginalis virus and the killer ds RNA viruses of S. cerevisiae, especially species L (3). Both S. cerevisiae ds RNA virus species L and T. vaginalis virus contain nonsegmented ds RNA genomes, and their shapes, sizes, buoyant densities, and nucleic acid and protein components are all quite similar (Table 1). They are transmitted within individually defined genetic systems for the host-i.e., they are infectious by heredity. Their propagation relies on cell division, a feature normally associated with the naturally occurring plasmids. One interesting possibility would be that both T. vaginalis and S. cerevisiae may infect the vaginal tract to allow exchange of the same ds RNA virus between the two species. Studies of protozoa have been hampered in the past 1. Xe;i t -Y;-m+ 0 0 ;'., + ;<0 '0;4 ""t;s ++V~~iaMf,,<w~~~~~~jr,; $ S ~ t :,;,,, 0 0 ';' vit''s> : si."1> '',i- dilit,'.?g~~~tf~~t ^ 4. ''' FIG. 5. Electron micrograph of the purified virus-like particles (T. vaginalis virus). (Bar = 100 nm.)

5 7960 Microbiology: Wang and Wang kda A _ _ 21.5 A_ FIG. 6. NaDodSO4/PAGE analysis of the protein profile of purified virus-like particles (T. vaginalis virus). Lanes: A, protein standards; B, T. vaginalis virus protein. The hazy band corresponding to 66 kda is a staining artefact from the silver stain in alkaline solution due to the inclusion of 2-mercaptoethanol (12). because of the lack of vectors capable of performing genetic transformation for the organisms. This deficiency has been particularly limiting on the investigations of the type of protozoa like T. vaginalis, which lacks sexual differentiation during its development and, thus, has no apparent means of genetic recombination. Our discovery of T. vaginalis virus may make genetic manipulation of T. vaginalis possible through viral transfection. The absence of a viral genomic sequence in the host DNA suggests a lack of reverse transcription and integration of the viral genome. In our previous investigation with the ds RNA purified directly from T. vaginalis by NACS-37 column chromatography and two cycles of agarose gel electrophoresis, some hybridization between the labeled RNA sample and T. vaginalis DNA fragments was observed on Southern blots (1). This was attributed to the presence of some labeled ribosomal Table 1. Comparisons of S. cerevisiae virus L* and T. vaginalis virus S. cerevisiae virus T. vaginali$ virus Shape Icosahedral Icosahedral Diameter, nm Density in CsCl ds RNA Lengtht, am Estimated size, kb t Major capsid protein, kda All information regarding S. cerevisiae virus is cited from ref. 3. *5. cereviziae ds RNA virus species L. tdetermined by electron microscopy. *This is calculated from electron microscopy data assuming 3 A for each base pair of ds RNA (18). B Proc. Natl. Acad. Sci. USA 83 (1986) RNA,which was removed from the purified T. vaginalis virus in the present studies. The T. vaginalis DNA fragments identified in the previous Southern hybridization experiments were cloned in the puc18 plasmid and identified as the T. vaginalis ribosomal gene by use of a cloned Schistosomal mansoni ribosomal gene (a gift from Philip Loverde, New York State University, Buffalo) as the probe for Southern hybridization (unpublished observation). Finally, in our recent investigations on another related anaerobic parasitic protozoan, Giardia lamblia, a similar but distinctive ds RNA virus specific to the parasite was identified, purified, and characterized (19). It is found in the two nuclei of G. lamblia trophozoite and appears as a sphere 33 nm in diameter. It contains a linear ds RNA equivalent to the size of a 7.0-kb DNA with little homology to T. vaginalis virus ds RNA, and it has one major 66-kDa protein. In contrast to T. vaginalis virus, this virus can be harvested from the culture medium of G. lamblia and used to infect the virus-free strains of G. lamblia. Thus, the prospect of finding a vector for genetic transformation in protozoa may soon come very close to reality. The authors would like to express their gratitude to Dr. Harold E. Varmus (University of California, San Francisco), Drs. Austin Newton and Noriko Ohta (Princeton University), and Dr. J. A. Bruenn (New York State University, Buffalo) for their interest in our studies, their stimulating discussions, and their helpful suggestions. Thanks also go to Dr. Philip LoVerde for his generous gift of cloned S. mansoni ribosomal gene, and Ms. Mei Lie Wong (University of California, San Francisco) for her invaluable help in preparing the electron micrographs of the virus-like particles. This work was supported by Grant AI from the National Institutes of Health. C.C.W. is a Burroughs-Wellcome Scholar in Molecular Parasitology. 1. Wang, A. L. & Wang, C. C. (1985) J. Biol. Chem. 260, Mflller, M. & Gorell, T. E. (1983) Antimicrob. Agents Chemother. 24, Bruenn, J. A. (1980) Annu. Rev. Microbiol. 34, Skipper, N. & Bussey, H. (1977) J. Bacteriol. 129, Diamond, L. S. (1957) J. Parasitol. 43, Wang, C. C. & Cheng, H.-W. (1983) Mol. Biochem. Parasitol. 10, Wang, A. L. & Wang, C. C. (1985) Mol. Biochem. Parasitol. 14, Haschmeyer, R. H. & Meyers, R. J. (1972) in Principles and Techniques of Electron Microscopy, ed. Hayat, M. A. (Van Nostrand-Reinhold, New York), Vol. 2, pp Blin, N. & Stafford, D. W. (1976) Nucleic Acids Res. 3, Southern, E. M. (1975) J. Mol. Biol. 98, Bruenn, J. & Brennan, V. (1980) Cell 19, Laemmli, U. K. (1970) Nature (London) 227, Wray, W., Boulikas, T., Wray, V. P. & Hancock, R. (1981) Anal. Biochem. 118, Mattern, C. F. T., Diamond, L. S. & Daniel, W. A. (1972) J. Virol. 9, Diamond, L. S., Mattern, C. F. T. & Bartgis, I. L. (1972) J. Virol. 9, Hruska, J. F., Mattern, C. F. T. & Diamond, L. S. (1974) J. Virol. 13, Diamond, L. S. & Mattern, C. F. T. (1976) Adv. Virus Res. 20, Langridge, R. & Gomatos, R. J. (1%3) Science 141, Wang, A. L. & Wang, C. C. (1986) Mol. Biochem. Parasitol., in press.