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Synthesis of simian rotavirus SA11 double-stranded RNA in a cell-free system
Yirw Research, 6 (1986/87) Elsevier
217
217-233
VRR 00300
Synthesis of simian rotavirus SA11 double-stranded RNA in a cell-free system John T. Patton Department
of Biology, University of South Florida, Tampa, FL 336220, U.S.A.
(Accepted
for,publication
1 August
1986)
Summary
A cell-free system was developed to study the replication of simian rotavirus SAll. The components of the system included (i) subviral particles prepared from infected cells to template the synthesis of viral RNA and (ii) an mRNA-dependent rabbit reticulocyte lysate to support protein synthesis. Based upon nuclease-sensitivity, approximately 20% of the RNA made in vitro was double-stranded (dsRNA) and 80% single-stranded (ssRNA). Electrophoretic analysis of the RNA products on polyacrylamide and low pH agarose gels showed that the system supported the synthesis of 11 dsRNAs and 11 positive-sense ssRNAs that corresponded in size to authentic viral RNAs. The synthesis of dsRNA in vitro was determined to be an asymmetrical process in which a nuclease-sensitive positive-strand RNA acted as a template for the synthesis of negative-strand RNA. The system also supported the initiation of negative-strand RNA using exogenous viral positive-strand RNA as a template. Finally, analysis of subviral particles recovered from reactions suggested that viral proteins made in vitro assembled into nucleoprotein complexes which were similar to those present in infected cells. Together, these results indicate that the cell-free system supported rotavirus RNA replication, transcription and the assembly of subviral particles. simian rotavirus SAll, cell-free replication system, negative-strand
RNA
The genome of the rotaviruses, members of the family Reoviridae, consists of eleven segments of double-stranded RNA (dsRNA) ranging in size from 0.4 to 2.0 x lo6 Da (see for review Estes et al., 1983). The positive-strand RNA present in 0168-1702/84/~03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
each segment is indistinguishable from viral mRNA; both contain 5’“terminal cap structures but lack 3’-terminal polydenylate sequences (Imai et al., 1983; McCrae and McCorquodale, 1983). Rotaviruses have a virion-associated RNA polymerase (transcriptase) capable of synthesizing viral mRNAs in vitro (Cohen, 1977; Mason et al., 1980; Flares et al., 1982). The virion consists of two icosahedral layers of protein (Petrie et al., 1981). For simian rotavirus SAll, the inner shell is made up of a core consisting of the viral proteins VP1 (125 kDa, 12X) and VP2 (94K) surrounded by the major inner shell protein VP6 (41K) (Ericson et al., 1982). The components of the outer shell include a 38K gly~oprotein fVP7) and a protease-sensitive 88K protein (VP31 (Espejo et al., 1981; Estes et al., 1981; Kalica et al., 19X3). Several virally induced nonstructural proteins are present in infected cells (Ericson et al., 1982); however, their role in virus replication is not certain. Examination of rotavirus-infected cells by electron microscopy indicates that viral replication takes place in the cytoplasm (Chasey, 1977). Numerous types of subviral particles have been identified in infected cells (Chasey, 1977; Altenburg et al., 1980; Petrie et al., 1981; Suzuki et al., 1984). These include (i) core particles in lysosomes, (ii) particles on the surface of the rough endoplasmic reticulum (RER), (iii) single-shelled particles (60-65 nm) in the cytoplasm and cisternae of the RER, (iv> membr~e-buund particles within the RER, and (v) double-shelled particles (70-75 nm> in the RER cistemae. It is not known which of these particles, if any, is responsible for the production of viral dsRNA and mRNA in infected cells. A method for assaying subviral particles for associated replicase (negative-strand RNA synthesis) or transcriptase (positive-strand RNA synthesis) activity would be useful for characterizing their role in infected cells. Towards that end, a cell-free system has been developed that supports the synthesis of rotavirus dsRNA, mRNA and protein in vitro. This system, modeled after a system developed by Davis and Wertz (1982) for vesicular stomatitis virus, consists of subviral particles isolated from rotavirus SAll-infected cells and an n-RNA-dependent rabbit reticulocyte lysate. As described in this report, the system supports the asymmetrical replication of rotavirus RNA including the initiation and elongation of viral negative-strand RNA.
Materials and Metltods Cell culture and virus
Monolayers of fetal rhesus monkey kidney cells (MA104) were grown in Eagle minimal essential media (MEM) containing 5% fetal calf serum and 5% newborn calf serum. Stock preparations of simian rotavirus SAll were propagated in MA104 cells infected with plaque-purified virus at a multiplicity of infection (m.o.i.) of < 0.01. Infected cultures were maintained in MEM without serum but containing 10 ,ug/ml trypsin (Difco 1: 250) until SO-90% of cells showed cytopathic effects (CFE; 5-7 days post infection, p-i.> (Mason et al., 1980). The cultures were then freezethawed three times and cellular debris removed by cent~fugatio~ at 12000 x g for
219 10 min at 4°C in a Sorvall SS-34 rotor. The supernatant (virus stock) was titered by plaque assay on MA104 cells and stored at - 70” C. Cells and virus were gifts from M. Sobsey. Virus purification Rotavirus was purified from infected cells essentially as described by Mason et al. (1980). Monolayers of MA104 cells were infected with virus stock at an m.o.i. of < 0.01. When 80-90% of cells exhibited CPE, cultures were freeze-thawed three times and homogenized with an equal volume of t~~hlorot~fluoroet~~e. The homogenate was then centrifuged at low speed until the aqueous and organic phases were separated. The aqueous phase was saved and the organic phase re-extracted with an equal volume of TN buffer (50 mM Tris-HCl, pH 7.5/150 mM NaCI). The aqueous phases were combined, adjusted to 10% polyethylene glycol, and left stirring overnight at 4°C. Virus was then pelleted from the aqueous phases by centrifugation at 65 000 x g for 30 min at 4” C in a Beckman Type 70Ti rotor and resuspended in TN buffer. The virus sample was then loaded onto 4 ml 20-40% linear CsCl gradients (w/w) in TN buffer which were centrifuged at 120000 X g for 18 h at 4°C in a Beckman SW50.1 rotor. Material banding at a density of 1.36 g/ml (double-shelled virions) was dialyzed overnight against TN buffer at 4OC. Preparation of subv~ralporticoes as te~~~ate~ for WA synthesis Intracellular subviral particles were prepared from infected cells by the method described by Davis and Wertz (1982) for vesicular stomatitis virus. Confluent monolayers of MA104 cells in lo-cm tissue culture dishes were infected with virus stock at an m.o.i. of 5-10. At 1 h p.i., the inoculum was removed and replaced with MEM containing 5 pg/ml actinomycin D. Plates were then incubated at 37°C until 6 h p.i. when the media was drawn off and the monolayers washed twice with hypotonic buffer (3 mM Tris-HCl, pH 8.1/0.5 mM MgC1,/3 mM NaCI). Afterwards, cells were scraped into the same buffer, incubated for 10 min on ice, and disrupted using 14 strokes of a Dounce homogenizer. Nuclei and large cellular debris were removed from the cell lysate by centrifugation at 12000 X g for 30 min at 4°C in a Sorvall SS-34 rotor. The supernatant was layered onto 3 ml 15-30% sucrose gradients (w/v) in TMN buffer (3 mM Tris-HCl, pH 8.1/3 mM MgCl,/66 mM NH,Cl/14 mM potassium acetate/l mM dithioerythritol (DTE)) and centrifuged at 200000 x g for 2 h at 4*C in a Beckman SW50.1 rotor. After removal of the supernatant, HGD buffer (10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-HCl, pH 7.6/10% glycerol/2 mM DTE) was placed on the pellets which were then allowed to resuspend by incubation overnight on ice. Subviral particles were resuspended at a final concentration of 5 ~1 of HGD buffer per starting tissue culture dish. Particles were used in reactions immediately or stored at - 70°C until needed. Freezing had no detectable effect on either the level of polymerase activity associated with or RNA products made by subviral particles (data not shown). In some instances, prior to centrifugation on 15-308 sucrose gradients, the nuclei-free supernatants were adjusted to 10 pg,/ml micrococcal nuclease, 50 mM
220 Tris-HCI, pH 8.1, and 1 mM CaCl,, and incubated for 10 n-tin at 23°C. Afterwards, the nuclease activity was inhibited by the addition of ethylene glycol-bis(~-a~noethyl ether)-&‘, N, N’, N’-tetraacetic acid (EGTA) to a concentration of 2 mM. Components of the ceil-free system In vitro reactions were carried out in a final volume of 25 pl which contained by volume 20% subviral-particle preparation and 70% micrococcal nuclease-treated rabbit reticulocyte lysate. Endogenous mRNA present in the reticulocyte lysate (Promega Biotec) was removed immediately prior to use by incubation for 10 min at 21°C in the presence of 30 FM hemin, 50 ,eg/ml creatine phosphokinase, 5.5 mM potassium acetate, 1 mM CaCl, and 10 ~g/ml micrococcal nuclease. The nuclease activity was then blocked by the addition of EGTA to a concentration of 2 mM (Davis and Wertz, 1982). Reactions also contained 50 mM HEPES-HCl, pH 7.7, 10 mM creatine phosphate, 1 mM ATP, 0.6 mM each CTP and GTP, 0.1 mM UTP, 0.05 mM each of the 20 amino acids except for methionine which was 1.25 ,uM, 14 mM potassium acetate, 2 mM DTE, 66 mM NH&l, 2 mM magnesium acetate, 1 pg of rabbit liver tRNA, 10 PCi of [35S]methionine (1110 Ci/mmol, New England Nuclear), and/or 25 PCi of [3H]UTP (40 Ci/mmol, ICN) (Davis and Wertz, 1982). Reactions were incubated for 90 min at 30°C. Incorporation of [ 3H]UMP into RNA and [35S]methionine into protein was assayed by counting radioactivity present in trichloroacetic acid precipitates of 2 pl samples taken from reactions during incubation (Davis and Wertz, 1982). To recover dsRNA product, a 15 ~1 portion of a reaction was adjusted to 2.75 mM CaCl, and 20 pg/ml micrococcal nuclease and incubated for 10 mm at 23°C. EGTA was then added to a concentration of 5 mM. The RNAs from the nucleasetreated portion as well as 5-8 ~1 of an untreated portion (total RNA) of the same reaction were then purified by phenol extraction and ethanol precipitation. Preparation of viral mRNA 3H-labeled viral mRNA was made in vitro by activated double-shelled virions (Cohen et al., 1972). Virus was activated by incubation in the presence of 5 mM EDTA for 20 min at 37OC followed by adjustment to 10 mM magnesium acetate. The transcription reactions contained the same components as described above for the cell-free system except that intracellular subviral particles were replaced with activated virions and no ~35S]met~o~ne was included. RNA was isolated from reactions by phenol extraction and ethanol precipitation. Electrophoresis RNA products were analyzed by electrophoresis on either 1.75% agarose gels containing 6 M urea and 0.025 M citrate buffer, pH 3 (Wertz and Davis, 1979) or 10% polyacrylamide gels containing sodium dodecyl sulfate (SDS) (Laemmli, I970). Agarose-urea gels were run at 150 V until the bromphenol blue dye marker migrated 22 cm, processed for fluorography and exposed to Kodak XAR-5 film (Wertz and Davis, 1979). 35S-labeled proteins were analyzed on 12% polyacrylamide gels containing SDS as described (Laemmli, 1970).
