Solubilization and Immunoprecipitation of Alphavirus Replication Complexes DAVID J

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JOURNAL OF VIROLOGY, Mar. 1991, p. 1496-1506 Vol. 65, No. 3 0022-538X/91/031496-11$02.00/0 Copyright © 1991, American Society for Microbiology Solubilization and Immunoprecipitation of Alphavirus Replication Complexes DAVID J. BARTON, STANLEY G. SAWICKI, AND DOROTHEA L. SAWICKI* Department of Microbiology, Medical College of Ohio, Toledo, Ohio 43699 Received 24 September 1990/Accepted 11 December 1990

Alphavirus replication complexes that are located in the mitochondrial fraction of infected cells which pellets at 15,000 x g (P15 fraction) were used for the in vitro synthesis of viral 49S genome RNA, subgenomic 26S mRNA, and replicative intermediates (RIs). Comparison of the polymerase activity in P15 fractions from Sindbis virus (SIN)- and Semliki Forest virus (SFV)-infected cells indicated that both had similar kinetics of viral RNA synthesis in vitro but the SFV fraction was twice as active and produced more labeled RIs than SIN. When assayed in vitro under conditions of high specific activity, which limits incorporation into RIs, at least 70% of the polymerase activity was recovered after detergent treatment. Treatment with Triton X-100 or with Triton X-100 plus deoxycholate (DOC) solubilized some prelabeled SFV RIs but little if any SFV or SIN RNA polymerase activity from large structures that also contained cytoskeletal components. Treatment with concentrations of DOC greater than 0.25% or with 1% Triton X-100-0.5% DOC in the presence of 0.5 M NaCl released the polymerase activity in a soluble form, i.e., it no longer pelleted at 15,000 x g. The DOC-solubilized replication complexes, identified by their polymerase activity in vitro and by the presence of prelabeled RI RNA, had a density of 1.25 g/ml, were 20S to 100S in size, and contained viral nsPl, nsP2, phosphorylated nsP3, nsP4, and possibly nsP34 proteins. Immunoprecipitation of the solubilized structures indicated that the nonstructural proteins were complexed together and that a presumed cellular protein of -120 kDa may be part of the complex. Antibodies specific for nsP3, and to a lesser extent antibodies to nsPl, precipitated native replication complexes that retained prelabeled RIs and were active in vitro in viral RNA synthesis. Thus, antibodies to nsP3 bound but did not disrupt or inhibit the polymerase activity of replication complexes in vitro.

Cells infected with alphaviruses such as Sindbis virus (ts) synthesis of 26S mRNA (19, 48, 55), which suggests that (SIN) or Semliki Forest virus (SFV) contain a virus-specific a domain of nsP2 affects recognition by the polymerase of RNA-dependent RNA polymerase activity that is responsi- the recently mapped internal promoter in the 49S negative- ble for the synthesis of three species of RNA: the viral strand template (15). No function has been demonstrated yet positive-strand 49S genome RNA and subgenomic 26S for nsP3, which is phosphorylated (32, 33, 42). However, a mRNA and the 49S negative-strand RNA that is the template mutation in nsP3 of SIN ts7 is responsible for its ts RNA- for synthesis of both species of positive-strand RNAs. The negative phenotype (19). Therefore, replication complexes formation of the polymerase activity requires translation of would be expected to contain the other nsPs, and perhaps the incoming genome RNA into nonstructural proteins host proteins, in addition to nsP4 or nsP34. (nsPs), which are numbered according to the positions of the The synthesis of viral RNA initiates with the synthesis of genes from the 5' end of the genome RNA (reviewed in the first negative strand. Negative strands in turn serve as reference 58). The nsPs are synthesized initially as polypro- the preferred templates in replication complexes that are teins that are subsequently cleaved to yield four mature stable once formed and that function to synthesize genome proteins, nsPl to nsP4. For certain alphaviruses, limited and subgenome mRNAs (49). Increasing numbers of repli- readthrough of an opal codon located between nsP3 and cation complexes are formed early in infection, coinciding nsP4 results in synthesis of the fusion protein nsP34 (56, 57). with the synthesis of increased numbers of negative strands nsPl, nsP2, and nsP3 are present in larger amounts in (49, 51). After this early period, synthesis of negative strands infected cells than is nsP4 or nsP34 (28, 33). Recent evidence indicates that nsP4, which contains the ceases, the rate of positive-strand synthesis changes from an exponentially increasing rate to a constant one, and the conserved X-Gly-Asp-Asp (XGDD) sequence common to number of replication complexes and replicative intermedi- many RNA-dependent polymerases (26, 43) and an autopro- ate RNAs (RIs) becomes constant. The alphavirus replica- tease activity (59), is an essential component of the alphavirus polymerase and functions in elongation and possibly tion complex is associated with cytoplasmic membranous structures that copurify with the mitochondrial fraction of initiation (4, 18, 47, 52). It is not known whether nsP34 has infected cells (10, 16, 35, 36, 38, 54). Infected cells were a function different from that of nsP4, as has been postulated (8, 21). nsPl has been implicated in negative-strand synthe- found also to contain cytopathic vacuoles whose numbers sis (19, 50, 60) and in the methylation and capping of positive increased with time after infection (10, 11), but their relationship to the viral replication complexes has not been strands (7, 37). nsP2 contains a conserved domain found in established. More recently, Clewley and Kennedy (5), Ranki nucleotide-binding and helicase proteins (14), possesses an and Kaariainen (46), and Gomatos et al. (13) reported the autoprotease activity (9, 23), and may interact directly with nsP4 purification of SFV replication complexes after solubiliza- (19). Mutations in nsP2 result in temperature-sensitive tion of the mitochondrial pellet (P15) fraction of infected cells with various detergents. These investigators found that there was loss of polymerase activity after detergent treat- * Corresponding author. ment and that some of the SFV replication complexes were

