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Journal of Virology, November 2002, p. 10766-10775, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10766-10775.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Complementation Analysis of the Flavivirus Kunjin NS3 and NS5 Proteins Defines the Minimal Regions Essential for Formation of a Replication Complex and Shows a Requirement of NS3 in cis for Virus Assembly{dagger}

Wen Jun Liu, Petra L. Sedlak, Natasha Kondratieva, and Alexander A. Khromykh*

Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, and Clinical Medical Virology Centre, University of Queensland, Brisbane, Queensland 4029, Australia

Received 29 April 2002/ Accepted 22 July 2002


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ABSTRACT
 
We have previously reported successful trans-complementation of defective Kunjin virus genomic RNAs with a range of large lethal deletions in the nonstructural genes NS1, NS3, and NS5 (A. A. Khromykh et al., J. Virol. 74:3253-3263, 2000). In this study we have mapped further the minimal region in the NS5 gene essential for efficient trans-complementation of genome-length RNAs in repBHK cells to the first 316 of the 905 codons. To allow amplification and easy detection of complemented defective RNAs with deletions apparently affecting virus assembly, we have developed a dual replicon complementation system. In this system defective replicon RNAs with a deletion(s) in the nonstructural genes also encoded the puromycin resistance gene (PAC gene) and the reporter gene for ß-galactosidase (ß-Gal). Complementation of these defective replicon RNAs in repBHK cells resulted in expression of PAC and ß-Gal which allowed establishment of cell lines stably producing replicating defective RNAs by selection with puromycin and comparison of replication efficiencies of complemented defective RNAs by ß-Gal assay. Using this system we demonstrated that deletions in the C-terminal 434 codons of NS3 (codons 178 to 611) were complemented for RNA replication, while any deletions in the first 178 codons were not. None of the genome-length RNAs containing deletions in NS3 shown to be complementable for RNA replication produced secreted defective viruses during complementation in repBHK cells. In contrast, structural proteins produced from these complemented defective RNAs were able to package helper replicon RNA. The results define minimal regions in the NS3 and NS5 genes essential for the formation of complementable replication complex and show a requirement of NS3 in cis for virus assembly.


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INTRODUCTION
 
Kunjin virus (KUN) is an Australian flavivirus closely related to other members of the Japanese encephalitis virus subgroup. The KUN genome consists of single-stranded RNA of positive polarity comprising 11,022 nucleotides (17) with one long open reading frame encoding 3,433 amino acids in three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (8). NS proteins are assumed to be involved primarily in the replication of viral RNA as a part of a replication complex (RC). Extensive analysis of the KUN RC in infected cells by immunogold labeling, radioimmunoprecipitation assays, and glutathione S-transferase (GST)-pull down assays identified a consensus composition of the RC which consists of NS1, NS2A, NS3, NS4A, and NS5 proteins (29, 41). Specific cell proteins, such as elongation factor 1 alpha, which interacts with the terminal stem-loop of the 3' untranslated region (3'UTR) of several flaviviruses, may also be a part of the flavivirus RC (2, 3).

NS5 protein of flaviviruses consists of 905 amino acids and contains seven motifs characteristic for RNA-dependent RNA polymerase situated in the C-terminal two-thirds of the protein (24) (Fig. 1) and two methyltransferase motifs situated in the N-terminal part of the protein (25) (Fig. 1). NS5 proteins of dengue 1 virus (34), West Nile virus (32), and KUN (13) were shown to possess nonspecific in vitro RNA-dependent RNA polymerase activity. Flavivirus NS3 is a multifunctional protein possessing protease, helicase, and RNA triphosphatase activities (31). The protease activity resides in the first 167 to 180 amino acids (5, 6, 10, 11, 40), while the C-terminal region commencing from codons 160 to 170 contains motifs for nucleoside triphosphatase, RNA helicase, and RNA-stimulated triphosphatase (4, 9, 12, 27, 38, 39). C-terminally truncated NS3 product (NS3' or p50), resulting from an alternative cleavage (QRR{downarrow}GR [arrow marks cleavage site]) have been detected in tick-borne encephalitis virus- and dengue virus-infected cells (1, 30, 35), but the function of this truncated protein is not known.



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  		FIG. 1. Schematic representation and complementation efficiencies of genomic RNAs containing progressive C-terminal deletions in the NS5 gene. Full-length KUN cDNA construct FLSDXHDVr was used to prepare all the NS5 deletion constructs. FLSDXHDVr was prepared from FLSDX (19) by adding the hepatitis delta virus antigenomic ribozyme sequence (HDVr)-simian virus 40 polyadenylation signal (pA) cassette (37) immediately downstream of the last nucleotide of the KUN sequence. Numbers in the full-length construct show amino acid positions in the KUN polyprotein, and numbers in the NS5 deletion constructs show amino acid positions in the NS5 protein (8). Abbreviations: SP6, SP6 RNA polymerase promoter; MT, methyltransferase domain; RDRP, RNA polymerase domain. a, b, and c represent highly conserved amino acid regions in the flavivirus NS5 proteins (20). Numbers shown in the column on the right-hand side show the titers of complemented secreted viruses recovered in the CFs of repBHK cells transfected with corresponding RNAs with deletions and harvested at 3 (superscript a), 4 (superscript b), and 6 (superscript c) days after transfection. The titers were detected as described in Materials and Methods.

