Nascent Flavivirus RNA Colocalized in Situ with Double-Stranded RNA in Stable Replication Complexes

Virology 258, 108–117 (1999) Article ID viro.1999.9683, available online at http://www.idealibrary.com on

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Nascent Flavivirus RNA Colocalized in Situ with Double-Stranded RNA in Stable Replication Complexes

Edwin G. Westaway,1 Alexander A. Khromykh, and Jason M. Mackenzie

Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital, Herston, Brisbane, Queensland 4029 Australia Received November 30, 1998; returned to author for revision January 20, 1999; accepted March 2, 1999

Incorporation of bromouridine (BrU) into viral RNA in Kunjin virus-infected Vero cells treated with actinomycin D was monitored in situ by immunofluorescence using antibodies reactive with Br-RNA. The results showed unequivocally that nascent viral RNA was located focally in the same subcellular site as dsRNA, the putative template for flavivirus RNA synthesis. When cells were labeled with BrU for 15 min, the estimated cycle period for RNA synthesis, the nascent Br-RNA was not digested in permeabilized cells by RNase A under high-salt conditions, in accord with our original model of flavivirus RNA synthesis (Chu, P. W. G., and Westaway, E. G., Virology 140, 68–79, 1985). The model assumes that there is on average only one nascent strand per template, which remains bound until displaced during the next cycle of RNA synthesis. The replicase complex located by BrU incorporation in the identified foci was stable, remaining active in incorporating BrU or [32P]orthophosphate in viral RNA after complete inhibition of protein synthesis in cycloheximide-treated cells. These results are in accord with our proposal that dsRNA detected in foci previously located by immunofluorescence or by immunogold labeling of induced vesicle packets is functioning as the true replicative intermediate (Westaway et al., J. Virol. 71, 6650–6661, 1997; Mackenzie et al., Virology 245, 203–215, 1998). Implications are that the replicase complex is able to recycle in the same membrane site in the absence of continuing protein synthesis and that possibly apart from uncleaved NS3–NS4A, it has no requirement for a polyprotein precursor late in infection. © 1999 Academic Press

INTRODUCTION fected by treatment with several ionic or nonionic detergents (Chu and Westaway, 1987, 1992; Grun and Brinton, The flavivirus Kunjin (KUN) replicates in cytoplasmic 1988). sites comprising virus factories identifiable by immuno- Because the flavivirus genome comprises one long fluorescence (IF) and by immunoelectron microscopy (Westaway et al., 1997a,b; Mackenzie et al., 1998). A open reading frame, all the gene products are translated replication model based on kinetic analysis of KUN RNA sequentially and continuously throughout the cycle of synthesis proposed that late in infection flavivirus RNA is infection. Of the seven nonstructural proteins KUN NS1, synthesized asymmetrically and semiconservatively in a NS2A, NS3, NS4A, and NS5 appeared to be associated replicative intermediate (RI) that uses the double- with the replication complex in biochemical assays and stranded RNA (dsRNA) or replicative form (RF) as a immunocytochemical analyses (Chu and Westaway, recycling template (Chu and Westaway, 1985, 1987, 1992). 1992; Westaway et al., 1997b; Mackenzie et al., 1998). The proposed dsRNA templates can be immunolabeled NS3 and NS5 contain domains associated with putative with antibodies to dsRNA (Ng et al., 1983) and late in helicase and RNA polymerase activities, respectively, in infection are located in induced membranes, which form addition to serine protease (NS3) and methyl transferase vesicle packets (Mackenzie et al., 1996). dsRNA is colo- (NS5) (Chambers et al., 1990a, 1991; Wengler and Weng- calized with most of the KUN nonstructural proteins by IF ler, 1991, 1993). Dengue-1 virus NS5 was shown to copy and by immunogold labeling of cryosections of infected RNA nonspecifically using an in vitro assay (Tan et al., cells (Westaway et al., 1997a,b; Mackenzie et al., 1998). 1996), and NTPase activity has been reported for NS3 of The immunofluorescent sites labeled with anti-dsRNA several related flavivirus species (Wengler and Wengler, antibodies are stable to disruption of membranes by 1991; Cui et al., 1998, and references therein). The re- Triton X-100 or by cytoskeletal disrupting agents (Ng et maining nonstructural proteins have no recognizable do- al., 1983; Westaway et al., 1997a,b), and the flavivirus mains associated with events of flavivirus replication. RNA-dependent RNA polymerase (RDRP) activity of in- However, in addition to our results on immunolabeling in fected cell lysates has been shown to be largely unaf- infected cells of NS1 and other nonstructural proteins associated with dsRNA in vesicle packets (Mackenzie et al., 1996; Westaway et al., 1997b), Lindenbach and Rice 1 To whom correspondence and reprint requests should be ad- (1997) showed that yellow fever virus RNA with a large dressed. Fax: (617) 3253-1401. E-mail: [email protected]. in-frame deletion of NS1 failed to replicate significant

amounts of RNA minus strands, but could be comple- mented in trans. In our previous studies on KUN replication, the analyses involved identification of the nonstructural proteins associated with RDRP activity or with putative sites of replication by radiolabeling, membrane purification, IF, or immunogold labeling of cryosections (Chu and West- away, 1992; Westaway et al., 1997a,b; Mackenzie et al., 1998). However, it was possible that the assumed sites of replication represented only sites of accumulation of dsRNA, rather than the actual sites of synthesis of viral RNA. Furthermore, because all the nonstructural proteins were continually being synthesized, it was not clear whether these newly synthesized proteins were required to maintain the active RDRP complex. In this paper we addressed these uncertainties by showing that nascent KUN RNA labeled during synthesis in situ was colocalized in foci with dsRNA and that these sites of replication retained replicative activity after all protein synthesis was inhibited.