1496 VOL. 65, 1991 ALPHAVIRUS REPLICATION COMPLEX 1497 released as 25S to 40S structures. We have continued these tion. Aliquots of the P15 fraction in RS buffer were mixed investigations and have determined conditions for the solu- with an equal volume of 2x detergent (either 2% Triton bilization of active SFV and SIN replication complexes. We X-100 [Pierce Chemical Co., Rockford, Ill.] plus 1% sodium also utilized antibodies that were obtained from Hardy and deoxycholate [DOC; Sigma Chemical Co., St. Louis, Mo.] in Strauss (22) and that are specific to the viral nsPs to identify RS buffer or 2% DOC in RS buffer), and the samples were the SIN nsPs and to demonstrate that they were present in immediately processed for centrifugation. To determine the solubilized replication complexes. We report that the anti- sizes of the detergent-treated replication complexes, the P15 bodies to nsP3 were the most effective in immunoprecipitat- was layered onto 15 to 30% glycerol gradients in 50 mM ing solubilized replicative complexes that retained their Tris-HCl, pH 7.8, 150 mM NaCI, 1 mM EDTA, and 0.1% RNA templates and polymerase activities after immunopre- Triton X-100 or 0.5% DOC, respectively, and centrifuged at cipitation. 4°C for 18 h at 25,500 rpm in the Beckman SW 28.1 rotor. Gradient fractions were collected from the bottom and MATERIALS AND METHODS analyzed through a UV monitor, with 60S and 40S ribosomal subunits serving as sedimentation markers. The buoyant Virus and cell cultures. Baby hamster kidney (BHK-21) density of the replication complexes was determined by cells were grown in Dulbecco modified Eagle minimum adjusting the sample to 30% sucrose and to 0.6 ml and essential medium supplemented with 6% newborn calf serum layering it on top of 0.5 ml of 50% sucrose and 1 ml of 60% and 5% tryptose phosphate broth (52). The heat-resistant sucrose (gradients without detergent were made with su- strain of SIN (SIN HR) has been described previously (50). crose in RS buffer, while detergent-treated samples were Infection and preparation of the P15 fraction. Monolayers analyzed on gradients of sucrose in RS and 0.1% Triton of BHK cells in 150-mm-diameter plastic petri dishes (-70 x X-100 or 0.5% DOC). Centrifugation was in the Beckman 106 cells per dish) were infected with a multiplicity of TLS-55 rotor (2.2-ml tubes) for 259,000 x g for 20 h at 4°C. infection of 30. At the end of a 1-h adsorption period, the Fractions (0.1 ml) were collected, and their densities were monolayers were refed with 37°C medium and incubated at determined from the refractive indices. 37°C until 5 h postinfection (p.i.), when the cells were Protein labeling and immunoprecipitation. Cultures of harvested in the cold and the P15 fraction was obtained as BHK-21 cells were infected with SIN HR at 37°C. Beginning described previously (4). Briefly, the cells were rinsed with at 2 h p.i., the cells were given a 2-h pulse-label in Dulbecco ice-cold phosphate-buffered saline (PBS), scraped from the modified Eagle minimum essential medium with 1% of the dish in PBS, and collected by centrifugation at 900 x g for 5 methionine and cysteine, 100 ,XCi of Trans 3"S-label per ml min at 4°C. The cells, typically from four 150-mm-diameter (1,150 Ci/mmole; ICN Radiochemicals), 2% dialyzed fetal dishes, were resuspended in 15 ml of RS buffer (10 mM Tris bovine serum, 2 ,ug of dactinomycin per ml, and 22 mM hydrochloride, pH 7.8, 10 mM NaCI) and were allowed to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH swell on ice for 15 min before being broken open in a Dounce 7.4, after which they were chased for 2 h in Dulbecco homogenizer. The nuclei were collected by centrifugation at modified Eagle minimum essential medium supplemented 900 x g for 5 min at 4°C and were resuspended in 15 ml of RS with 3% newborn calf serum and 5% tryptose phosphate buffer on ice. The postnuclear supernatant was centrifuged broth before harvest and fractionation. For direct analysis of at 15,000 x g for 20 min at 4°C in the Beckman JS13.1 rotor, the labeled proteins, aliquots of the cellular fractions were and both the supernatant (S15 fraction) and the pellet (P15 adjusted to 1% LDS, heated at 100°C for 3 min, and rapidly fraction) were collected. The P15 fraction was suspended in cooled before electrophoresis on 5 to 10% polyacrylamide storage buffer (10 mM Tris hydrochloride, pH 7.8, 10 mM gels with buffers described by Laemmli (30). NaCl, 15% glycerol) at 1 to 4 mg of protein per ml and stored For immunoprecipitation, the denatured proteins in 1% in aliquots at -80°C. LDS were diluted to 0.1% LDS in RIPA by the addition of 9 In vitro transcription reaction. The amount of P15 fraction volumes of modified radioimmunoprecipitation (RIPA) used in a typical reaction was equivalent to 2.5 x 105 cells. buffer (1) that contained 1% Triton X-100, 1% DOC, 10 mM Equal volumes of P15 and a 2x reaction mixture (100 mM Tris hydrochloride, pH 8.1, and 150 mM NaCl but lacked Tris hydrochloride, pH 7.8; 100 mM KCl; 7 mM MgCl2; 20 LDS. Equal aliquots of cellular fractions that had not been mM dithiothreitol; 20 ,ug of dactinomycin per ml; 10 mM denatured with LDS and heat were diluted in modified RIPA creatine phosphate; 50,g of creatine phosphokinase [Calbi- buffer. The solutions were preabsorbed with group G strep- ochem, San Diego, Calif.] per ml; 4 mM [each] ATP, GTP, tococcal cells (100 ,ul of a 10% solution of heat-killed and UTP and 0.4 mM CTP for low-specific-activity reactions streptococcal G cells per solubilized P15 from 5 x 105 and no unlabeled CTP for high-specific-activity reactions; 3 infected cells), followed by end-over-end mixing for 20 min mCi of [ca-32P]CTP [ICN Radiochemicals, Irvine, Calif., or at room temperature. The bacterial cells were pelleted at Dupont NEN, Wilmington, Del.] per ml; 400 to 800 U of 10,000 x g for 5 min, and the supernatant was divided into RNasin [Promega Biotec, Madison, Wis.] per ml) were 600-pu aliquots for immunoprecipitation, essentially as de- incubated at 30°C for 30 min or as indicated in the text. scribed previously (22). The immunoprecipitates were re- Reactions were terminated by the addition of 5% lithium solved on S to 10% polyacrylamide gels in Laemmli buffer. dodecyl sulfate (LDS) containing 100,ug of proteinase K per ml when RNA products were being assayed. After phenol RESULTS and chloroform extraction and ethanol precipitation of the samples, the RNA was analyzed by separation on 0.8% Characterization of the in vitro polymerase activity. The agarose gels in TBE (89 mM Tris base; 89 mM boric acid; 2 P15 fraction from SIN- or SFV-infected cells was enriched in mM EDTA) or 0.8% agarose gels containing 2.2 M formal- viral replication complexes and contained about 80% of the dehyde in morpholinepropanesulfonic acid (MOPS) buffer polymerase activity (Fig. 1 and Table 1). The nuclear and the (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0) cytoplasmic supernatant (S15) fractions each contained and was visualized by autoradiography of the dried gels. about 10% of the polymerase activity (Table 1). RNA Solubilization and velocity and buoyant-density centrifuga- synthesized in vitro migrated on agarose gels at the positions 1498 BARTON ET AL. J. VIROL. A B Low Specific Activity High Specific Activity _ C) LO O , o , I- z (In EL - 7z LO L