223 Results
Kinetics of RNA and protein synthesis in vitro At 6 h p-i., rotavirus SAll RNA replication, transcription, protein synthesis and virion production have begun in infected cells (Estes et al., 1979; McCrae and Faulkner-Valle, 1982; Ericson et al., 1982). The ratio of viral positive- to negativestrand RNA synthesis at this time is nearly 1: 1. By 12 h p.i., this value changes to approximately 10 : 1 (Stacy-Phipps and Patton, manuscript in preparation). To develop a cell-free system for the study of rotavirus, subviral particles were isolated from infected cells at 6 h p-i. so as to rna~~ze the ratio of particles obtund with replicase activity to transcriptase activity. This avoided the possibility that high levels of transcriptase activity associated with subviral particles isolated later in infection would interfere with the detection of the relatively lesser amounts of associated replicase activity. To prepare subviral particles with associated polymerase activity, rotavirus-infected cells were harvested, swollen in a h~otonic buffer and lysed by Dounce homogenization. Nuclei and large cellular debris were removed from the lysate by centrifugation. Subviral particles were then collected from the lysate by centrifugation through a 15-30% sucrose gradient and resuspended in a 10% glycerol buffer.
Minutes Fig. 1. Kinetics of RNA and protein synthesis. Two identical reactions, one cont~~ng [ 3H]UTP and the other [35S]methionine, were prepared as described in Materials and Methods. During incubation, 2 ~1 samples were removed and assayed for total acid-precipitable 3H- or 35S-labeled material. Another 2 pl sample from the reaction containing [‘H]UTP was diluted to 100 ~1 with 10 mM HEPES/Z mM MgClz/2 mM DTE/66 mM NH,C1/14 mM potassium acetate/O.1 mM EGTA, pH 7.7, adjusted with an additional 5 mM CaCl, and 12.5 kg/ml micrococcal nuclease, and incubated for 15 min at 23°C. EGTA was then added to a concentration of 5 mM and the sample assayed for acid-precipitable radiolabeled material. [ 3HJUMP incorporated into total (0) and nuclease-resistant (0) RNA and [35S]methionine into protein (0) per microliter of reaction mixture.
222 The ability of the purified subviral particles to template the synthesis of RNA in vitro was examined in reactions containing by volume 20% sub~ral-particle preparation and 70% nuclease-treated rabbit reticulocyte lysate. Newly synthesized RNA and protein were labeled with [ 3H]UTP and [ 35S]methionine, respectively. The incorporation of isotope was assayed by acid precipitation of samples taken from reaction mixtures during incubation. As shown in Fig. 1, the majority of 3H-labeled total RNA was made in reactions during the first 30 mm of incubation with synthesis completed by 60 min. Similarly, the synthesis of radiolabeled nuclease-resistant RNA was completed by 60 mm post incubation. Comparison of the amount of total and nuclease-resistant products made after 90 min of incubation indicated that approximately 20% of the total product was nuclease-resistant. The synthesis of 35S-labeled protein in vitro was usually maximal during the first 30 min of incubation and completed by 60 min (Fig. 1). Cell-free synthesis of viral single- and double-stranded RNA The production of 3H-labeled nuclease-resistant RNA in vitro suggested that the system supported the synthesis of rotavirus dsRNA. To test for the presence of dsRNA product, 3H-labeled total and nuclease-resistant RNAs made in the system were purified by phenol extraction and analyzed by electrophoresis on a 10% polyacrylamide gel containing SDS. A fluorograph of the gel is shown in Fig. 2. The position of the eleven viral genome segments in the gel was determined by electrophoresis of virion-derived dsRNA in a parallel lane followed by staining with ethidium bromide (not shown). Electrophoresis of the total RNA products shows that eleven 3H-labeled RNAs were synthesized in vitro that were identical in size to virion-derived dsRNAs (lane 1). These RNAs were resistant to digestion by micrococcal nuclease (lane 2) and, therefore, double-stranded. The system also supported the synthesis of several poorly resolved RNAs (lane 1) that were nuclease-sensitive and corresponded in size to mRNAs made in vitro by activated virions (lane 3). The total RNAs made in the cell-free system were also examined by electrophoresis on low pH agarose gels containing 6 M urea (Wertz and Davis, 1979). The relative migration rates of the positive and negative strands of the eleven genome segments of rotavirus SAll on agarose-urea gels have been determined (Patton and Stacy-Phipps, 1986). Analysis of total RNA product on agarose-urea gels showed that approximately 25 different RNAs were synthesized by the cell-free system (Fig. 3, lane 3). Products co~esponding in size to all eleven viral positive- and negativestrand RNAs were detected. The lack of exact co-ovation between newly synthesized RNAs (lane 3) and marker RNAs derived from virions {lane 2) in regions of the gel containing segments 1 to 4 and 6 was due to the presence of large amounts of ribosomal RNA which also migrated to these regions (data not shown). Several species of RNAs made in reactions containing subviral particles (lane 3) or activated virions (lane 1) did not co-migrate with any of the positive or negative strands of the eleven genome segments (lane 2). The origin of these RNAs is unknown. Examination of the nuclease-resistant RNA products of the system by electrophoresis on agarose-urea gels indicated that ten or eleven nuclease-resistant RNAs
223
Fig. 2. ~ectrophoretic analysis of RNA products. After incubation, a 15 ~1 portion of a reaction treated with micrococcal nuclease and an untreated portion (8 ~1) were each deprote~~d by phenol extraction. The ‘H-labeled RNA products were then analyzed by electrophoresis on a 10% polyacryiamide gel containing SDS. The positions of the eleven genome segments of rotavirus SAll are labeled. Total (lane 1) and nuclease-resistant (lane 2) RNA products of the cell-free s&tern; mRNA made in vitro by activated virions (lane 3).
were made in vitro (Fig. 3, lane 6). Each of the nuclease-resistant RNA products comigrated with one of the rotavirus negative-strand RNAs. (The uncertainty in the number of nuclease-resistant RNA products stems from the co-migration of the negative strands of genome segments 7 and 8 on agarose-urea gels; Patton and Stacy-Phipps, 1986). No newly synthesized nuclease-resistant positive-strand RNAs that were full-length could be detected. Together, these data show that the cell-free system supports the synthesis of full-length viral positive- and negative-strand RNA and, therefore, supports rotavirus RNA replication and transcription. However, these results also indicate that although the system promotes the synthesis of rotavirus dsRNA, it is only the synthesis of the negative strand of this product that can be detected. Effect of protein synthesis on replication in vitro The effect of protein synthesis on the production of RNA in the cell-free system was analyzed by comparing RNA products made in an unmodified reaction with products made in reactions either containing anisomycin and cycloheximide or
224
Fig. 3. Agarose-urea gel electrophoresis of RNA prepared from reactions as described in Materials 1.75% agarose gel containing 6 M urea, pH 3.0, negative-strand for each of the genome segments is mRNA made in vitro by activated Cons (lane 1); with [ 3H]uridine (lane 2). ‘H-labeled total and unmodified reaction (lanes 3 and 6), in a reaction mide (lanes 4 and 7), and in a reaction containing
products. Total and nuclease-resistant RNAs were and Methods and analyzed by electrophoresis on a and fluorography. The position of the positive- and indicated (Patton and Stacy-Phipps, 1986). 3H-labeled virion dsRNA derived from cells continuously labeled nuclease-resistant RNA, respectively, made in an containing 40 pg/ml each anisomycin and cyclohexino reticulocyte lysate (lanes 5 and 8).