RESULTS Stability of immunofluorescent foci of KUN replication sites Dual-labeled foci of NS3 (or of several other nonstructural proteins) and dsRNA were prominent by IF and by immunogold labeling of cryosections of KUN-infected cells by 24 h and were detectable at 16 h (Westaway et al., 1997b; Mackenzie et al., 1998). While such foci appeared to be stable, there are no data on whether they continue to function for long periods as replication sites or whether nonstructural flavivirus proteins in the replication complex remain active for many cycles of replication. We therefore continuously treated KUN-infected cells from 24 h with cycloheximide (CHXM) using a dose of 10Ϫ3.5 M (90 ␮g/ml), known to completely inhibit all protein synthesis (see Fig. 2), and examined the distribution by IF of the putative replication sites. We then measured the effects of CHXM treatment on viral RNA synthesis and yield of infectious virus. Figure 1 shows that perinuclear foci dual-labeled with anti-NS3 and anti-dsRNA antibodies appeared similar to those in untreated infected cells after treatment with CHXM from 24 to 28 h (compare a, b and c, d) or from 24

coverslips were treated at 23 h with actinomycin D and then 10Ϫ3.5 M CHXM was added from 24 to 28 h (c, d) or from 24 to 40 h (e, f). Parallel cultures were also treated with CHXM from 24 to 40 h, the drug was removed, and the cells were reincubated in normal medium for4h(g, h). No CHXM was added to the control infected cultures harvested at 24 h (a, b) and 40 h (i, j). All cell monolayers were fixed in cold acetone and dual-labeled with rabbit and guinea pig antibodies to NS3 and dsRNA, respectively. The secondary antibodies were conjugated to FIG. 1. Lack of effect of CHXM treatment in KUN-infected Vero cells fluorescein isothiocyanate or to Texas red, and dual label in d (only) on distribution of immunofluorescent foci of NS3 and dsRNA. Cells on shows coincidental foci as a yellow hue. 110 WESTAWAY, KHROMYKH, AND MACKENZIE

FIG. 2. Effects of CHXM treatment on protein synthesis (A) and viral RNA synthesis (B) in KUN-infected Vero cells treated and maintained in ACD as in Fig. 1. In (A) mock-infected and infected cells were radiolabeled from 24 to 27 h with [35S]methionine/cysteine in medium that included 10Ϫ3.5 M CHXM only for the cells represented in lanes 2 (mock) and 5 (KUN). Cells were either harvested in SDS immediately for gel electrophoresis (lanes 1 to 5) or chased for3hinmedium with excess methionine/cysteine included, either unsupplemented (lane 7) or supplemented with CHXM (lane 6), and then harvested. Cells represented in lane 3 and lane 4 (shorter exposure time of dried gel than for lane 3) were radiolabeled continuously from 24 to 30 h as a reference sample. Virus-specified proteins are identified in lane 7. Electrophoresis was in a 12.5% SDS discontinuous– polyacrylamide gel. In (B), KUN-infected cells were radiolabeled from 24 to 28 h with [32P]orthophosphate in phosphate-deficient medium, and the phenol-extracted RNA was electrophoresed in a 1% agarose gel (lanes 1, 2) or ina7Murea–polyacrylamide gel (lanes 3, 4), as described previously (Chu and Westaway, 1985). The viral RNA species are replicative form (RF), replicative intermediate (RI), and genomic-sized RNA (vRNA). The RI is retained completely at the origin in the urea–polyacrylamide gel. Autoradiographs were prepared from dried gels. to 40 h (compare e, f and i, j). The extent of clear fully reversible (not shown) and CHXM treatment had no coincidence of label in major foci was striking. As re- effect on the gel profile of viral proteins radiolabeled ported previously, staining by anti-NS3 antibodies also prior to treatment (compare lanes 3, 4, 6, and 7 in Fig. extended from the perinuclear region into a pattern of 2A). RNA was extracted from 32P-labeled cells and elec- dense endoplasmic reticulum (Westaway et al., 1997b). trophoresed in parallel with RNA from non-CHXM-treated We concluded that continuing protein synthesis (either cells (Fig. 2B); the gel profiles revealed only a small viral or cellular) was not essential for preserving the decrease in radiolabeled viral RNA synthesized in the apparent architecture of the replication sites and the CHXM-treated cells, in agreement with a measured de- colocalization of NS3 and dsRNA. Following CHXM treat- crease of about 25% in incorporated radiolabel. We con- ment of cells from 24 to 40 h, removal of the drug caused cluded that viral RNA synthesis did not require continu- no obvious rearrangement of the dual-labeled foci during ing protein synthesis and, in view of the IF data in Fig. 1, the next 4 h (Figs. 1g and 1h). Similarly no apparent that the viral replication complexes apparently continued rearrangement of foci occurred after reversal for 3 h to function at the same equivalent sites in CHXM-treated following CHXM treatment from 24 to 28 h (results not and untreated cells. shown). Supernatant culture fluids were also collected at the To assess the effect of protein synthesis inhibition on end of the radiolabeling periods and the virus yields viral RNA synthesis, KUN-infected cells were treated were compared by plaque assay. As found for RNA continuously from 23 h with ACD and CHXM and radio- synthesis, the inhibition of protein synthesis by CHXM labeled from 24 to 28 h either with [32P]orthophosphate treatment had a relatively small effect on virus yield, in phosphate-deficient medium or with [35S]methionine/ which was reduced relative to untreated control cultures cysteine. SDS–polyacrylamide gel electrophoresis of (107 PFU/ml) by about 50 and 90% after 4 h (24–28 h) or 35S-labeled cell lysates showed that all protein synthesis 16 h (24–40 h) of treatment, respectively. However, some was blocked by the CHXM treatment (Fig. 2A, lanes 2 visible CPE appeared during the 16-h treatment. Presum- and 5). The inhibitory effect on protein synthesis was ably, the released virus represented virus preformed NASCENT FLAVIVIRUS RNA IN STABLE REPLICATION COMPLEXES 111