RI -u-W

49S-~- 46ss- _

26S --o

FIG. 1. Active replication complexes were enriched in the P15 fraction, and transcription in vitro required all four nucleoside triphosphates. (A) SIN-infected BHK cells were harvested at 5 h p.i. and fractionated as described in the text. Equal proportions of the total-cell lysate and the nuclear, S15, and P15 fractions (equivalent to 2.5 x 105 cells) were incubated in vitro at 30°C for 30 min in either low-specific-activity or high-specific-activity (no supplementation of the radioactive CTP) reaction conditions before the products were analyzed on 0.8% agarose gels in TBE buffer. (B) Aliquots of the P15 fraction from SIN-infected BHK cells were incubated in vitro for 30 min at 30°C in either the complete low-specific-activity reaction mixture containing 32P-CTP and all four unlabeled ribonucleoside triphosphates or in reaction mixtures lacking (W/O) one or three of the unlabeled nucleoside triphosphates. The products were deproteinized and analyzed by electrophoresis on 0.8% agarose gels in MOPS-formaldehyde buffer. of the viral RIs and the two viral single-stranded RNAs, the when one or more of the unlabeled nucleoside triphosphates genomic 49S RNA and the subgenomic 26S mRNA (Fig. were omitted (data not shown). The small amount of labeling 1A). As reported recently (3), radiolabel appears after 1 min of RIs and 26S RNA molecules in reactions lacking one or of incubation in the viral RIs and subsequently appears and more triphosphates was probably due to the limited elonga- accumulates in the two single-stranded RNAs. Treatment of tion or release of almost-completed chains, because the the radiolabeled RNAs with pancreatic RNase before elec- amounts varied inversely with the base composition (1 G, 15 trophoresis yielded the expected RNase-resistant replicative A, and 28 U residues) of the 3' heteropolymeric sequence of forms (RFs) of the RIs, the RFI, RFII, and RFIII molecules 26S and 49S RNA. In addition to the usual low-specific- (data not shown) (3, 53). The polymerase activity required all activity labeling conditions (in which the radiolabeled CTP four nucleoside triphosphates and specifically incorporated was with radiolabeled CTP into all viral RNA species. Little or no supplemented 200 ,uM unlabeled CTP), reactions label was incorporated into viral RNAs under conditions in were carried out also at high specific activity (the radiola- which one or more of the four triphosphates were omitted beled CTP was not supplemented with unlabeled CTP). The (Fig. 1B), whereas labeling of tRNA occurred maximally high-specific-activity reactions yielded predominantly radiolabeled RI RNA and little or no radiolabeled 49S and 26S RNAs. Some single-stranded RNA, mostly 26S mRNA, was released in the whole (total) lysates, probably because of TABLE 1. P15 fraction enriched in SIN polymerase activity endogenous unlabeled nucleoside triphosphates. About 30 pmol of CMP incorporated/mg of proteina times more RNA was synthesized under low-specific-ac- Fraction Low tivity labeling conditions than under conditions of limited specific activity High specific activity CTP concentrations (Table 1); however, more 32P-CMP was Total 22 0.4 found in the RI and RF RNA under high-specific-activity Nuclear 9 0.3 conditions. We have shown previously (4) that the RNA S15 5 0.1 pulse-labeled under conditions of high specific activity is P15 81 3.0 chased into single-stranded 49S and 26S viral RNA after the a Incorporation of radiolabel into viral RNA was determined by Cerenkov addition of unlabeled nucleoside triphosphates. counting the RI, 49S, and 26S RNAs after electrophoresis on an agarose gel. We also monitored nucleoside triphosphatase and RNase The amount of protein in the cell fractions was determined with the BCA reagent (Pierce Chemical Co., Rockford, Ill.). The specific activity of CMP in activities in the P15 fractions. The amount of radiolabeled the low-specific-activity reaction was 17,000 cpm/pmol and in the high-specific CTP that was converted to CDP, CMP, and Pi during the activity reaction was 6.8 x 106 cpm/pmol. reactions was analyzed by thin-layer chromatography and VOL. 65, 1991 ALPHAVIRUS REPLICATION COMPLEX 1499 A B Activity Pre-label Prelabel SIN SFV SIN SFV cJ cJ v 16 60 0~~~~~~~~~) ) 0 55 E X _ F .FN-a. , o ° 12 50 CLo-IE 45O3soU UL80 8 cn 40 RI -_