containing no reticulocyte lysate. Protein synthesis was inhibited in the reaction containing anisomycin and cycloheximide by 95% (data not shown). The 3H-labeled total and nuclease-resistant RNA products of the system were examined by electrophoresis on agarose-urea gels and fluorography. As shown in Fig. 3, the polarity (negative-strand RNA), number and sizes of nuclease-resistant RNA products were the same for the unmodified reaction (lane 6) the reaction containing protein synthesis inhibitors (lane 7), and the reaction lacking reticulocyte lysate (lane 8). Thus, negative-strand RNA synthesis in the system was not dependent on protein synthesis or the presence of the reticulocyte lysate. No difference was detected in the 3H-labeled total RNAs made in the unmodified reaction (Fig. 3, lane 3) and in the reaction containing protein synthesis
225 inhibitors (lane 4). However, the products of these reactions included several RNAs that were not present in the total products of a reaction lacking reticulocyte lysate (lane 5). These RNAs did not corn&rate on agarose-urea gels with either the positive- or negative-strand RNAs of any of the eleven genome segments (lane 2) and, based on their absence in the nuclease-resistant product (lanes 6 and 7), were single-stranded in nature. Similar RNAs were synthesized in reactions that contained activated virions instead of subviral particles (lane 1). ~uc~effse-se~si?ivi~
of the template for dsRNA synthesis
Previous studies have shown that the template for reovirus dsRNA synthesis is a positive-strand RNA (Acs et al., 1971; Sakuma and Watanabe, 1971). To examine
Fig. 4. Sensitivity of template for RNA replication to nuclease digestion. Nuclease-treated subviral particles were prepared by treating a nuclei-free lysate of infected cells with micrococcal nuclease in the presence of CaCl,. After incubakon for 10 min at 23*C, EGTA was added and the subvirai particles recovered from the lysate by centrifugation through a 15-308 sucrose gradient. Total (lane 1) and n&ease-resistant (lane 3) RNA products of a reaction containing untreated subviral particles. Total (lane 2) and nucfease-resistant (lane 4) RNA products of a reaction containing nuclease-treated subviral particles.
226 the nature of the template for rotavirus dsRNA synthesis, subviral particles were treated with micrococcal nuclease before addition to the cell-free system to remove ssRNAs that might serve as templates for RNA replication. The total and nucleaseresistant RNA products from a reaction containing nuclease-treated subviral particles and from a control reaction containing untreated particles were then analyzed by electrophoresis on a 1.75% agarose-urea gel (Fig. 4). Treated particles did not support the synthesis of nuclease-resistant RNA (lane 4); however, the same particles did support the synthesis of rotavirus positive-strand RNAs (lane 2). These data show that the template for rotavirus RNA replication, i.e. negative-strand RNA synthesis, is a nuclease-sensitive, positive-strand RNA. In addition, since nuclease treatment should destroy any nascent ssRNAs associated with subviral particles, the synthesis of full-sized positive-strand RNA by treated particles (lane 2) provides evidence that the cell-free system supported both the initiation and complete elongation of positive-strand RNA.
Synthesis of dsRNA in association with subviral particles To determine whether the nuclease-resistant RNA produced by the cell-free system remained associated with subviral particles upon its synthesis, a portion of a reaction was layered onto a 15-30% sucrose gradient formed over a cushion of saturated sucrose. After centrifugation, fractions from the gradient were assayed for
FRACTION
Fig. 5. Association of dsRNA product with subviral particles. A 22 ~1 portion of a reaction was diluted to 1 ml with TMN buffer and layered onto a 3 ml gradient of 15-30% sucrose in TMN buffer formed over a 1 ml cushion of saturated sucrose. After centrifugation for 2 h at 200000 x g in a Beckman SW50.1 rotor at 4’C, 0.2 ml fractions from the gradient were collected and 25 ~1 of each assayed for acid-precipitable 3H-labeled RNA product (0). To determine the % RNase resistant product, samples (25 ~1) of a fraction were placed into each of two tubes containing 1 ml of 0.3 M NaCI, 0.15 M Tris-HCl, pH 7.4, and 3 mM EDTA. After addition of 10 pg/ml RNase A to one sample, both were incubated for 30 min at 37OC. Samples were adjusted to 0.5% SDS and 20 pg/ml bovine serum albumin and assayed for acid precipitable RNA product. % RNase resistant = (cpm of rmclease-resistant RNA product/cpm of total RNA product) X 100.
227 3H-labeled total and nuclease-resistant RNA. As shown in Fig. 5, two major peaks of radiolabeled total RNA were detected. One peak was found at the interface above the sucrose cushion (fractions 4-5); this also corresponds to the position at which single-shelled and double-shelled virus and subviral particles with associated polymerase activity sediment in these gradients (fractions 4-5) (data not shown). The second peak of RNA product was found at an intermediate position in the gradient (fractions 12-13); this position corresponds to the position at which purified viral mRNA and dsRNA sediment. Analysis of the peak fractions of the RNA product by nuclease digestion showed that appro~mately 40% of the radiolabeled material at the interface was nucleaseresistant. In contrast, less than 5% of the RNA product that constituted the siower migrating peak (fractions 12-13) was nuclease-resistant. Thus, after its synthesis, dsRNA product in the cell-free system remained associated with subviral particles.
123456
Fig. 6. Initiation of negative-strand RNA. 3H-labeled mRNA made in vitro by activated Cons was purified by phenol extraction and added to four parallel reactions containing no [3H]UTP. After incubation, each reaction was treated with 20 pg/ml of micrococcal nuclease to remove ssRNAs. Nuclease-resistant RNAs were then recovered by phenol extraction and ethanol precipitation and analyzed by electrophoresis on a 10% polyacrylamide gel containing SDS and fluorography. Lane 1: 3H-labeled dsRNA purified from virions; lane 2: nuclease-resistant RNA products made in an unmodified reaction containing [3H]UTP; lanes 3-6: 3H-labeled nuclease-resistant RNAs recovered from reactions containing 50000 cpm/reaction of ‘H-labeled mRNA but no 13H]UTP. Reactions containing reticulocyte lysate with (lane 3) and without (lane 4) subviral particles; reactions containing no reticulocyte lysate with (lane 5) and without (lane 6) subviral particles.
228 rniti~tion of lenitive-strong RNA in vitro Analysis of dsRNA product showed that the system supported the synthesis of full-length negative-strand RNA. To test the ability of the system to support the initiation of negative-strand RNA, rotavirus 3H-labeled mRNA was added to reactions containing no added radiolabeled nucleotides. The products were then analyzed by polyacrylamide gel electrophoresis for 3H-labeled dsRNA product; its presence would indicate that the exogenous positive-strand RNA acted as a template for the initiation and elongation of negative-strand RNA in vitro. The results are shown in Fig. 6. The radiolabeled products of a reaction containing subviral 3H-labeled mRNA but no [ 3H]UTP included reticulocyte lysate, particles, nuclease-resistant radiolabeled RNAs (lane 3) that comigrated with v&ion-derived dsRNAs (lane 1). Hence, in addition to elongation, the system supported the initiation of negative-strand RNA. The conversion of exogenous mRNA to dsRNA required subviral particles; 3H-labeled nuclease-resistant RNA was not detected in reactions lacking these particles (lane 4). Reactions that lacked reticulocyte lysate, regardless of the presence of subviral particles, also were unable to support the conversion of positive-strand RNA into dsRNA (lanes 5 and 6). Synthesis of viral proteins in vitro To examine the composition of 35S-labeled products made in the cell-free system, samples were taken from reactions during incubation and analyzed by electrophore-
Fig. 7. Synthesis of viral proteins in vitro. 1 ~1 samples were removed during incubation from a reaction containing [35S]methionine and subjected to electrophoresis on a 12% polyacrylamide gel containing SDS. The gel was processed for fluorography and exposed to Kodak XAR-5 film. Cytoplasmic lysate (lane 1) and subviral particles (lane 2) prepared at 6 h pi. from infected cells continuously labeled with [35S]methionine. Protein products in samples taken from reactions at 0 (lane 3), 15 (lane 4), 30 (lane 5), 60 (lane 61, and 90 (lane 7) min post incubation. Structural and nonstructural proteins as reported by Petrie et al. (1984) are indicated.
229
sis on 12% polyacqlamide gels and fluorography. Cytoplasmic lysate (lane 1) and subviral particles (lane 2) prepared from infected cells continuously labeled with [3”S]methionine were electrophoresed in parallel lanes with the samples as protein markers (Fig. 7). In agreement with Fig, 1, the synthesis of protein in reactions was greatest during the first 30 min of incubation (lanes 3-5); the maximal level of radiolabcled protein product was reached by 60 min post incubation (lane 6). The structural viral protein products identified in reactions included VP1 (125K), VP2 (94K& VP3 (88K) and VP6 (41K); the nonstructural viral protein products included NS53 (53K), NS35, NS34 and NS29 (Petrie et al., 1984).