FIG. 3. Incorporation and subcellular location shown by IF of pulsed-labeled BrU in KUN-infected Vero cells. (A) ACD-treated infected cells at 24 h postinfection were incubated for 60 min without (a, b) or with (c, d) 40 mM BrUTP in cationic liposomes prepared with DOTAP as described under Materials and Methods, fixed in cold acetone, and dual-labeled with antibodies to detect NS3, dsRNA, or incorporated BrU, as indicated. Mock-infected cells not treated with ACD were incubated for 20 min with 40 mM BrU and 40 mM BrUTP in liposomes and fixed in cold acetone (e) or in formaldehyde–methanol (f) (Westaway et al., 1997a), followed by labeling to detect incorporated BrU. (B) ACD-treated infected cells at 24 h postinfection were pulsed for 15 min (a–f) or for 60 min (g, h) with 100 mM BrU in cell culture medium, fixed in cold acetone, and dual-labeled with anti-BrdU and anti-dsRNA antibodies. Three fields of cells for both pulse periods were photographed to show reproducibility of coincidental staining. The secondary antibodies were conjugated to FITC or to Texas red. prior to CHXM treatment or newly assembled virus en- lian cells, it is essential to permeabilize them with strep- capsidated by a pool of structural proteins that had tolysin O (Jackson et al., 1993) or Triton X-100 (Wansink et accumulated prior to CHXM treatment. al., 1993; Pombo et al., 1994) or to use cationic liposomes (Haukenes et al., 1997; van der Meer et al., 1998). The Subcellular location of nascent KUN RNA first two methods involve disruption of membranes, incorporating short pulses of BrU which may perturb any membrane sites of flavivirus nas- The bromo-substituted nucleotide 5-bromouridine 5Ј- cent RNA synthesis, and the last entails a delay in uptake triphosphate (BrUTP) can be incorporated into RNA dur- of BrUTP. In initial experiments we delivered BrUTP by ing synthesis in cells and the incorporated analog can be transfection into KUN cells at 24 h postinfection using detected in subcellular sites by IF using primary antibod- DOTAP (see Materials and Methods). Incorporation of ies to 5-bromodeoxyuridine (BrdU; Jackson et al., 1993; BrUTP in the ACD-treated infected cells was detected by Wansink et al., 1993; Pombo et al., 1994; Restrepo- IF after 60 min of incorporation, and in dual-labeled cells Hartwig and Ahlquist, 1996; Van der Meer et al., 1998). many of the sites of incorporation were coincidental with This labeling procedure provided the opportunity to com- foci labeled with anti-dsRNA (Fig. 3A, panels c–d). No pare the site of incorporation of nascent flavivirus RNA incorporation was detected in cytoplasmic components with that of dsRNA, the proposed template, by dual- of ACD-treated mock-infected cells after a 20-min incu- labeled IF. In order to allow entry of BrUTP into mamma- bation with bromouridine (BrU) and BrUTP (results not 112 WESTAWAY, KHROMYKH, AND MACKENZIE shown), but if pretreatment with ACD was omitted, strong NS3 engaged in other events of virus replication (com- incorporation in newly synthesized cellular Br-RNA was pare the IF patterns in Fig. 3B with those in Fig. 1). Some detected in the nucleus (Fig. 3A, panels e, f). Interest- diffuse staining of dsRNA probably represents early de- ingly, the nucleoplasm was clearly stained only when the velopment of new sites of replication as observed during nuclear membrane was permeabilized for antibody ac- the latent period (Westaway et al., 1997b; Mackenzie et cess using formaldehyde/methanol fixation (Fig. 3A, al., 1998) and that were beyond the detection limit of our panel f), and the nucleolus was stained only after ace- antibodies to BrdU. tone fixation although in a rare possibly damaged cell the nucleoplasm was also stained (Fig. 3A, panel e). Confirmation that pulsed BrU is incorporated in an Similar patterns of nuclear staining were observed pre- RNase-resistant form into foci coincident viously in KUN-infected cells using the same fixation with double-stranded viral RNA conditions and antibodies to KUN core protein, which contains a bipartite nuclear localization signal and a Our model of flavivirus RNA synthesis proposed that possible nucleolar localization signal (Westaway et al., on average only a single strand of nascent RNA is bound 1997a). to the putative dsRNA template and forms the RI (Chu Because of the relatively slow uptake into cells of and Westaway, 1985; see calculation below). However, if BrUTP over several hours using DOTAP (see Boehringer rapid reinitiation involving synthesis of several nascent Mannheim 1998 Biochemicals catalogue) it was desir- strands on the same template is occurring (in conflict able to use a faster delivery of bromo-substituted uridine with our model), the period for which each nascent RNA in order to detect its incorporation into viral Br-RNA is fully bound would be quite brief, hence exposing some during short pulses. We showed previously that the cycle or most of these nascent strands to possible RNase of viral RNA synthesis to complete one full-length strand digestion as each is displaced by a newly initiated in KUN-infected Vero cells at 20 h is complete in about 15 strand. We have established that KUN core protein is not min (Chu and Westaway, 1985), similar