35 30 49S -.- Fraction Activity 26S o-

8 0 , 1.25g/cc 60 55 (A UM0 50 L-

45 U)

40 35

30 2 4 6 8 10 12 14 16 18 Fraction FIG. 2. SIN and SFV replication complexes have a density of 1.25 g/ml after treatment with 1% DOC or with 1% Triton X-100-0.5% DOC. Representative results are shown. (A) The P15 fraction from SIN-infected cells was incubated in vitro under high-specific-activity labeling conditions (Prelabel) and was repelleted before treatment with 1% DOC and buoyant-density centrifugation as described in Materials and Methods. An aliquot of each gradient fraction was acid precipitated directly. To identify polymerase activity (Activity), P15 fractions were treated directly with 1% DOC before centrifugation. Every second gradient fraction was incubated in vitro at 30°C for 30 min in high-specific-activity reactions, and the products were deproteinized before electrophoresis on 0.8% agarose-TBE gels. The incorporation in viral RIs was determined by cutting and counting that area of the gel. (B) Fluorogram of a 0.8% agarose gel in TBE showing that the RNA polymerase activity causing the radiolabeling of RI RNA was in the 1.25-g/ml fractions from SIN- and SFV-infected cells and that the prelabeled RIs were also in the same fraction.

indicated that the P15 fractions prepared from mock- or activity was affected by treatment of the P15 fraction with virus-infected cells contained similar levels of phosphatase Triton X-100 and DOC, used singly or in combination and at activity and that this activity was partially responsible for concentrations of each detergent above its critical micelle the cessation of incorporation (data not shown). Viral RNAs concentration. Detergent-treated fractions had increased continued to be synthesized in vitro as long as the nucleoside levels of RNase (data not shown) that most likely were triphosphate concentration did not decrease to less than 50% responsible for the observed low recovery of single-stranded of the initial concentration, i.e., less than 0.05 to 0.1 mM. RNAs when the polymerase activity was assayed under Use of less concentrated P15 or the addition of more low-specific-activity labeling conditions. Under high-specif- nucleoside triphosphates increased the time over which ic-activity labeling conditions, RI RNAs were radiolabeled incorporation continued. Endogenous RNases also contrib- to high levels and at least 70% of the original polymerase uted to a failure to continue linear incorporation beyond 90 activity remained after exposure to the highest concentra- min and to a loss of previously synthesized single-stranded tions of detergent (data not shown). RNA. RNase activity was detected by incubating radiola- In the absence of detergent treatment, the polymerase beled transcripts of the infectious clone of SIN in various activity in SIN and SFV replication complexes in the P15 reactions and determining the amount of intact labeled RNA fraction was associated with 1.16 g/ml smooth membranes. at the end of the incubation period (data not shown). These After treatment with either 1% DOC alone (Fig. 2A) or 1% nucleases were not inhibited by RNasin (Promega, Madison Triton X-100 plus 0.5% DOC (Fig. 2B), which solubilized Wis.). Lower incubation temperatures (25 to 30°C) and lipids and 80% of the membrane proteins, active SIN repli- shorter incubation times (15 to 30 min) gave the highest cation complexes had a density of 1.25 g/ml. The radiolabel recovery of polymerase products in vitro. incorporated by the SIN and SFV polymerases was in viral DOC efficiently solubilized the SIN and SFV replication RIs and RFs (Fig. 2B). Prelabeled SIN and SFV RIs, which complexes. We determined the extent to which polymerase were obtained by incubation in vitro and repelleting of the 1500 BARTON ET AL. J. VIROL.