The synthesis of radiolabeled dsRNA in vitro using exogenous ‘H-labeled mRNA as a template showed that viral protein is associated with the positive-strand RNA. This suggested that the system supported the assembly of subviral particles with replicase activity. To examine the possibility that subviral particles were formed in vitro from 35S-labeled protein products, after incubation particles were
Fraction Fig. 8. Equilibrium density gradient centrifugation
of protein products. A 20 ~1 sample of a reaction containme, [“5S]methionine was diluted to 2 ml with hypotonic buffer and centrifuged through a 3 ml 1%30% sucrose gradient as described in Materials and Methods. The pellet was resuspended in 25 ~1 HGD buffer, diluted to 1 ml in TMN buffer and layered onto a 4 ml gradient of 20-45s CsCl (w/w) and centrifuged at iZOooOX g for 17 b at 4OC in a Beckman SW50.1 rotor (upper panel). A parallel gradient contained subviral particles labeled in viva with [‘sS]methionine (lower panel). Fractions (ct.2 ml) from the gradients were assayed for density (A) and for acid-~rec~p~table product in 20 ~1 {e)_ The pellet of the gradient was resuspended in 0.2 ml of TMN buffer and a 25 gl portion assayed for radioactivity (I).
230 recovered from a reaction by cent~ifn~atio~ through a 15-30X sucrose gradient and tested for the presence of newly synthesized protein. Approximately 25% of the acid-precipitable 35S-labeled material detected in the gradient was faund in the pellet, which contains subviral particles. The pellet was resuspended and, in parallel with subviral particles labeled in vivo with [35S]methionine; ce~t~i~~ged to equilibrium on 20-45% CsCl gradients. Fractions collected from the gradients were assayed for density and acid-precipitable radiolabeled material (Fig. 8). The majority of the ‘“S-labeled material derived from the c&-free system banded at densities in the gradient between 1.18 and 1.38 gfrnr of CsCf with a peak at 1.25 @ml (upper panel). Most of the rad~o~abe~ed rnate~~ in the subviral particles prepared from infected cells (lower panel) banded near 1.24 g/ml of CsCf with minor peaks of material found at 1.18 and 1.34,gJmk Studies have indicated that subviral particles with associated tranxxiptase activity when centrifuged on CsCl gradients band primarily at a density of 1.38 g/ml
231
and consist of large amounts of the viral proteins VP2 and VP6 with lesser amounts of VPl, NS53, NS35 and NS34 protein (Helmberger-Jones and Patton, manuscript in preparation). The pellets of these gradients are enriched for particles with replicase activity and contain large amounts of VP2 and NS34 protein and lesser amounts of VPI, VP6, NS53, and NS35 protein. The possibility that similar particles were assembled in vitro from newly made protein was examined by electrophoretic analysis of radiolabeled proteins in fractions obtained by centrifugation of reaction mixtures on CsCl gradients. As found for particles labeled in vivo with [3SS]-methionine (Fig. 9, lower panel), the products of the cell-free system included material that banded at a density of 1.38 g/ml which consisted of newly made VP2 and VP6 protein and lesser amounts of VPl, NS53, NS35 and NS34 protein (upper panel, lane 21). The pellet of gradients cont~ning products of the system included a large mount of NS34 and lesser amounts of VPl, VP2, VP6, NS53 and NS3.5 proteins (upper panel, lane P). Several other fractions of the CsCl gradient also contained newly made proteins similar to proteins found at the same density in gradients of subviral particles labeled in infected cells. These included fractions 11 (density 1.20 g/ml), 13 (1.24 g/ml) and 19 (1.34 g/ml). Thus, proteins synthesized in the cell-free system associated into structures that were similar to subviral particles present in infected cells.
Discussion
A cell-free system was developed that supports several processes that occur during the replication of the rotaviruses. The system contained subviral particles prepared at 6 h p-i. from cells infected with simian rotavirus SAll and an mRNA-dependent rabbit reticulocyte lysate. Characterization of the system showed that the following processes were supported in vitro: (i) the synthesis by subviral particles of eleven dsRNAs identical in size to those derived from virions, (ii) the initiation and elongation of both negative- and positive-strand RNA into full-sized product, and (iii) the assembly of newly made protein into complexes similar to subviral particles found in infected cells. The ability of the system to support the initiation of negative-strand RNA using exogenous positive-strand RNA as a template provides a method to study the specificity of viral proteins in recognition and replication of rotavirus mRNAs. Treatment of subviral particles with micrococcal nuclease prevented the synthesis of negative-strand RNA. This shows that rotavirus RNA replication, like that of the reoviruses (Sakuma and Watanabe, 1971; Acs et al., 1971), is an asymmetrical process whereby a positive-strand RNA acts as a template for the synthesis of a complementary, negative-strand RNA to produce dsRNA. The exact nature of the particle in which dsRNA synthesis occurs is not known. Results obtained to date indicate that particles with associated replicase activity (replicase particles), like those with transcriptase activity, consist of the core proteins VP1 and VP2. In addition, the replicase particles appear to contain smaller amounts of the inner shell protein, VP6, and greater amounts of the nonstructural protein, NS34, relative to
232 the transcriptase particles (Helmberger-Jones and Patton, manuscript submitted). The susceptibility of the positive-strand RNA in replicase particles to nuclease digestion provides evidence that dsRNA synthesis does not occur within a tightly enclosed protein structure. This agrees with the results of similar studies by Acs et al. (1971) for reovirus. The fact that most of the dsRNA made in the cell-free system co-migrated with subviral particles in sucrose gradients indicates that, also like reovirus (Acs et al., 1971; Zweerink et al., 1972), rotavirus dsRNA remains associated with subviral particles upon its synthesis. The initiation of negative-strand RNA using exogenous positive-strand RNA as a template demonstrates that the cell-free system supported the assembly of a ~bonu~leoprot~in complex which had associated replicase activity. This process occurred only in reactions containing subviral particles and reticulocyte lysate; no initiation was detected in reactions containing subviral particles but lacking reticulocyte lysate. These results provide evidence that initiation was dependent on protein synthesis in the cell-free system. The inability of reactions containing reticulocyte lysate and no subviral particles to convert mRNA to dsRNA suggests that particles were required for initiation of negative-strand RNA. However, because the overall level of protein synthesis was several-fold higher in reactions containing subviral particles than in those without (data not shown), it is possible that the level of protein synthesis in reactions containing only mRNA and reticulocyte lysate was insuffjc~ent to support the initiation of negative-strand RNA at detectable levels. The elongation of negative-strand RNA in vitro to give rise to full-length dsRNA occurred both in reactions containing inhibitors of protein synthesis and in reactions containing no reticulocyte lysate. Although initiation of negative-strand RNA may require protein synthesis, these data suggest that the elongation of nascent negative-strand RNAs associated with subviral particles does not. This is consistent with the results of several studies where the in vitro synthesis of reovirus dsRNA was accomplished in the absence of newly made protein or added soluble protein (Acs et al., 1971; Sakuma and Watanabe, 1971; 1972; Zweerink et al., 1972). In summary, a cell-free system has been developed for simian rotavirus SAl 1 that supports the asy~et~ca~ RNA replication and the assembly of functional subviral particles. This system should prove useful in describing the mo~hogenesis of the rotaviruses by providing a method for assaying the different types of subviral particles in infected ceIIs for associated replicase and transcriptase activity.
Acknowledgements Special thanks to Nancy L. Davis for critical reading of this manuscript. This work was supported by Public Health Service grant AI21478 from the National Institutes of Health, Biomedical Research Support Grant No. RR07121 from the National Institutes of Health and a grant from the LJSF Research and Creative Scholarship Program.