to the cycle for present at the site of RNA synthesis, i.e., in vesicle dengue-2 virus (Cleaves et al., 1981), and according to packets (Westaway et al., 1997a,b), and hence it is un- our model of RNA synthesis, the nascent and recently likely to protect by encapsidation any partially released completed viral RNA remains bound in the recycling nascent RNA strands. The calculation of an average of dsRNA template until displaced during the next round of only about one nascent strand of flavivirus RNA per RNA synthesis (Chu and Westaway, 1985). In such ex- active RI is based on application of the formula devised periments, incorporation of [5-3H]uridine into RI and RF for poliovirus by Baltimore (1968): Percentage RNase- could be detected after a pulse of only 10 min. We resistance in the totally labeled RI ϭ (2/[2 ϩ N/2]) ϫ 100, reasoned that pulse incorporation of BrU in KUN-infected where the average length of each nascent strand is cells could be detected similarly by IF using anti-BrdU assumed to be one-half of a complete molecule, N/2 is antibodies as recently employed for detection of newly the average molecule equivalents of nascent strands N, made RNA in nuclei of HeLa cells (Jackson et al., 1998). and 2/(2 ϩ N/2) represents the ratio of labeled complete Hence there would be a brief window of time during (ϩ) and (Ϫ) strands (2) to the total number of labeled which truly nascent KUN RNA could be detected in rep- strands (2 ϩ N/2). However, in flavivirus RI only about lication sites in cells, without the delay or membrane 10% of incorporated radiolabel is in the RNA(Ϫ) strand disruption associated with use of BrUTP. Accordingly, late in infection (Cleaves et al., 1981), and virtually all of infected cells were treated with ACD and incubated with the RNA(ϩ) strands would be labeled late in infection. BrU for 15- and 60-min pulses (Fig. 3B). These results The estimate for ratio of RNase-resistant label in flavivi- showed unequivocal staining of nascent viral RNA by rus RI should therefore be 1.1/(1.1 ϩ N/2). Thus when the anti-BrdU antibodies after the 15-min pulse of BrU, and Baltimore formula is modified accordingly, the calculated dual-labeling with anti-dsRNA antibodies showed that N for number of nascent strands in flavivirus RI is 1.0 if the foci containing Br-RNA were prominently and consis- RNase resistance of the RI is 70% as for KUN after a tently coincident with the foci of dsRNA. After the longer 60-min pulse (Chu and Westaway, 1985) and 70% also for 60-min pulse, a similar pattern of dual-staining was ob- dengue-2 virus after a 10-min pulse (Cleaves et al., 1981). served, confirming the proposed stability of the sites of Thus there is an expectation that RNase treatment of replication. These results established that nascent RNA flavivirus replication complexes in situ would have very was incorporated directly into the proposed sites of fla- little if any observable effect on detectable BrU incorpo- vivirus replication indicated by the IF pattern of dsRNA, of rated during a 15-min pulse in the IF foci of dsRNA late which most if not all were actively engaged in viral RNA in infection, in accord with the cartoon of RI pulse- synthesis. Because the pattern of IF after a pulse label labeled with BrU in Fig. 4. with BrU specifically labels the sites of replicating viral To test the above prediction, we pulsed infected cells RNA, the coincidence of label with that of dsRNA is with BrU, permeabilized these cells with Triton X-100 to superior to that involving nonstructural proteins such as allow penetration of subsequently added RNase, and NASCENT FLAVIVIRUS RNA IN STABLE REPLICATION COMPLEXES 113

FIG. 4. Model of semiconservative and asymmetric synthesis of flavivirus RNA (Chu and Westaway, 1985) showing the expected effect of RNase treatment in high salt on KUN RI pulse-labeled in cells with BrU for 15 min. The depicted structure of RI assumes that it contains an average of only about one nascent RNA strand, which is base-paired on the template strand until displaced, and that the time for completion of synthesis of full-length RNA is about 15 min (see text). The solid lines represent unlabeled RNA, and the dotted lines represent pulse-labeled RNA. Of the label incorporated in RI late in flavivirus infection, only about 10% is in minus strand (Cleaves et al., 1981). observed by IF the effects of RNase digestion on incor- ously identified foci of dsRNA rather than in other unsus- porated BrU (Fig. 5A). In the control cells (i.e., no RNase), pected sites. Therefore KUN-infected cells were treated BrU was incorporated after a 15- or 60-min pulse into IF at 24 h with CHXM for 4 h and pulsed with BrU, as foci dual-labeled by anti-BrdU and anti-dsRNA antibodies previously, in order to detect by dual-labeled IF the loca- as previously (compare Fig. 5A, panels a, b and g, h, with tion of nascent Br-RNA. The results were unequivocal. Fig. 3B, panels a, b and g, h, respectively), and the Triton Foci of Br-RNA were readily identified after 15- and 60- X-100 treatment had no apparent effect on Br-RNA label- min pulses of BrU, and these were coincident with the ing, indicating the stability to detergent of the newly majority of foci labeled by anti-dsRNA antibodies (Fig. labeled Br-RNA foci. RNase digestion under high-salt 5B, a–d). It was therefore concluded that viral RNA syn- conditions also had virtually no discernible effect on the thesis occurring after CHXM switch-off of all protein dual-labeled IF patterns of Br-RNA and dsRNA after a 15- synthesis was associated with and located in the same or a 60-min pulse of BrU (Fig. 5A, panels