vitro incubation of the P15 fraction. Thus, DOC efficiently 41310 released both the SIN and SFV replication complexes as small ribonucleoprotein structures that retained polymerase 800X activity and RI templates after the detergent treatment and 0~ after their release from membranes. 600 > Distribution of SIN nsPs. Figure 4 shows the distribution of w labeled proteins in SIN- or mock-infected cell fractions from _400-0 cultures that had been pulse-labeled from 2 to 4 h p.i. and m chased from 4 to 6 h p.i. before harvest. Because replication in - 200< complexes were being formed at an exponential rate early w infection, nsPs synthesized at this early time were more I. likely to be components of replication complexes than were 0 5 10 15 20 25 30 35 proteins synthesized late in infection after the rate of posi- Fraction tive-strand synthesis had reached its maximum. SIN nsP2 was clearly enriched in the nuclear fraction (see also refer- FIG. 3. The solubilized SIN replication complexes were of het- ence 41), which contained about 9% of the total incorpora- erogenous size. The 1% DOC-generated 1.25-g/ml replication com- and plexes were analyzed by velocity centrifugation. After centrifugation. The S15 fraction had 74% of the total incorporation tion, the pellets were resuspended in a volume of buffer equal to one contained labeled proteins of the size of the viral nsPs as well fraction. Prelabeled replication complexes were identified by deter- as labeled host proteins. The P15 fraction, which contained mining the acid-insoluble radioactivity in each fraction. Active 0.5% of the total incorporation, was enriched in proteins of replication complexes were identified by assaying the pellet and the size of the nsPs and contained proteins migrating in a every fourth fraction for polymerase activity by using high-specific- broad band in the 50-kDa region of the gel where the viral activity conditions, separating the products by electrophoresis on envelope proteins would migrate. After DOC treatment, the agarose gels, and cutting and counting the areas of the gels contain- 1.25-g/ml fraction retained only 20% of the labeled P15 ing the viral RIs and RFs. proteins but was enriched in proteins of the size of the nsPs. Among the labeled cellular proteins, the presence of cytoskeletal proteins actin (-43 kDa), myosin (-200 kDa), and P15 fraction before detergent treatment, yielded an identical tubulin (-50 kDa) in both the mock and the SIN DOC- enrichment of prelabeled RIs in the 1.25-g/ml fraction after generated 1.25-gIml fractions was apparent from the finding detergent treatment (Fig. 2). Our analysis also indicated that of labeled proteins of their respective sizes and from the SFV P15 fractions were about twice as active as SIN P15 immunoprecipitation of myosin with monospecific antiserum fractions, which was probably due to increased numbers of (data not shown). Of particular note was the presence in the SFV replication complexes since there were more labeled 1.25-g/ml fractions from mock- and SIN-infected cells of SFV RIs than SIN RIs (Fig. 2B) (3). Increased radiolabeling proteins of -120 kDa in size. of SFV RIs compared with radiolabeling of SIN RIs has been Immunoprecipitation of SIN nsPs and replication com- observed in experiments that radiolabeled these molecules in plexes. We utilized immunoprecipitation for definitive iden- vivo (data not shown). That some of the increased numbers tification of the SIN nsPs. Each antiserum reacted only with of SFV RI molecules were RFs was suggested by the heavier its specific nsP in the total infected-cell lysate when the labeling of molecules that migrated faster on gels, at the proteins were denatured before immunoprecipitation (Fig. position of the RNase-generated RFI structures. Similar SA). In this gel, 10 times more anti-nsP3 and anti-nsP4 results were found after treatment of the P15 or 1.16-g/ml immunoprecipitates than anti-nsPl and anti-nsP2 immuno- membrane fractions, although greater levels of activity were precipitates were loaded onto the lanes to facilitate their recovered with the P15 fraction. Therefore, we used the P15 detection. Phosphorylated forms of SIN nsP3 and nsP34 fraction as the source of the viral replication complexes to were precipitated by the nsP3 antibodies. Radiolabeled determine which detergents efficiently released the replica- nsP34 and nsP4 were present in the infected-cell lysates and tion complexes from the P15 pellet. precipitated with the nsP4 antibodies. By immunoprecipita- We found that DOC at concentrations of 0.25% or higher tion, significant amounts of all the nsPs were present in both released substantially all of the polymerase activity in a form the P15 fraction that contained the majority of the polymer- that no longer pelleted at 15,000 x g (data not shown). The ase activity in vitro and the S15 fraction that contained little presence of high concentrations of DOC was inhibitory, but polymerase activity (Fig. SB). More nsPs were found in the significant amounts of polymerase activity remained even S15 fraction from SIN-infected cells than were reported for when assayed in the presence of 0.5% DOC. Treatment with the S15 fraction from SFV-infected cells (40). 1% Triton X-100 or 1% Triton X-100-0.5% DOC failed to Portions of the DOC-solubilized SIN 1.25-g/ml fraction release the polymerase activity or the prelabeled SIN ribo- (equivalent to that from -106 cells) that had been labeled nucleoproteins, although some of the prelabeled SFV ribo- with 35S-methionine-cysteine from 2 to 4 h p.i. and chased nucleoproteins were released from the fraction pelleted at from 4 to 6 h p.i. before harvest were reacted separately with 15,000 x g. However, the addition of 0.5 M NaCI to the 5 ,ul of each antiserum or of preimmune serum. When the Triton X-100 solutions released the polymerase activity into proteins were denatured before immunoprecipitation, each the supernatant fraction centrifuged at 15,000 x g (data not nsP was immunoprecipitated specifically and little if any shown). After DOC treatment, the SIN polymerase activity coprecipitation of the other nsPs was detected (Fig. 6A). was associated with replication complexes that were 40S to Consistent with these results was the finding that superna- 100S in size (Fig. 3), although most of the prelabeled tants after immunoprecipitation were reduced in the specific replication complexes were smaller and sedimented from nsP being recognized, although no evaluation could be made about 20S to 40S (Fig. 3). Their smaller sizes probably result for nsP4 because nsP4 could not be directly visualized on from the loss of nascent chains and proteins either from autoradiograms (data not shown). The results indicated that elongation and release or from nuclease digestion during in after DOC treatment the 1.25-g/ml fraction retained nsPl, VOL. 65, 1991 ALPHAVIRUS REPLICATION COMPLEX 1501

Sindbis infected cells Mock infected cells

u ._ Q~~~~~~~. ._ o~~~~~~.