233 References Acs, G., Klett, H., Schonberg, M., Christman, J., Levin, D.H. and Silverstein, SC. (1971) Mechanism of reovirus double-stranded ribonucleic acid synthesis in vivo and in vitro. J. Virol. 8, 684-689. Altenburg, B.C., Graham, D.Y. and Estes, M.K. (1980) Ultrastructural study of rotavirus replication in cultures cells. J. Gen. Virol. 46, 75-85. Chasey, D. (1977) Different particle types in tissue culture and intestinal epithelium infected with rotavirus. J. Gen. Virol. 37, 443-451. Cohen, J. (1977) Ribonucleic acid polymerase activity associated with purified calf rotavirus. J. Gen. Virol. 36, 395-402. Cohen, J., Laporte, J., Charpilienne, A. and Scherrer, R. (1979) Activation of rotavirus RNA polymerase by calcium chelation. Arch. Virol. 60, 177-186. Davis, N.L. and Wertz, G.W. (1982) Synthesis of vesicular stomatitis virus negative-strand RNA in vitro: dependence on viral protein synthesis. J. Virol. 41, 821-832. Ericson, B.L., Graham, D.Y., Mason, B.B. and Estes, M.K. (1982) Identification, synthesis, and modifications of simian rotavirus SAll polypeptides in infected cells. J. Virol. 42, 825-839. Espejo, R.T., Lopez, S. and Arias, C. (1981) Structural polypeptides of simian rotavirus SAll and the effect of trypsin. J. Virol. 37, 156-160. Estes, M.K.. Graham, D.Y., Gerba, C.P. and Smith, E.M. (1979) Simian rotavirus SAll replication in cell cultures. J. Virol. 31, 810-815. Estes, M.K., Graham, D.Y. and Mason, B.B. (1981) Proteolytic enhancement of rotavirus infectivity: molecular mechanisms. J. Virol. 39, 879-888. Estes, M.K., Palmer, E.L. and Obijeski, J.F. (1983) Rotaviruses: A review. In: Current Topics in Microbiology and Immunology, Vol. 105 (Cooper, M. et al., eds.), pp. 123-184. Springer-Verlag, Berlin. Flores, J., Myslinski, J., Kalica, A.R., Greenberg, H.B., Wyatt, R.G., Kapikian, A.Z. and Chanock, R.M. (1982) In vitro transcription of two human rotavimses. J. Virol. 43, 1032-1037. Imai, M., Kantani, K.A., Ikegami, N. and Fumichi, Y. (1983) Capped and conserved terminal structures in human rotavirus genome double-stranded RNA segments. J. Virol. 47, 125-136. Kalica, A.R., Flores, J. and Greenberg, H.B. (1983) Identification of the rotaviral gene that codes for hemagglutination and protease-enhanced plaque formation. Virology 125, 194-205. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 277, 685. Mason, B.B., Graham, D.Y. and Estes, M.K. (1980) In vitro transcription and translation of simian rotavirus SAll gene products. J. Virol. 33, 1111-1121. McCrae, M.A. and McCorquodale, J.G. (1983) Molecular biology of rotaviruses. V. Terminal structure of viral RNA species. Virology 126, 204-212. McCrae, M.A. and Faulkner-Valle, G.P. (1981) Molecular biology of rotaviruses. I. Characterization of basic growth parameters and pattern of macromolecular synthesis. J. Virol. 39, 490-496. Patton, J.T. and Stacy-Phipps, S. (1986) Electrophoretic separation of the plus and minus strands of rotavirus SAll double-stranded RNAs. J. Virol. Methods (in press). Petrie, B.L., Graham, D.Y. and Estes, M.K. (1981) Identification of rotavirus particle types. Intervirology 16, 20-28. Petrie, B.L., Greenberg, H.B., Graham, D.Y. and Estes, M.K. (1984) Ultrastructural localization of rotavirus antigens using colloidal gold. Virus Res. 1, 133-152. Sakuma, S. and Watanabe, Y. (1971) Unilateral synthesis of reovirus double-stranded ribonucleic acid by a cell-free replicase system. J. Virol. 8, 190-196. Sakuma, S.V. and Watanabe, Y. (1972) Reovints replicase-directed synthesis of double-stranded ribonucleic acid. J. Virol. 10, 628-638. Suzuki, H., Konno, T., Kitaoka, S., Sato, T., Ebina, T. and Ishida, N. (1984) Further observations on the morphogenesis of human rotavirus in MA104 cells. Arch. Virol. 79, 147-159. Wertz, G.W. and Davis, N.L. (1979) RNase III cleaves vesicular stomatitis virus genome-length RNAs but fails to cleave viral mRNAs. J. Virol. 30, 108-115. Zweerink, H.J., Ito, Y. and Matsuhisa, T. (1972) Synthesis of reovirus double-stranded RNA within virion-like particles. Virology 50, 349-358. (Manuscript
received
14 January
1986)
217
217-233
VRR 00300
Synthesis of simian rotavirus SA11 double-stranded RNA in a cell-free system John T. Patton Department
of Biology, University of South Florida, Tampa, FL 336220, U.S.A.
(Accepted
for,publication
1 August
1986)
Summary
A cell-free system was developed to study the replication of simian rotavirus SAll. The components of the system included (i) subviral particles prepared from infected cells to template the synthesis of viral RNA and (ii) an mRNA-dependent rabbit reticulocyte lysate to support protein synthesis. Based upon nuclease-sensitivity, approximately 20% of the RNA made in vitro was double-stranded (dsRNA) and 80% single-stranded (ssRNA). Electrophoretic analysis of the RNA products on polyacrylamide and low pH agarose gels showed that the system supported the synthesis of 11 dsRNAs and 11 positive-sense ssRNAs that corresponded in size to authentic viral RNAs. The synthesis of dsRNA in vitro was determined to be an asymmetrical process in which a nuclease-sensitive positive-strand RNA acted as a template for the synthesis of negative-strand RNA. The system also supported the initiation of negative-strand RNA using exogenous viral positive-strand RNA as a template. Finally, analysis of subviral particles recovered from reactions suggested that viral proteins made in vitro assembled into nucleoprotein complexes which were similar to those present in infected cells. Together, these results indicate that the cell-free system supported rotavirus RNA replication, transcription and the assembly of subviral particles. simian rotavirus SAll, cell-free replication system, negative-strand
RNA
The genome of the rotaviruses, members of the family Reoviridae, consists of eleven segments of double-stranded RNA (dsRNA) ranging in size from 0.4 to 2.0 x lo6 Da (see for review Estes et al., 1983). The positive-strand RNA present in 0168-1702/84/~03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
each segment is indistinguishable from viral mRNA; both contain 5’“terminal cap structures but lack 3’-terminal polydenylate sequences (Imai et al., 1983; McCrae and McCorquodale, 1983). Rotaviruses have a virion-associated RNA polymerase (transcriptase) capable of synthesizing viral mRNAs in vitro (Cohen, 1977; Mason et al., 1980; Flares et al., 1982). The virion consists of two icosahedral layers of protein (Petrie et al., 1981). For simian rotavirus SAll, the inner shell is made up of a core consisting of the viral proteins VP1 (125 kDa, 12X) and VP2 (94K) surrounded by the major inner shell protein VP6 (41K) (Ericson et al., 1982). The components of the outer shell include a 38K gly~oprotein fVP7) and a protease-sensitive 88K protein (VP31 (Espejo et al., 1981; Estes et al., 1981; Kalica et al., 19X3). Several virally induced nonstructural proteins are present in infected cells (Ericson et al., 1982); however, their role in virus replication is not certain. Examination of rotavirus-infected cells by electron microscopy indicates that viral replication takes place in the cytoplasm (Chasey, 1977). Numerous types of subviral particles have been identified in infected cells (Chasey, 1977; Altenburg et al., 1980; Petrie et al., 1981; Suzuki et al., 1984). These include (i) core particles in lysosomes, (ii) particles on the surface of the rough endoplasmic reticulum (RER), (iii) single-shelled particles (60-65 nm) in the cytoplasm and cisternae of the RER, (iv> membr~e-buund particles within the RER, and (v) double-shelled particles (70-75 nm> in the RER cistemae. It is not known which of these particles, if any, is responsible for the production of viral dsRNA and mRNA in infected cells. A method for assaying subviral particles for associated replicase (negative-strand RNA synthesis) or transcriptase (positive-strand RNA synthesis) activity would be useful for characterizing their role in infected cells. Towards that end, a cell-free system has been developed that supports the synthesis of rotavirus dsRNA, mRNA and protein in vitro. This system, modeled after a system developed by Davis and Wertz (1982) for vesicular stomatitis virus, consists of subviral particles isolated from rotavirus SAll-infected cells and an n-RNA-dependent rabbit reticulocyte lysate. As described in this report, the system supports the asymmetrical replication of rotavirus RNA including the initiation and elongation of viral negative-strand RNA.
Materials and Metltods Cell culture and virus
Monolayers of fetal rhesus monkey kidney cells (MA104) were grown in Eagle minimal essential media (MEM) containing 5% fetal calf serum and 5% newborn calf serum. Stock preparations of simian rotavirus SAll were propagated in MA104 cells infected with plaque-purified virus at a multiplicity of infection (m.o.i.) of < 0.01. Infected cultures were maintained in MEM without serum but containing 10 ,ug/ml trypsin (Difco 1: 250) until SO-90% of cells showed cytopathic effects (CFE; 5-7 days post infection, p-i.> (Mason et al., 1980). The cultures were then freezethawed three times and cellular debris removed by cent~fugatio~ at 12000 x g for