c, d and i, j, subcellular location as the proposed dsRNA template respectively). The coincidence of BrU-labeled foci with employed during normal replication. These results con- most of the dsRNA foci was retained, and the size and firm independently the biochemical assay showing con- intensity of the Br-RNA foci appeared to be unaffected by tinuation of viral RNA synthesis by radiolabeling after this RNase treatment. When all RNA including dsRNA CHXM treatment (Fig. 2B). was rendered susceptible to digestion by RNase in low salt, both patterns of IF labeling were virtually eliminated Reproducibility of the subcellular location of as expected (Fig. 5A, panels e, f, k, l). Also as expected, immunofluorescent foci containing nascent nascent single-stranded Br-RNA in mock-infected cells Br-RNA using a KUN replicon C20SDrep was totally digested by RNase under high- and low-salt transfected in BHK cells conditions (Fig. 5A, panels n and o, respectively). C20SDrep RNA is a highly efficient KUN replicon derived We therefore concluded that nascent KUN RNA la- from the full-length KUN cDNA clone FLSD (Khromykh et al., beled after short pulses of BrU was strongly protected 1998) by deletion of the sequence of the structural genes from RNase digestion in situ where it was colocalized by (C.prM.E) except for the first 20 codons of the C gene and IF with dsRNA and that these results are thus in accord the last 21 codons of the E gene. Its replication was mon- with the above predictions based on our model of flavi- itored initially by detection of expressed NS3 using IF at virus RNA synthesis. 24 h posttransfection (results not shown). Under the same conditions, the transfected BHK cells were pulsed for 15 Confirmation by IF of the persistence of viral RNA min with 100 mM BrU and processed for IF using antibod- synthesis at BrU-labeled replication sites in the ies to BrdU and dsRNA, as previously (Fig. 5C). The ob- absence of protein synthesis served foci showed virtually complete coincidence of the In view of the earlier result showing that viral RNA dual label and must represent the sites of synthesis of synthesis continued in the absence of protein synthesis nascent replicon RNA in BHK cells. Because the replicon (Fig. 2) it was of interest to establish that under these lacks the structural genes, these sites are completely dis- conditions nascent RNA was incorporated in the previ- sociated from virus assembly and maturation. Thus this 114 WESTAWAY, KHROMYKH, AND MACKENZIE

FIG. 5. Immunofluorescent foci of nascent Br-RNA (A) after RNase treatment or (B) after synthesis in the absence of protein synthesis in KUN-infected Vero cells and (C) in replicon-transfected BHK cells. In (A) ACD-treated infected cells were pulse-labeled with 100 mM BrU for 15 min (a–f) or for 60 min (g–l); cells were immediately permeabilized by treatment with Triton X-100, fixed in cold acetone, and either left untreated (a, b, g, h) or incubated with RNase A in high salt (HS; c, d, i, j) or in low salt (LS; e, f, k, l) as described under Materials and Methods. Mock-infected Vero cells not treated with ACD, as in Fig. 3A, were pulse-labeled with BrU for 20 min and left untreated (m) or treated with RNase A in high salt (n) or low salt (o) as for infected cells, after prior treatment with Triton X-100 and permeabilization of the nuclear membrane with 95% methanol for 10 min at 20°C. Cells were dual-labeled with antibodies to detect incorporated BrU and dsRNA. The secondary antibodies were conjugated to FITC or to Texas red. Photographic exposure times and processing procedures were equivalent for untreated and RNase-treated cells. In (B), ACD-treated infected cells were incubated with 10Ϫ3.5 M CHXM from 25 to 28 h and then pulsed in the presence of CHXM with added 100 mM BrU for 15 min (a, b) or for 60 min (c, d), followed immediately by processing for dual-label IF as in A. In (C), BHK21 cells were electroporated with a high-efficiency KUN replicon C20SDrep (see text), pulsed for 15 min with 100 mM BrU at 24 h, and dual-labeled for IF as in A. result was obtained independent of the previous experi- In summary, our results have established the suitabil- ments, using a different cell line, a different RNA, and a ity of BrU for pulse labeling nascent viral RNA and anal- different route for entry of RNA. ysis by IF and shown that nascent KUN RNA colocalized NASCENT FLAVIVIRUS RNA IN STABLE REPLICATION COMPLEXES 115 by IF in an RNase-resistant form with the putative dsRNA infection. The implication from the CHXM experiments template in virus replication complexes. must be that the viral replicase complex recycles the same protein components or that after each cycle of RNA DISCUSSION synthesis the complex disperses and is reassembled from a relatively abundant pool of previously translated The results presented have established that recently proteins. Several considerations favor the first alternative synthesized KUN RNA is associated with cytoplasmic rather than the second. Thus the putative dsRNA tem- foci identifiable by dual-labeled IF using antibodies to plate was recycled about every 15 min (Chu and West- dsRNA and to incorporated BrU (Figs. 3 and 5). Our away, 1985, 1987), and RNA synthesis and the distribu- previous studies on the sites of KUN replication as- tion of postulated dsRNA templates were not signifi- sumed that the presence of dsRNA in discrete foci, cantly affected by CHXM treatment (Figs. 1 and 2). identified by both IF and immunogold labeling of vesicle Furthermore, the RDRP activity was shown to be unaf- packets in cryosections of infected cells, indicated