X U) o .10 Z LoLo LO~ o " Z X _ I-z cn XLO _

nsPl -w

* kD

FIG. 4. Distribution after fractionation of SIN nsPs and host proteins. SIN-infected or mock-infected BHK cells were pulsed-labeled with Trans 35S-label from 2 to 4 h p.i. and chased from 4 to 6 h p.i. before harvest and fractionation. Samples of each fraction were adjusted to 1% LDS before electrophoresis in 5 to 10% polyacrylamide Laemmli gels. An equal amount of acid-insoluble radioactivity was applied to each lane. The locations of coelectrophoresed, prestained proteins.:'.:.&rmarkers and the viralJinsPs*are ~~~-25.7shown. nsP2, nsP3, nsP4, and some nsP34. Compared with total Immunoprecipitation of active replication complexes. We infected-cell lysates (Fig. 5A), the 1.25-g/ml fraction was next asked whether replication complexes that contained RI enriched in the highly phosphorylated nsP3 and was en- RNA and were active in RNA synthesis could be immuno- riched in nsP4 relative to nsP34 (Fig. 6A). precipitated. We performed two kinds of experiments. The The next experiment demonstrated that the nsPs were first tested whether the RIs in the replication complexes associated in a complex. When proteins in the 1.25-g/ml would be coprecipitated by the nsP antibodies. P15 replica- fraction were immunoprecipitated directly, without denatur- tion complexes were prelabeled before treatment with 1% ation, all of the viral nsPs coprecipitated (Fig. 6B). Compar- DOC. The 1.25-glml fraction was obtained, and aliquots ison of the four reactions indicated that each immunoprecip- (equivalent to that from about 106 cells) were incubated itate contained large amounts of the specific nsP being directly with 5 ,ul of one of the nsP antisera or of the recognized by that antibody (i.e., 50 to 90% of the amount preimmune serum. Nucleic acids were recovered from the precipitated after denaturation) and different amounts of the immunoprecipitates and were electrophoresed on agarose other nsPs. In particular, antibodies to nsP3 coprecipitated gels (Fig. 7A). Prelabeled RIs were detected in immunopre- significant amounts of nsPl, nsP2, and nsP4, and antibodies cipitates from the anti-nsP3 and anti-nsPl antibody reac- to nsPl coprecipitated significant amounts of nsP4 but little tions. The finding that the labeled RIs were recovered as RFI nsP2 and nsP3. In addition, antibodies to nsPl coprecipi- and RFIII RNAs after the assay was not unexpected: limited tated an -120-kDa protein that is most likely a cellular elongation in vitro of 49S and 26S nascent chains selectively protein because it was enriched in the 1.25-g/ml fraction enriched the radiolabeling of these parts of the RI molecules obtained from DOC-treated, mock-infected cell extracts (52), and the extensive handling and incubation conditions (Fig. 4) and because, once denatured, it failed to precipitate led to cleavage of the RIs. Antibodies to nsP3 immunopre- with any of the anti-nsP antibodies. We concluded from cipitated 13% of the prelabeled RNA in the aliquot assayed, these experiments that coprecipitation of the nsPs was due and nsPl antibodies precipitated 2% of the RNA. Antibodies to their association in a complex and, unexpectedly, that specific for nsP2 or for nsP4 precipitated only twice as much anti-nsP3 antiserum was most effective in immunoprecipitat- prelabeled RNA as the background level (0.1%) found in the ing the complex. preimmune serum reaction. Consistent with these results, 1502 BARTON ET AL. J. VIROL. A B Total S15 P15 Pl 2 3 4 r4 s P 2 3 14 2 34 2 -3

-nsP34

- nsP4 nsP1-

FIG. 5. Immunoprecipitation of the viral nsPs. (A) Identification of SIN nsPs by immunoprecipitation with monospecific antisera. SIN-infected cells were labeled with Trans 35S-label from 2 to 4 h p.i. and chased from 4 to 6 h p.i. before harvest as described in Materials and Methods. Samples were immunoprecipitated, and the precipitated proteins were electrophoresed in 5 to 10% polyacrylamide gels. Ten times more anti-nsP3 and anti-nsP4 precipitates than anti-nsPl and nsP2 precipitates were loaded. (B) nsPs were present in both the S15 and the P15 fractions. Aliquots of the total-cell extract, the S15 fraction, and the P15 fraction were incubated with each of the four monospecific anti-nsP antibodies. The precipitated proteins were electrophoresed on 5 to 10%o polyacrylamide gels and visualized by fluorography. only the supernatant from the anti-nsP3 reaction was signif- corporate in vitro radiolabeled CTP into viral RNA. Portions icantly reduced in prelabeled RNA (data not shown). of an unlabeled SIN 1.25-g/ml fraction were reacted sepa- The second approach we used determined whether the rately with each antiserum or preimmune serum, and the immunoprecipitated replication complexes were able to in- resulting immunoprecipitates (including the streptococcus G

A B Pi (LI ( (x3 (X.4 Pl cdI 2tO 3 u4 m 4, E S S 5 ~ t f 200 kDa

4. lfR W|I| I .