219 10 min at 4°C in a Sorvall SS-34 rotor. The supernatant (virus stock) was titered by plaque assay on MA104 cells and stored at - 70” C. Cells and virus were gifts from M. Sobsey. Virus purification Rotavirus was purified from infected cells essentially as described by Mason et al. (1980). Monolayers of MA104 cells were infected with virus stock at an m.o.i. of < 0.01. When 80-90% of cells exhibited CPE, cultures were freeze-thawed three times and homogenized with an equal volume of t~~hlorot~fluoroet~~e. The homogenate was then centrifuged at low speed until the aqueous and organic phases were separated. The aqueous phase was saved and the organic phase re-extracted with an equal volume of TN buffer (50 mM Tris-HCl, pH 7.5/150 mM NaCI). The aqueous phases were combined, adjusted to 10% polyethylene glycol, and left stirring overnight at 4°C. Virus was then pelleted from the aqueous phases by centrifugation at 65 000 x g for 30 min at 4” C in a Beckman Type 70Ti rotor and resuspended in TN buffer. The virus sample was then loaded onto 4 ml 20-40% linear CsCl gradients (w/w) in TN buffer which were centrifuged at 120000 X g for 18 h at 4°C in a Beckman SW50.1 rotor. Material banding at a density of 1.36 g/ml (double-shelled virions) was dialyzed overnight against TN buffer at 4OC. Preparation of subv~ralporticoes as te~~~ate~ for WA synthesis Intracellular subviral particles were prepared from infected cells by the method described by Davis and Wertz (1982) for vesicular stomatitis virus. Confluent monolayers of MA104 cells in lo-cm tissue culture dishes were infected with virus stock at an m.o.i. of 5-10. At 1 h p.i., the inoculum was removed and replaced with MEM containing 5 pg/ml actinomycin D. Plates were then incubated at 37°C until 6 h p.i. when the media was drawn off and the monolayers washed twice with hypotonic buffer (3 mM Tris-HCl, pH 8.1/0.5 mM MgC1,/3 mM NaCI). Afterwards, cells were scraped into the same buffer, incubated for 10 min on ice, and disrupted using 14 strokes of a Dounce homogenizer. Nuclei and large cellular debris were removed from the cell lysate by centrifugation at 12000 X g for 30 min at 4°C in a Sorvall SS-34 rotor. The supernatant was layered onto 3 ml 15-30% sucrose gradients (w/v) in TMN buffer (3 mM Tris-HCl, pH 8.1/3 mM MgCl,/66 mM NH,Cl/14 mM potassium acetate/l mM dithioerythritol (DTE)) and centrifuged at 200000 x g for 2 h at 4*C in a Beckman SW50.1 rotor. After removal of the supernatant, HGD buffer (10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-HCl, pH 7.6/10% glycerol/2 mM DTE) was placed on the pellets which were then allowed to resuspend by incubation overnight on ice. Subviral particles were resuspended at a final concentration of 5 ~1 of HGD buffer per starting tissue culture dish. Particles were used in reactions immediately or stored at - 70°C until needed. Freezing had no detectable effect on either the level of polymerase activity associated with or RNA products made by subviral particles (data not shown). In some instances, prior to centrifugation on 15-308 sucrose gradients, the nuclei-free supernatants were adjusted to 10 pg,/ml micrococcal nuclease, 50 mM
220 Tris-HCI, pH 8.1, and 1 mM CaCl,, and incubated for 10 n-tin at 23°C. Afterwards, the nuclease activity was inhibited by the addition of ethylene glycol-bis(~-a~noethyl ether)-&‘, N, N’, N’-tetraacetic acid (EGTA) to a concentration of 2 mM. Components of the ceil-free system In vitro reactions were carried out in a final volume of 25 pl which contained by volume 20% subviral-particle preparation and 70% micrococcal nuclease-treated rabbit reticulocyte lysate. Endogenous mRNA present in the reticulocyte lysate (Promega Biotec) was removed immediately prior to use by incubation for 10 min at 21°C in the presence of 30 FM hemin, 50 ,eg/ml creatine phosphokinase, 5.5 mM potassium acetate, 1 mM CaCl, and 10 ~g/ml micrococcal nuclease. The nuclease activity was then blocked by the addition of EGTA to a concentration of 2 mM (Davis and Wertz, 1982). Reactions also contained 50 mM HEPES-HCl, pH 7.7, 10 mM creatine phosphate, 1 mM ATP, 0.6 mM each CTP and GTP, 0.1 mM UTP, 0.05 mM each of the 20 amino acids except for methionine which was 1.25 ,uM, 14 mM potassium acetate, 2 mM DTE, 66 mM NH&l, 2 mM magnesium acetate, 1 pg of rabbit liver tRNA, 10 PCi of [35S]methionine (1110 Ci/mmol, New England Nuclear), and/or 25 PCi of [3H]UTP (40 Ci/mmol, ICN) (Davis and Wertz, 1982). Reactions were incubated for 90 min at 30°C. Incorporation of [ 3H]UMP into RNA and [35S]methionine into protein was assayed by counting radioactivity present in trichloroacetic acid precipitates of 2 pl samples taken from reactions during incubation (Davis and Wertz, 1982). To recover dsRNA product, a 15 ~1 portion of a reaction was adjusted to 2.75 mM CaCl, and 20 pg/ml micrococcal nuclease and incubated for 10 mm at 23°C. EGTA was then added to a concentration of 5 mM. The RNAs from the nucleasetreated portion as well as 5-8 ~1 of an untreated portion (total RNA) of the same reaction were then purified by phenol extraction and ethanol precipitation. Preparation of viral mRNA 3H-labeled viral mRNA was made in vitro by activated double-shelled virions (Cohen et al., 1972). Virus was activated by incubation in the presence of 5 mM EDTA for 20 min at 37OC followed by adjustment to 10 mM magnesium acetate. The transcription reactions contained the same components as described above for the cell-free system except that intracellular subviral particles were replaced with activated virions and no ~35S]met~o~ne was included. RNA was isolated from reactions by phenol extraction and ethanol precipitation. Electrophoresis RNA products were analyzed by electrophoresis on either 1.75% agarose gels containing 6 M urea and 0.025 M citrate buffer, pH 3 (Wertz and Davis, 1979) or 10% polyacrylamide gels containing sodium dodecyl sulfate (SDS) (Laemmli, I970). Agarose-urea gels were run at 150 V until the bromphenol blue dye marker migrated 22 cm, processed for fluorography and exposed to Kodak XAR-5 film (Wertz and Davis, 1979). 35S-labeled proteins were analyzed on 12% polyacrylamide gels containing SDS as described (Laemmli, 1970).
223 Results
Kinetics of RNA and protein synthesis in vitro At 6 h p-i., rotavirus SAll RNA replication, transcription, protein synthesis and virion production have begun in infected cells (Estes et al., 1979; McCrae and Faulkner-Valle, 1982; Ericson et al., 1982). The ratio of viral positive- to negativestrand RNA synthesis at this time is nearly 1: 1. By 12 h p.i., this value changes to approximately 10 : 1 (Stacy-Phipps and Patton, manuscript in preparation). To develop a cell-free system for the study of rotavirus, subviral particles were isolated from infected cells at 6 h p-i. so as to rna~~ze the ratio of particles obtund with replicase activity to transcriptase activity. This avoided the possibility that high levels of transcriptase activity associated with subviral particles isolated later in infection would interfere with the detection of the relatively lesser amounts of associated replicase activity. To prepare subviral particles with associated polymerase activity, rotavirus-infected cells were harvested, swollen in a h~otonic buffer and lysed by Dounce homogenization. Nuclei and large cellular debris were removed from the lysate by centrifugation. Subviral particles were then collected from the lysate by centrifugation through a 15-30% sucrose gradient and resuspended in a 10% glycerol buffer.
Minutes Fig. 1. Kinetics of RNA and protein synthesis. Two identical reactions, one cont~~ng [ 3H]UTP and the other [35S]methionine, were prepared as described in Materials and Methods. During incubation, 2 ~1 samples were removed and assayed for total acid-precipitable 3H- or 35S-labeled material. Another 2 pl sample from the reaction containing [‘H]UTP was diluted to 100 ~1 with 10 mM HEPES/Z mM MgClz/2 mM DTE/66 mM NH,C1/14 mM potassium acetate/O.1 mM EGTA, pH 7.7, adjusted with an additional 5 mM CaCl, and 12.5 kg/ml micrococcal nuclease, and incubated for 15 min at 23°C. EGTA was then added to a concentration of 5 mM and the sample assayed for acid-precipitable radiolabeled material. [ 3HJUMP incorporated into total (0) and nuclease-resistant (0) RNA and [35S]methionine into protein (0) per microliter of reaction mixture.
222 The ability of the purified subviral particles to template the synthesis of RNA in vitro was examined in reactions containing by volume 20% sub~ral-particle preparation and 70% nuclease-treated rabbit reticulocyte lysate. Newly synthesized RNA and protein were labeled with [ 3H]UTP and [ 35S]methionine, respectively. The incorporation of isotope was assayed by acid precipitation of samples taken from reaction mixtures during incubation. As shown in Fig. 1, the majority of 3H-labeled total RNA was made in reactions during the first 30 mm of incubation with synthesis completed by 60 min. Similarly, the synthesis of radiolabeled nuclease-resistant RNA was completed by 60 mm post incubation. Comparison of the amount of total and nuclease-resistant products made after 90 min of incubation indicated that approximately 20% of the total product was nuclease-resistant. The synthesis of 35S-labeled protein in vitro was usually maximal during the first 30 min of incubation and completed by 60 min (Fig. 1). Cell-free synthesis of viral single- and double-stranded RNA The production of 3H-labeled nuclease-resistant RNA in vitro suggested that the system supported the synthesis of rotavirus dsRNA. To test for the presence of dsRNA product, 3H-labeled total and nuclease-resistant RNAs made in the system were purified by phenol extraction and analyzed by electrophoresis on a 10% polyacrylamide gel containing SDS. A fluorograph of the gel is shown in Fig. 2. The position of the eleven viral genome segments in the gel was determined by electrophoresis of virion-derived dsRNA in a parallel lane followed by staining with ethidium bromide (not shown). Electrophoresis of the total RNA products shows that eleven 3H-labeled RNAs were synthesized in vitro that were identical in size to virion-derived dsRNAs (lane 1). These RNAs were resistant to digestion by micrococcal nuclease (lane 2) and, therefore, double-stranded. The system also supported the synthesis of several poorly resolved RNAs (lane 1) that were nuclease-sensitive and corresponded in size to mRNAs made in vitro by activated virions (lane 3). The total RNAs made in the cell-free system were also examined by electrophoresis on low pH agarose gels containing 6 M urea (Wertz and Davis, 1979). The relative migration rates of the positive and negative strands of the eleven genome segments of rotavirus SAll on agarose-urea gels have been determined (Patton and Stacy-Phipps, 1986). Analysis of total RNA product on agarose-urea gels showed that approximately 25 different RNAs were synthesized by the cell-free system (Fig. 3, lane 3). Products co~esponding in size to all eleven viral positive- and negativestrand RNAs were detected. The lack of exact co-ovation between newly synthesized RNAs (lane 3) and marker RNAs derived from virions {lane 2) in regions of the gel containing segments 1 to 4 and 6 was due to the presence of large amounts of ribosomal RNA which also migrated to these regions (data not shown). Several species of RNAs made in reactions containing subviral particles (lane 3) or activated virions (lane 1) did not co-migrate with any of the positive or negative strands of the eleven genome segments (lane 2). The origin of these RNAs is unknown. Examination of the nuclease-resistant RNA products of the system by electrophoresis on agarose-urea gels indicated that ten or eleven nuclease-resistant RNAs
223
Fig. 2. ~ectrophoretic analysis of RNA products. After incubation, a 15 ~1 portion of a reaction treated with micrococcal nuclease and an untreated portion (8 ~1) were each deprote~~d by phenol extraction. The ‘H-labeled RNA products were then analyzed by electrophoresis on a 10% polyacryiamide gel containing SDS. The positions of the eleven genome segments of rotavirus SAll are labeled. Total (lane 1) and nuclease-resistant (lane 2) RNA products of the cell-free s&tern; mRNA made in vitro by activated virions (lane 3).