the fected by treatment of flavivirus-infected cell lysates with putative site of viral RNA synthesis late in infection a range of detergents or by sedimentation away from (Westaway et al., 1997a,b; Mackenzie et al., 1998). Our soluble constituents (Grun and Brinton, 1987, 1988; Chu present results are in accord with this assumption. IF foci and Westaway, 1987, 1992). Dual enrichment of dsRNA were shown to contain BrU incorporated directly into and specific nonstructural proteins in or on individual truly nascent viral RNA synthesized during 15-min pulses vesicles within vesicle packets, as detected by immuno- and were precisely coincident with nearly all the dsRNA gold labeling of cryosections of KUN-infected cells, indi- foci (Figs. 3B and 5). The resistance of this short-pulsed cates that the replicase complex is in a relatively stable BrU-RNA in IF foci to RNase digestion in high salt (Fig. arrangement or configuration (Westaway et al., 1997b; 5A) fits well with the prediction under Results based on Mackenzie et al., 1998). Several of the components are our model of flavivirus RNA replication (Chu and West- probably bound strongly to specific membranes or lo- away, 1985, 1987). Furthermore, the IF sites of active cated within the lumen of the ER, e.g., the hydrophobic replication were stable despite the complete block in proteins NS2A and NS4A, and glycoprotein NS1, respec- translation by CHXM treatment (Figs. 1 and 5B) and were tively (Coia et al., 1988; Westaway et al., 1997b; Macken- readily identified also by pulse-labeling with BrU of BHK zie et al., 1998). Membranes possibly act as a scaffold for cells transfected with KUN replicon RNA (Fig. 5C). Syn- assembly of the RDRP that remains cohesive and active thesis of viral RNA in the presence of CHXM (Fig. 2) even after solubilization (Chu and Westaway, 1992). indicates that continuing cell or viral protein synthesis is Characterization of the flavivirus replication complex is not required late in infection. Although newly synthesized still incomplete. In addition to the present results and viral RNA has been detected previously by IF in mem- those showing the relationship between RF and RI (Chu brane-associated replication complexes after a 1-h pulse and Westaway, 1985, 1987, 1992), our major contributions of Br-UTP in liposomes in BHK21 and RK13 cells infected have been to establish that all the KUN nonstructural with equine arteritis virus (Van der Meer et al., 1998), or proteins except NS2B and NS4B are associated with after briefer pulses of BrUTP in brome mosaic virus- dsRNA in induced vesicle packets, the postulated site of infected barley protoplasts (Restrepo-Hartwig and Ahl- RNA replication (Westaway et al., 1997a,b; Mackenzie et quist, 1996), we believe our results represent the first al., 1998). We also showed previously that the structural demonstration for RNA viruses of BrU incorporation in proteins were not involved in the active KUN replication nascent viral RNA achieved using a short pulse of BrU. complex (Chu and Westaway, 1992; Khromykh and West- Although minor amounts of uncleaved NS3-NS4A away, 1997), as is again evident in the replicon experi- polyprotein have been reported for several flaviviruses ment (Fig. 5C). An essential role for NS5 was evident (Chambers et al., 1991; Lobigs, 1992; Zhang and Padma- because in transfected cells, KUN RNA with deletions in nabhan, 1993), posttranslational cleavage of the flavivi- NS5 of the RNA polymerase motif GDD or the S-adeno- rus polyprotein appears to be otherwise complete in sylmethionine binding site was not viable, but was com- minutes rather than in hours, as well documented by plemented in trans by KUN replicon RNA (Khromykh et kinetic studies of the processing of pulse labeled viral al., 1998). In replication studies in vitro with other flavivi- proteins for KUN and yellow fever virus infections ruses, purified dengue-1 virus NS5 copied nonspecific (Schrader and Westaway, 1988; Chambers et al., 1990b). template RNA (Tan et al., 1996), NS3 and NS5 were In view of this rapid proteolytic processing, and the lack implicated in flavivirus RNA replication by the demon- of significant reduction in RNA synthesis during several strated inhibitory effects of specific antibodies in the hours of CHXM inhibition (Fig. 2), it seems unlikely that RDRP assay for dengue-2 and Japanese encephalitis polyprotein precursors were involved in synthesis of viruses (Bartholomeusz and Wright, 1993; Edward and KUN RNA and hence like the similar poliovirus strategy Takegami, 1993), and both proteins of the latter virus (Novak and Kirkegaard, 1994), coupling between trans- bound to the terminal stem loop in the 3Ј UTR of genomic lation of viral RNA and replication does not occur late in RNA (Chen et al., 1997). Despite this progress, the puta- 116 WESTAWAY, KHROMYKH, AND MACKENZIE tive roles of NS3 and NS5 as RNA helicase and RNA Immunofluorescence and antibodies polymerase, respectively, in specific synthesis of flavivi- Coverslip cultures were washed, fixed in acetone at rus RNA have not yet been directly demonstrated. Ϫ20°C for 30 s, and processed for IF as described Our accumulated evidence on KUN replication previously (Westaway et al., 1997a,b). Rabbit anti-KUN strongly implicates the RF as the template for flavivirus NS3 antibodies have been described previously (West- RNA synthesis in RI, both being reactive with antibodies away et al., 1997b). Guinea pig anti-dsRNA antibodies to dsRNA. RF and RI were pulse-labeled with [3H]U were a generous gift from Dr. Jia Yee Lee (Macfarlane within about 10 min, radiolabeled genomic RNA