92.5 kDa

68 kDa

FIG. 6. Immunoprecipitation of the solubilized replication complexes. The 1.25-g/ml fraction obtained after treatment of the P15 from SIN-infected cells either was treated with 1% LDS in 1 mM EDTA, heated to 100'C for 3 min, and fast cooled (A) or was left untreated (B) before both were aliquoted into RIPA buffer without LDS and incubated with preimmune serum or one of the four monospecific nsP antisera (22). The immunoprecipitates were obtained and analyzed by electrophoresis as described in Materials and Methods. VOL. 65, 1991 ALPHAVIRUS REPLICATION COMPLEX 1503 A B SerUm: PI i1 (t2 uo3 (4 1 2 3 4 RFI RFI I,. RFIII RFIII

FIG. 7. Replication complexes in immunoprecipitated with anti-nsP3 retain polymerase activity. (A) Immunoprecipitation of prelabeled replication complexes. SIN P15 was incubated in vitro to prelabel the viral RI RNA before solubilization with 1% DOC and preparation of the 1.25-g/ml complexes. After incubation with preimmune serum (PI) or one of the nsP antibodies, the immunoprecipitates were collected with streptococcus G cells and washed twice with 1 M NaCl-RIPA buffer. RNA was extracted after incubation of the precipitates with sodium dodecyl sulfate and proteinase K and was analyzed by electrophoresis in 0.8% agarose-TBE gels. The positions of the viral RFI and RFIII cores of the RIs are indicated. (B) Immunoprecipitation of polymerase activity. The 1.25-g/ml fraction was reacted with preimmune serum (lane 3) or with anti-nsPl (lane 4), anti-nsP2 (lane 5), anti-nsP3 (lane 6), or anti-nsP4 (lane 7) antibodies. Immunoprecipitates were collected with streptococcus G cells and washed extensively, and a portion was added to an equal volume of 2x high-specific-reactivity reaction mixture and incubated at 30°C for 30 min. The RNAs were isolated and analyzed by electrophoresis on 0.8% agarose-TBE gels. One-seventh as much 1.25-g/ml material was incubated in vitro directly in the absence (lane 1) or in the presence of an equivalent amount of streptococcus G cells (lane 2). Products were also obtained from reactions performed in the absence of streptococcus G cells (lane 8), in the absence of nsP antibody (lane 9), or in the absence of both (lane 10). cells) were incubated in vitro under high-specific-activity polymerase activity. Both the SFV and SIN replication labeling conditions. The results are shown in Fig. 7B. complexes were present on smooth membranes that had a Replication complexes in the 1.25-g/ml fraction gave high density of 1.16 g/ml. When released from membranes follow- levels of incorporation into viral RIs (lane 1) that were not ing exposure to 1% DOC, the viral replication complexes greatly affected (less than twofold) by the presence of had a density of 1.25 g/ml, were heterogeneous, and sedi- streptococcus G cells (lane 2). Only two of the immunopre- mented at 20S to 100S. This heterogeneity suggests that cipitates gave significant levels of incorporation of radiolabel some of the replication complexes may not be completely into viral RFI and RFIII RNAs. Replication complexes in dissociated from larger structures or that they contain dif- the anti-nsP3 precipitate (lane 6) had 16% of the polymerase ferent amounts of proteins or nascent RNA chains, as activity that would have been present in this amount of previously reported for poliovirus replication complexes 1.25-g/ml fraction incubated in the presence of streptococcus (12). Replication complexes retained polymerase activity G cells. The anti-nsPl precipitate (lane 4) had a lower level after disruption of the membranes with detergents when the of incorporation but a level higher than that in the preim- in vitro assay kept the radiolabeled viral RNA in a double- mune (lane 3), anti-nsP2 (lane 5), or anti-nsP4 (lane 7) stranded form, protecting it from nuclease degradation. precipitates. Furthermore, radiolabeled viral RNA was not Thus, our results suggest that the previous failure (5, 13, 46) detected in other control reactions that were identical except to detect polymerase activity after detergent treatment re- that they lacked streptococcus G cells (lane 8) or serum (lane sulted from the degradation of the newly synthesized single- 9) or both (lane 10). The polymerase activity profile closely stranded RNAs by detergent-released nucleases. Although resembled that observed for the precipitation of prelabeled the distribution of the SIN nsPs did not mimic precisely the replication complexes (Fig. 7A). Thus, antibodies directed distribution of the viral polymerase activity and therefore against epitopes in nsP3 were particularly effective in bind- to the ing to the DOC-solubilized replication complexes and in could not be used alone track replication complex immunoprecipitating replication complexes that retained during purification, fractions enriched in polymerase activity contained significant amounts of all four nsPs. polymerase activity. Recovery of the majority of the SFV and SIN replication complexes as small ribonucleoproteins required incubation DISCUSSION of the P15 membranes in the ionic detergent DOC or in The results of our studies indicated that SFV-infected cells Triton X-100 solutions supplemented with 0.5 M NaCl contained about twice as many replication complexes as (see also references 5 and 47). Other nonionic (dodecyl-13- SIN-infected cells, which had correspondingly lower in vitro D-maltoside, n-octylglucoside, Nonidet P-40, and Triton 1504 BARTON ET AL. J. VIROL.