were made in vitro (Fig. 3, lane 6). Each of the nuclease-resistant RNA products comigrated with one of the rotavirus negative-strand RNAs. (The uncertainty in the number of nuclease-resistant RNA products stems from the co-migration of the negative strands of genome segments 7 and 8 on agarose-urea gels; Patton and Stacy-Phipps, 1986). No newly synthesized nuclease-resistant positive-strand RNAs that were full-length could be detected. Together, these data show that the cell-free system supports the synthesis of full-length viral positive- and negative-strand RNA and, therefore, supports rotavirus RNA replication and transcription. However, these results also indicate that although the system promotes the synthesis of rotavirus dsRNA, it is only the synthesis of the negative strand of this product that can be detected. Effect of protein synthesis on replication in vitro The effect of protein synthesis on the production of RNA in the cell-free system was analyzed by comparing RNA products made in an unmodified reaction with products made in reactions either containing anisomycin and cycloheximide or
224
Fig. 3. Agarose-urea gel electrophoresis of RNA prepared from reactions as described in Materials 1.75% agarose gel containing 6 M urea, pH 3.0, negative-strand for each of the genome segments is mRNA made in vitro by activated Cons (lane 1); with [ 3H]uridine (lane 2). ‘H-labeled total and unmodified reaction (lanes 3 and 6), in a reaction mide (lanes 4 and 7), and in a reaction containing
products. Total and nuclease-resistant RNAs were and Methods and analyzed by electrophoresis on a and fluorography. The position of the positive- and indicated (Patton and Stacy-Phipps, 1986). 3H-labeled virion dsRNA derived from cells continuously labeled nuclease-resistant RNA, respectively, made in an containing 40 pg/ml each anisomycin and cyclohexino reticulocyte lysate (lanes 5 and 8).
containing no reticulocyte lysate. Protein synthesis was inhibited in the reaction containing anisomycin and cycloheximide by 95% (data not shown). The 3H-labeled total and nuclease-resistant RNA products of the system were examined by electrophoresis on agarose-urea gels and fluorography. As shown in Fig. 3, the polarity (negative-strand RNA), number and sizes of nuclease-resistant RNA products were the same for the unmodified reaction (lane 6) the reaction containing protein synthesis inhibitors (lane 7), and the reaction lacking reticulocyte lysate (lane 8). Thus, negative-strand RNA synthesis in the system was not dependent on protein synthesis or the presence of the reticulocyte lysate. No difference was detected in the 3H-labeled total RNAs made in the unmodified reaction (Fig. 3, lane 3) and in the reaction containing protein synthesis
225 inhibitors (lane 4). However, the products of these reactions included several RNAs that were not present in the total products of a reaction lacking reticulocyte lysate (lane 5). These RNAs did not corn&rate on agarose-urea gels with either the positive- or negative-strand RNAs of any of the eleven genome segments (lane 2) and, based on their absence in the nuclease-resistant product (lanes 6 and 7), were single-stranded in nature. Similar RNAs were synthesized in reactions that contained activated virions instead of subviral particles (lane 1). ~uc~effse-se~si?ivi~
of the template for dsRNA synthesis
Previous studies have shown that the template for reovirus dsRNA synthesis is a positive-strand RNA (Acs et al., 1971; Sakuma and Watanabe, 1971). To examine
Fig. 4. Sensitivity of template for RNA replication to nuclease digestion. Nuclease-treated subviral particles were prepared by treating a nuclei-free lysate of infected cells with micrococcal nuclease in the presence of CaCl,. After incubakon for 10 min at 23*C, EGTA was added and the subvirai particles recovered from the lysate by centrifugation through a 15-308 sucrose gradient. Total (lane 1) and n&ease-resistant (lane 3) RNA products of a reaction containing untreated subviral particles. Total (lane 2) and nucfease-resistant (lane 4) RNA products of a reaction containing nuclease-treated subviral particles.
226 the nature of the template for rotavirus dsRNA synthesis, subviral particles were treated with micrococcal nuclease before addition to the cell-free system to remove ssRNAs that might serve as templates for RNA replication. The total and nucleaseresistant RNA products from a reaction containing nuclease-treated subviral particles and from a control reaction containing untreated particles were then analyzed by electrophoresis on a 1.75% agarose-urea gel (Fig. 4). Treated particles did not support the synthesis of nuclease-resistant RNA (lane 4); however, the same particles did support the synthesis of rotavirus positive-strand RNAs (lane 2). These data show that the template for rotavirus RNA replication, i.e. negative-strand RNA synthesis, is a nuclease-sensitive, positive-strand RNA. In addition, since nuclease treatment should destroy any nascent ssRNAs associated with subviral particles, the synthesis of full-sized positive-strand RNA by treated particles (lane 2) provides evidence that the cell-free system supported both the initiation and complete elongation of positive-strand RNA.
Synthesis of dsRNA in association with subviral particles To determine whether the nuclease-resistant RNA produced by the cell-free system remained associated with subviral particles upon its synthesis, a portion of a reaction was layered onto a 15-30% sucrose gradient formed over a cushion of saturated sucrose. After centrifugation, fractions from the gradient were assayed for
FRACTION
Fig. 5. Association of dsRNA product with subviral particles. A 22 ~1 portion of a reaction was diluted to 1 ml with TMN buffer and layered onto a 3 ml gradient of 15-30% sucrose in TMN buffer formed over a 1 ml cushion of saturated sucrose. After centrifugation for 2 h at 200000 x g in a Beckman SW50.1 rotor at 4’C, 0.2 ml fractions from the gradient were collected and 25 ~1 of each assayed for acid-precipitable 3H-labeled RNA product (0). To determine the % RNase resistant product, samples (25 ~1) of a fraction were placed into each of two tubes containing 1 ml of 0.3 M NaCI, 0.15 M Tris-HCl, pH 7.4, and 3 mM EDTA. After addition of 10 pg/ml RNase A to one sample, both were incubated for 30 min at 37OC. Samples were adjusted to 0.5% SDS and 20 pg/ml bovine serum albumin and assayed for acid precipitable RNA product. % RNase resistant = (cpm of rmclease-resistant RNA product/cpm of total RNA product) X 100.
227 3H-labeled total and nuclease-resistant RNA. As shown in Fig. 5, two major peaks of radiolabeled total RNA were detected. One peak was found at the interface above the sucrose cushion (fractions 4-5); this also corresponds to the position at which single-shelled and double-shelled virus and subviral particles with associated polymerase activity sediment in these gradients (fractions 4-5) (data not shown). The second peak of RNA product was found at an intermediate position in the gradient (fractions 12-13); this position corresponds to the position at which purified viral mRNA and dsRNA sediment. Analysis of the peak fractions of the RNA product by nuclease digestion showed that appro~mately 40% of the radiolabeled material at the interface was nucleaseresistant. In contrast, less than 5% of the RNA product that constituted the siower migrating peak (fractions 12-13) was nuclease-resistant. Thus, after its synthesis, dsRNA product in the cell-free system remained associated with subviral particles.
123456
Fig. 6. Initiation of negative-strand RNA. 3H-labeled mRNA made in vitro by activated Cons was purified by phenol extraction and added to four parallel reactions containing no [3H]UTP. After incubation, each reaction was treated with 20 pg/ml of micrococcal nuclease to remove ssRNAs. Nuclease-resistant RNAs were then recovered by phenol extraction and ethanol precipitation and analyzed by electrophoresis on a 10% polyacrylamide gel containing SDS and fluorography. Lane 1: 3H-labeled dsRNA purified from virions; lane 2: nuclease-resistant RNA products made in an unmodified reaction containing [3H]UTP; lanes 3-6: 3H-labeled nuclease-resistant RNAs recovered from reactions containing 50000 cpm/reaction of ‘H-labeled mRNA but no 13H]UTP. Reactions containing reticulocyte lysate with (lane 3) and without (lane 4) subviral particles; reactions containing no reticulocyte lysate with (lane 5) and without (lane 6) subviral particles.