became Burnet Centre for Medical Research, Melbourne, Austra- prominent during a 10- to 20-min chase period, and the lia). Mouse monoclonal IgG antibodies to BrdU, which labeled RF appeared to recycle rather than accumulate can react with BrdU incorporated in DNA and cross-react as a “dead end” product (Chu and Westaway, 1985). with Br-RNA (Jackson et al., 1993; Pombo et al., 1994), Similar results were obtained during in vitro RDRP as- were supplied by Boehringer Mannheim and by Sigma says (Chu and Westaway, 1987). Antibodies to dsRNA Chemical Co. Dual-labeling experiments used FITC- and coprecipitated the same nonstructural proteins shown to Texas red-conjugated species-specific anti-IgG (Edward be associated with dsRNA by cryo-IEM in induced ves- Keller, Australia). All photographs were taken with ASA icle packets, the only cytoplasmic location where enrich- 400 or ASA 1600 film, the color slides were scanned on ment of dsRNA was observed (Westaway et al., 1997b; an AGFA Arcus II Color Scanner, and images were pro- Mackenzie et al., 1998). In this paper, a 15-min pulse of cessed for presentation using an IBM computer and BrU was incorporated in nascent RNA convincingly co- Adobe and Powerpoint software. For ribonuclease diges- incident with dsRNA in IF foci and was obviously resis- tion of Br-RNA in situ cells on coverslips were pulse- tant to RNase digestion, as predicted by our model. The labeled with BrU and immediately permeabilized by source of the flavivirus-induced membranes and the pretreatment with 0.05% Triton X-100 on ice for 4 min. Cells cise relationships of the replicase components to one were washed briefly in cold PBS, fixed in cold acetone, another have not yet been defined. Our present charac- and incubated with 50 ␮g/ml RNase A (Sigma Chemical terization of the postulated site of RNA synthesis and Co.) for2hat37°C in 0.01ϫ SSC buffer (1ϫ SSC is 150 some of its properties should contribute significantly to mM NaCl, 15 mM sodium citrate, pH 7.0) or in 2ϫ SSC future studies on flavivirus replication. buffer, and then processed for IF. MATERIALS AND METHODS ACKNOWLEDGMENTS Cells and virus We are grateful to Mark Kenney for excellent assistance. This work Vero cells were grown in Medium 199 (Gibco) supple- was supported by a grant from the National Health and Medical mented with 5% fetal calf serum. In maintenance medium Research Council of Australia. This is SASVRC Publication No. 95. (Eagle’s minimum essential medium), serum was re- placed with 0.1% bovine serum albumin. Cells were in- REFERENCES fected at 37°C using a m.o.i. of 2 to 10 and treated with Baltimore, D. (1968). Structure of the poliovirus replicative intermediate 10 ␮g/ml actinomycin D (ACD; Merck, Sharpe and RNA. J. Mol. Biol. 32, 359–368. Dohme) for1hat23h;thereafter the ACD concentration Bartholomeusz, A. L., and Wright, P. J. (1993). Synthesis of dengue virus was reduced to 3 ␮g/ml (MEM/ACD medium). For the RNA in vitro: Initiation and the involvement of proteins NS3 and NS5. experiment presented in Fig. 5C only, BHK21 cells were Arch. Virol. 128, 111–121. Chambers, T. J., Hahn, C. S., Galler, R., and Rice, C. M. (1990a). electroporated with a KUN replicon described under Re- Flavivirus genome organization, expression, and replication. Annu. sults. Cells for IF were grown on coverslips, and for Rev. Microbiol. 44, 649–688. radiolabeling the cells were grown in 60-mm plastic petri Chambers, T. J., McCourt, D. W., and Rice, C. M. (1990b). Production of dishes. yellow fever virus proteins in infected cells: Identification of discrete polyprotein species and analysis of cleavage kinetics using region- Incorporation of bromo-substituted uridine in nascent specific polyclonal antisera. Virology 177, 159–174. Chambers, T. J., Grakoui, A., and Rice, C. M. (1991). Processing of the RNA yellow fever virus nonstructural polyprotein: A catalytically active Bromo-uridine (Sigma Chemical Co.) was added to NS3 proteinase domain and NS2B are required for cleavages at dibasic sites. J. Virol. 65, 6042–6050. infected cells at 24 h, as specified under Results. For Chen, C.-J., Kuo, M.-D., Chien, L.-J., Hsu, S.-L., Wang, Y.-M., and Lin, J.-H. incorporation using BrUTP (Sigma Chemical Co.), the (1997). RNA–protein interactions: Involvement of NS3, NS5, and 3Ј procedure of Haukenes et al. (1997) was used. Briefly, noncoding regions of Japanese encephalitis virus genomic RNA. BrUTP with or without BrU was incubated with an equal J. Virol. 71, 3466–3473. volume of DOTAP Liposomal Transfection Reagent Chu, P. W. G., and Westaway, E. G. (1985). Replication strategy of Kunjin ␮ virus: Evidence for recycling role of replicative form RNA as a tem- (Boehringer Mannheim) for 10 min at 20°C, and 10 lof plate in semiconservative and asymmetric replication. Virology 140, the mixture was added to 300 ␮l MEM/ACD on coverslip 68–79. cultures for specified periods. Chu, P. W. G., and Westaway, E. G. (1987). Characterization of Kunjin NASCENT FLAVIVIRUS RNA IN STABLE REPLICATION COMPLEXES 117