X-114) and zwitterionic {3-[(3-cholamidopropyl)-dimethyl- that nsP3 may not play an essential role in viral RNA ammonio]-1-propanesulfonate} detergents did not release the synthesis. So far only one ts, RNA-negative mutant of SIN, SIN or SFV replication complexes (data not shown). In this ts7, has been mapped to nsP3 (19). This mutant is defective regard, our results are similar to those of Grun and Brinton in the formation of the replication complexes at 40°C. (17), who found that DOC efficiently solubilized the flavi- However, ts7 replication complexes formed at 30°C function virus West Nile replication complex and retained its activity, normally at 40°C (27, 50). Recent studies by Hardy et al. (21) and of Girard and Baltimore (12), who found that DOC identified no major defects in polyprotein synthesis or proc- solubilized the poliovirus replication complex. Since re- essing by ts7. If a peptide domain which is functionally moval of lipids and of at least 80% of the proteins in the P15 analogous to alphavirus nsP3 exists in plant viruses belong- membranes with the other detergents tested was insufficient ing to the SIN superfamily, its amino acid sequence would to release the replication complex, it may be attached to a be specific for each virus and might play a role in determin- nonlipid and relatively insoluble component of the P15 ing the host range of the virus. The anti-nsP3 antiserum used fraction. That the replication complex was part of a lipid- in this study recognized SIN nsP3 whether it was extensively containing structure originally was indicated by its buoyant phosphorylated or not (Fig. SA), although it may have a density of 1.16 g/ml before detergent treatment, by its preference for epitopes in the phosphorylated, carboxy- change in density to 1.25 g/ml after detergent treatment, and terminal part of nsP3 (32), since the anti-nsP3 antibody, by the finding that incubation in 0.5 M NaCl without deter- unlike the anti-nsPl, anti-nsP2, and anti-nsP4 antibodies, did gent treatment did not release the replication complex as not cross-react with SFV nsPs. Complexes precipitated by small ribonucleoproteins. The Triton X-100-DOC-treated antibodies to nsPl, nsP2, or nsP4 also contained proteins of replication complex had the same size, density, and deter- the size of the phosphorylated nsP3. The anti-nsP3 antibod- gent resistance as the cytoskeletal framework (44). We found ies precipitated more SIN replication complexes than the that cytoskeletal components actin, tubulin, and myosin other anti-nsP antisera, whether measured by prelabeling the were present in the 1.25-g/ml fractions; also, a cell protein of RIs or the nsPs or by determining the levels of polymerase the size of actin was found previously in partially purified activity. We do not know why the other nsP antibodies were SFV replication complexes (5). We do not know the signif- not as effective as the anti-nsP3 antibodies. It may be due to icance of finding the replication complexes associated with an unavailability or altered conformation of particular nsP the cytoskeletal framework (3). However, others have re- epitopes when they are in a native conformation, or it may ported that integral membrane proteins or mRNAs (20, 34) indicate inhibition or disruption of the replication complex are associated with the cytoskeletal framework and that the after reaction with certain antisera. Thus, our present results interaction of viral proteins with the cytoskeletal framework and the genotype of ts7 argue that nsP3 plays an essential is necessary for assembly or for viral polymerase activity role in the replication complex, perhaps to enable the (25, 34, 39). In this regard, the relationship of the alphavirus interaction of the nsPs with one another or with a host replication complex to the cytopathic vacuoles identified by component. Grimley et al. (16) and more recently studied by Froshauer et Because host cell proteins were precipitated by antibodies al. (11) is intriguing, especially because such structures specific for each of the viral nsPs, they might also be part of might contain cytoskeletal components. However, it re- the replication complex. The -120-kDa host protein was mains to be shown that the accumulation of cytopathic precipitated with anti-nsPl, but only if the proteins were not vacuoles during infection follows the accumulation of repli- first denatured, which demonstrated also that it was not a cation complexes and that cytopathic vacuoles contain viral precursor to nsPl. Because the amount of the -120-kDa RI templates and are active in viral RNA synthesis. protein that precipitated with the viral nsPs was proportional The alphavirus RI and nsPl, nsP2, nsP3, and nsP4 were to the amount of nsPl, it may be specifically associated with present in a complex with sufficient stability to withstand nsPl. nsPl has been implicated in methyltransferase activity disruption of the membrane and immunoprecipitation. That (37) as well as in negative-strand synthesis (19, 50, 60). The the four nsPs were complexed together was not unexpected. possible role of host proteins in alphavirus RNA synthesis Analysis of the alphavirus ts RNA-negative mutants indi- has been suggested previously by the sensitivity of alphavi- cated that the four nsPs were required for viral RNA rus replication to dactinomycin pretreatment (2, 6) and by synthesis and that their synthesis initially as polyproteins the isolation of ts host-range mutants of SIN (29) that were would favor their interaction. Replication complexes iso- mapped (31) to amino acid changes in nsP4. Other host lated at 6 h p.i. at 37°C, when positive strands are actively proteins recognize sequences near the 3' end of the genome and exclusively being made (50), contained nsPs synthesized RNA of rubella virus (40) or copurify with the polymerase of between 2 and 4 h p.i. that included nsP4. Interpretation of brome mosaic virus (45) or with the polymerase of cucumber the presence of nsP34 in the SIN replication complex was mosaic virus (24). The ability to solubilize the alphavirus complicated by the inability to distinguish between this replication complex with detergents while retaining polymer- possibility and its separate immunoprecipitation by anti- ase activity will facilitate the identification of the viral and nsP3 antibodies. Thus, our present results agree with previ- host components of the alphavirus replication complex. ous studies (references 49, 51, and 60 and references cited therein) that indicated that the majority of the replication complexes were formed before 4 h p.i. and were stable once ACKNOWLEDGMENTS formed. What was surprising was that the SIN replication com- We especially thank W. Reef Hardy and James Strauss, California plexes which were precipitated by antibodies to nsP3 re- Institute of Technology, for their generous gift of polyclonal SIN nsP antibodies. We also acknowledge that the group G streptococcal tained polymerase activity. The fact that protein sequences cells were a generous gift from Ervin Faulmann, Medical College of homologous to nsP3 were not encoded in the genome of Ohio; mouse polyclonal antibodies to SIN AR339 were a gift from plant RNA viruses that were members of the SIN superfam- Robert Johnson, University of North Carolina, Chapel Hill; and ily and that did encode proteins homologous to the other monoclonal antibodies to actin, tubulin, and myosin were a gift from three alphavirus nsPs (reviewed in reference 58) suggested R. Lane, Medical College of Ohio. VOL. 65, 1991 ALPHAVIRUS REPLICATION COMPLEX 1505

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