228 rniti~tion of lenitive-strong RNA in vitro Analysis of dsRNA product showed that the system supported the synthesis of full-length negative-strand RNA. To test the ability of the system to support the initiation of negative-strand RNA, rotavirus 3H-labeled mRNA was added to reactions containing no added radiolabeled nucleotides. The products were then analyzed by polyacrylamide gel electrophoresis for 3H-labeled dsRNA product; its presence would indicate that the exogenous positive-strand RNA acted as a template for the initiation and elongation of negative-strand RNA in vitro. The results are shown in Fig. 6. The radiolabeled products of a reaction containing subviral 3H-labeled mRNA but no [ 3H]UTP included reticulocyte lysate, particles, nuclease-resistant radiolabeled RNAs (lane 3) that comigrated with v&ion-derived dsRNAs (lane 1). Hence, in addition to elongation, the system supported the initiation of negative-strand RNA. The conversion of exogenous mRNA to dsRNA required subviral particles; 3H-labeled nuclease-resistant RNA was not detected in reactions lacking these particles (lane 4). Reactions that lacked reticulocyte lysate, regardless of the presence of subviral particles, also were unable to support the conversion of positive-strand RNA into dsRNA (lanes 5 and 6). Synthesis of viral proteins in vitro To examine the composition of 35S-labeled products made in the cell-free system, samples were taken from reactions during incubation and analyzed by electrophore-
Fig. 7. Synthesis of viral proteins in vitro. 1 ~1 samples were removed during incubation from a reaction containing [35S]methionine and subjected to electrophoresis on a 12% polyacrylamide gel containing SDS. The gel was processed for fluorography and exposed to Kodak XAR-5 film. Cytoplasmic lysate (lane 1) and subviral particles (lane 2) prepared at 6 h pi. from infected cells continuously labeled with [35S]methionine. Protein products in samples taken from reactions at 0 (lane 3), 15 (lane 4), 30 (lane 5), 60 (lane 61, and 90 (lane 7) min post incubation. Structural and nonstructural proteins as reported by Petrie et al. (1984) are indicated.
229
sis on 12% polyacqlamide gels and fluorography. Cytoplasmic lysate (lane 1) and subviral particles (lane 2) prepared from infected cells continuously labeled with [3”S]methionine were electrophoresed in parallel lanes with the samples as protein markers (Fig. 7). In agreement with Fig, 1, the synthesis of protein in reactions was greatest during the first 30 min of incubation (lanes 3-5); the maximal level of radiolabcled protein product was reached by 60 min post incubation (lane 6). The structural viral protein products identified in reactions included VP1 (125K), VP2 (94K& VP3 (88K) and VP6 (41K); the nonstructural viral protein products included NS53 (53K), NS35, NS34 and NS29 (Petrie et al., 1984).
The synthesis of radiolabeled dsRNA in vitro using exogenous ‘H-labeled mRNA as a template showed that viral protein is associated with the positive-strand RNA. This suggested that the system supported the assembly of subviral particles with replicase activity. To examine the possibility that subviral particles were formed in vitro from 35S-labeled protein products, after incubation particles were
Fraction Fig. 8. Equilibrium density gradient centrifugation
of protein products. A 20 ~1 sample of a reaction containme, [“5S]methionine was diluted to 2 ml with hypotonic buffer and centrifuged through a 3 ml 1%30% sucrose gradient as described in Materials and Methods. The pellet was resuspended in 25 ~1 HGD buffer, diluted to 1 ml in TMN buffer and layered onto a 4 ml gradient of 20-45s CsCl (w/w) and centrifuged at iZOooOX g for 17 b at 4OC in a Beckman SW50.1 rotor (upper panel). A parallel gradient contained subviral particles labeled in viva with [‘sS]methionine (lower panel). Fractions (ct.2 ml) from the gradients were assayed for density (A) and for acid-~rec~p~table product in 20 ~1 {e)_ The pellet of the gradient was resuspended in 0.2 ml of TMN buffer and a 25 gl portion assayed for radioactivity (I).
230 recovered from a reaction by cent~ifn~atio~ through a 15-30X sucrose gradient and tested for the presence of newly synthesized protein. Approximately 25% of the acid-precipitable 35S-labeled material detected in the gradient was faund in the pellet, which contains subviral particles. The pellet was resuspended and, in parallel with subviral particles labeled in vivo with [35S]methionine; ce~t~i~~ged to equilibrium on 20-45% CsCl gradients. Fractions collected from the gradients were assayed for density and acid-precipitable radiolabeled material (Fig. 8). The majority of the ‘“S-labeled material derived from the c&-free system banded at densities in the gradient between 1.18 and 1.38 gfrnr of CsCf with a peak at 1.25 @ml (upper panel). Most of the rad~o~abe~ed rnate~~ in the subviral particles prepared from infected cells (lower panel) banded near 1.24 g/ml of CsCf with minor peaks of material found at 1.18 and 1.34,gJmk Studies have indicated that subviral particles with associated tranxxiptase activity when centrifuged on CsCl gradients band primarily at a density of 1.38 g/ml
231
and consist of large amounts of the viral proteins VP2 and VP6 with lesser amounts of VPl, NS53, NS35 and NS34 protein (Helmberger-Jones and Patton, manuscript in preparation). The pellets of these gradients are enriched for particles with replicase activity and contain large amounts of VP2 and NS34 protein and lesser amounts of VPI, VP6, NS53, and NS35 protein. The possibility that similar particles were assembled in vitro from newly made protein was examined by electrophoretic analysis of radiolabeled proteins in fractions obtained by centrifugation of reaction mixtures on CsCl gradients. As found for particles labeled in vivo with [3SS]-methionine (Fig. 9, lower panel), the products of the cell-free system included material that banded at a density of 1.38 g/ml which consisted of newly made VP2 and VP6 protein and lesser amounts of VPl, NS53, NS35 and NS34 protein (upper panel, lane 21). The pellet of gradients cont~ning products of the system included a large mount of NS34 and lesser amounts of VPl, VP2, VP6, NS53 and NS3.5 proteins (upper panel, lane P). Several other fractions of the CsCl gradient also contained newly made proteins similar to proteins found at the same density in gradients of subviral particles labeled in infected cells. These included fractions 11 (density 1.20 g/ml), 13 (1.24 g/ml) and 19 (1.34 g/ml). Thus, proteins synthesized in the cell-free system associated into structures that were similar to subviral particles present in infected cells.
Discussion
A cell-free system was developed that supports several processes that occur during the replication of the rotaviruses. The system contained subviral particles prepared at 6 h p-i. from cells infected with simian rotavirus SAll and an mRNA-dependent rabbit reticulocyte lysate. Characterization of the system showed that the following processes were supported in vitro: (i) the synthesis by subviral particles of eleven dsRNAs identical in size to those derived from virions, (ii) the initiation and elongation of both negative- and positive-strand RNA into full-sized product, and (iii) the assembly of newly made protein into complexes similar to subviral particles found in infected cells. The ability of the system to support the initiation of negative-strand RNA using exogenous positive-strand RNA as a template provides a method to study the specificity of viral proteins in recognition and replication of rotavirus mRNAs. Treatment of subviral particles with micrococcal nuclease prevented the synthesis of negative-strand RNA. This shows that rotavirus RNA replication, like that of the reoviruses (Sakuma and Watanabe, 1971; Acs et al., 1971), is an asymmetrical process whereby a positive-strand RNA acts as a template for the synthesis of a complementary, negative-strand RNA to produce dsRNA. The exact nature of the particle in which dsRNA synthesis occurs is not known. Results obtained to date indicate that particles with associated replicase activity (replicase particles), like those with transcriptase activity, consist of the core proteins VP1 and VP2. In addition, the replicase particles appear to contain smaller amounts of the inner shell protein, VP6, and greater amounts of the nonstructural protein, NS34, relative to
232 the transcriptase particles (Helmberger-Jones and Patton, manuscript submitted). The susceptibility of the positive-strand RNA in replicase particles to nuclease digestion provides evidence that dsRNA synthesis does not occur within a tightly enclosed protein structure. This agrees with the results of similar studies by Acs et al. (1971) for reovirus. The fact that most of the dsRNA made in the cell-free system co-migrated with subviral particles in sucrose gradients indicates that, also like reovirus (Acs et al., 1971; Zweerink et al., 1972), rotavirus dsRNA remains associated with subviral particles upon its synthesis. The initiation of negative-strand RNA using exogenous positive-strand RNA as a template demonstrates that the cell-free system supported the assembly of a ~bonu~leoprot~in complex which had associated replicase activity. This process occurred only in reactions containing subviral particles and reticulocyte lysate; no initiation was detected in reactions containing subviral particles but lacking reticulocyte lysate. These results provide evidence that initiation was dependent on protein synthesis in the cell-free system. The inability of reactions containing reticulocyte lysate and no subviral particles to convert mRNA to dsRNA suggests that particles were required for initiation of negative-strand RNA. However, because the overall level of protein synthesis was several-fold higher in reactions containing subviral particles than in those without (data not shown), it is possible that the level of protein synthesis in reactions containing only mRNA and reticulocyte lysate was insuffjc~ent to support the initiation of negative-strand RNA at detectable levels. The elongation of negative-strand RNA in vitro to give rise to full-length dsRNA occurred both in reactions containing inhibitors of protein synthesis and in reactions containing no reticulocyte lysate. Although initiation of negative-strand RNA may require protein synthesis, these data suggest that the elongation of nascent negative-strand RNAs associated with subviral particles does not. This is consistent with the results of several studies where the in vitro synthesis of reovirus dsRNA was accomplished in the absence of newly made protein or added soluble protein (Acs et al., 1971; Sakuma and Watanabe, 1971; 1972; Zweerink et al., 1972). In summary, a cell-free system has been developed for simian rotavirus SAl 1 that supports the asy~et~ca~ RNA replication and the assembly of functional subviral particles. This system should prove useful in describing the mo~hogenesis of the rotaviruses by providing a method for assaying the different types of subviral particles in infected ceIIs for associated replicase and transcriptase activity.
Acknowledgements Special thanks to Nancy L. Davis for critical reading of this manuscript. This work was supported by Public Health Service grant AI21478 from the National Institutes of Health, Biomedical Research Support Grant No. RR07121 from the National Institutes of Health and a grant from the LJSF Research and Creative Scholarship Program.
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