virus RNA-dependent RNA polymerase: Reinitiation of synthesis in proteinase: Accumulation of a NS3–4A precursor which requires vitro. Virology 157, 330–337. mature NS3 for efficient processing. J. Gen. Virol. 73, 2305–2312. Chu, P. W. G., and Westaway, E. G. (1992). Molecular and ultrastructural Mackenzie, J. M., Jones, M. K., and Young, P. R. (1996). Immunolocal- analysis of heavy membrane fractions associated with the replica- ization of the dengue virus nonstructural glycoprotein NS1 suggests tion of Kunjin virus RNA. Arch. Virol. 125, 177–191. a role in viral RNA replication. Virology 220, 232–240. Cleaves, G. R., Ryan, T. E., and Schlesinger, R. W. (1981). Identification Mackenzie, J. M., Khromykh, A. A., Jones, M. K., and Westaway, E. G. and characterization of type 2 dengue virus replicative intermediate (1998). Subcellular localization and some biochemical properties of and replicative form RNAs. Virology 111, 73–85. the flavivirus Kunjin nonstructural proteins NS2A and NS4A. Virology Coia, G., Parker, M. D., Speight, G., Byrne, M. E., and Westaway, E. G. 245, 203–215. (1988). Nucleotide and complete amino acid sequences of Kunjin Ng, M. L., Pedersen, J. S., Toh, B. H., and Westaway, E. G. (1983). virus: Definitive gene order and characteristics of the virus-specified Immunofluorescent sites in Vero cells infected with the flavivirus proteins. J. Gen. Virol. 69, 1–21. Kunjin. Arch. Virol. 78, 177–190. Cui, T., Sugrue, R. J., Xu, Q., Lee, A. K. V., Chan, Y.-C., and Fu, J. (1988). Novak, J. E., and Kirkegaard, K. (1994). Coupling between genome Recombinant dengue virus type 1 NS3 protein exhibits specific viral translation and replication in an RNA virus. Genes Dev. 8, 1726–1737. RNA binding and NTPase activity regulated by the NS5 protein. Pombo, A., Ferreira, J., Bridge, E., and Carmo-Fonseca, M. (1994). Virology 246, 409–417. Adenovirus replication and transcription sites are spatially separated Edward, Z., and Takegami, T. (1993). Localization and functions of in the nucleus of infected cells. EMBO J. 13, 5075–5085. Japanese encephalitis virus nonstructural proteins NS3 and NS5 for Restrepo-Hartwig, M. A., and Ahlquist, P. (1996). Brome mosaic virus viral RNA synthesis in the infected cell. Microbiol. Immunol. 37, helicase- and polymerase-like proteins co-localize in the endoplas- 239–243. mic reticulum at sites of viral RNA synthesis. J. Virol. 70, 8908–8916. Grun, J. B., and Brinton, M. A. (1986). Characterization of West Nile virus Schrader, A. P., and Westaway, E. G. (1988). Translation mapping with RNA-dependent RNA polymerase and cellular terminal adenylyl and the flavivirus Kunjin: Gene order and anomalies in translation of NS5. uridylyl transferases in cell-free extracts. J. Virol. 60, 1113–1124. Virus Res. 9, 323–333. Grun, J. B., and Brinton, M. A. (1987). Dissociation of NS5 from cell Tan, B.-H., Fu, J., Sugrue, R. J., Yap, E.-H., Chan, Y.-C., and Tan, Y. H. fractions containing West Nile virus-specific polymerase activity. J. Vi- (1996). Recombinant dengue type 1 virus NS5 protein expressed in rol. 61, 3641–3644. Escherichia coli exhibits RNA-dependent RNA polymerase activity. Grun, J. B., and Brinton, M. A. (1988). Separation of functional West Nile Virology 216, 317–325. virus replication complexes from intracellular membrane fragments. Van der Meer, Y., van Tol, H., Locker, J. K., and Snijder, E. J. (1998). J. Gen. Virol. 69, 3121–3127. ORF1a-encoded replicase subunits are involved in the membrane Haukenes, G., Szilvay, A.-M., Brokstad, K. A., and Kalland, K.-H. (1997). association of the arterivirus replication complex. J. Virol. 72, 6689– Labelling of RNA transcripts of eukaryotic cells in culture with BrUTP 6698. using a liposome transfection reagent (DOTAP). BioTechniques 22, Wansink, D. G., Schul, W., van der Kraan, I., van Steensel, B., van Driel, 308–312. R., and de Jong, L. (1993). Fluorescent labelling of nascent RNA Jackson, D. A., Hassan, A. B., Errington, R. J., and Cook, P. R. (1993). reveals transcription by RNA polymerase II in domains scattered Visualization of focal sites of transcription within human nuclei. throughout the nucleus. J. Cell Biol. 122, 283–293. EMBO J. 12, 1059–1065. Wengler, G., and Wengler, G. (1991). The carboxy-terminal part of the Jackson, D. A., Iborra, F. J., Manders, E. M. M., and Cook, P. R. (1998). NS3 protein of the West Nile flavivirus can be isolated as a soluble Numbers and organization of RNA polymerases, nascent transcripts, protein after proteolytic cleavage and represents an RNA-stimulated and transcription units in HeLa nuclei. Mol. Biol. Cell 9, 1523–1536. NTPase. Virology 184, 707–715. Khromykh, A. A., and Westaway, E. G. (1997). Subgenomic replicons of Wengler, G., and Wengler, G. (1993). The NS3 nonstructural protein of the flavivirus Kunjin: Construction and applications. J. Virol. 71, 1497– flavivirus contains an RNA triphosphatase activity. Virology 197, 265– 1505. 273. Khromykh, A. A., Kenney, M. T., and Westaway, E. G. (1998). Trans- Westaway, E. G., Khromykh, A. A., Kenney, M. T., Mackenzie, J. M., and complementation of flavivirus RNA polymerase gene NS5 by using Jones, M. K. (1997a). Proteins C and NS4B of the flavivirus Kunjin Kunjin virus replicon-expressing BHK cells. J. Virol. 72, 7270–7279. translocate independently into the nucleus. Virology 234, 31–41. Lai, M. M. C. (1998). Cellular factors in the transcription and replication Westaway, E. G., Mackenzie, J. M., Kenney, M. T., Jones, M. K., and of viral RNA genomes: A parallel to DNA-dependent RNA transcrip- Khromykh, A. A. (1997b). Ultrastructure of Kunjin virus-infected cells: tion. Virology 244, 1–12. Co-localization of NS1 and NS3 with double-stranded RNA and of Lindenbach, B. D., and Rice, C. M. (1997). Trans-complementation of NS2B with NS3, in virus-induced membrane structures. J. Virol. 71, yellow fever virus NS1 reveals a role in early RNA replication. J. Virol. 6650–6661. 71, 9608–9617. Zhang, L., and Padmanabhan, R. (1993). Role of protein conformation in Lobigs, M. (1992). Proteolytic processing of a Murray Valley encepha- the processing of dengue virus type 2 nonstructural prolyprotein litis virus nonstructural polyprotein segment containing the viral procursor. Gene 129, 197–205.