Our previous studies demonstrated that replication of the defective KUN genomic RNA with NS5 protein truncated to retain only the first 397 amino acids was efficiently complemented by the helper RC produced from the KUN replicon (subgenomic) RNA in repBHK cells, while genomic RNA with a further truncation leaving only the first 227 amino acids in NS5 was complemented very inefficiently (20). Amino acid sequence comparisons of the N-terminal region of NS5 proteins from different flaviviruses revealed the presence of three small (10 to 20 amino acids) highly conserved domains, which we named a (KUN amino acids 141 to 151), b (KUN amino acids 203 to 223), and c (KUN amino acids 343 to 365), and we proposed that the retention of these domains was essential for efficient trans-complementation. In similar complementation studies with genomic RNAs from KUN with NS3 deletions, we showed that deletions encompassing the region between amino acids 178 and 567 of NS3 protein were complemented in repBHK cells (21). We also showed that KUN genomic RNAs with combined large in-frame deletions in NS1, NS3, and NS5 proteins could be complemented (21). Interestingly, while complementation of KUN RNAs with deletions in NS1 and NS5 allowed formation of the defective secreted virions, complementation of any RNAs containing deletions in NS3 did not permit recovery of the defective secreted viruses. It was suggested from these results that full-length NS3 protein was required in cis for virus assembly and/or release.

The absence of amplification of complemented genomic RNAs with deletions in NS3 via spread of complemented viruses made further complementation mapping of the NS3 gene difficult. Here we report the development of a dual replicon complementation system in which a defective KUN replicon RNA encoding the puromycin resistance gene (PAC gene) is used for complementation in repBHK cells (originally established by selection with G418), and a cell population producing both the complemented defective and the helper replicon RNAs is established by selection with two antibiotics. Complementation systems using helper repBHK cells and defective genomic or replicon RNAs were used in this study to define further minimal complementable regions in the KUN NS5 and NS3 proteins and to show the requirement of full-length NS3 in cis for virus assembly.


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MATERIALS AND METHODS
 
Cells. BHK-21 cells were maintained in Dulbecco's modification of minimal essential medium supplemented with 10% fetal bovine serum. repBHK cells containing stably replicating KUN replicon RNA (19) were maintained in the same medium supplemented with 0.5 to 1 mg of G418 (Geneticin; Invitrogen, San Diego, Calif.) per ml.

Construction of plasmids. All deletion constructs were prepared from our previously described KUN full-length cDNA clone FLSDX (19), KUN replicon vectors (18, 19, 36, 37), and appropriate intermediate plasmids (20, 21, 23) by using PCR amplification of small cDNA fragments with high-fidelity Pfu DNA polymerase (Stratagene, La Jolla, Calif.) and primers containing appropriate restriction sites. All deletions were designed to maintain an open reading frame. The details of the plasmid constructs can be obtained from the corresponding author upon request.

RNA transcription and transfection, IF, and Northern blotting. All RNA transcripts were prepared with SP6 RNA polymerase from XhoI-linearized plasmid DNAs, and ~10 µg of each RNA was electroporated into 2 x 106 BHK or repBHK cells as described previously (19). Detection of replication and expression of complemented defective genomic KUN RNAs in transfected repBHK cells was performed by immunofluorescence (IF) analysis with KUN anti-E antibodies and by Northern blot analysis with 32P-labeled cDNA probe complementary to the prM region as described previously (19-21).

Determination of the titers of defective secreted viruses. The titers of the defective secreted viruses (in infectious units [IU] per milliliter) were determined by counting E-positive foci of repBHK cells at 2 days after infection with serial dilutions of the culture fluids (CFs) collected from repBHK cells transfected with defective full-length RNAs.

Selection and characterization of cells stably producing complemented defective KUN replicon RNAs. Thirty-six to forty-eight hours after electroporation of repBHK cells with defective KUN replicon RNAs encoding PAC and ß-galactosidase (ß-Gal) (see Fig. 4), puromycin (5 µg/ml; Sigma-Aldrich, Castle Hill, New South Wales, Australia) was added and cells were allowed to grow in the medium with puromycin for an additional 5 to 9 days. The puromycin-containing medium was replaced every 2 to 3 days to ensure continuous selection of puromycin-resistant cells.



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  		FIG. 4. Replicon deletion constructs and their relative complementation efficiencies. (A) KUN replicon constructs with single and combined deletions in the NS1, NS3, and NS5 genes. The repPACß-gal construct represents the parental replicon used for preparation of the deletion constructs. Numbers show corresponding amino acid positions in the KUN polyprotein (8). Two copies of foot-and-mouth disease virus 2A autoprotease sequence (FMDV2A) upstream and downstream of the ß-Gal gene allow cytoplasmic release of PAC and ß-Gal gene products upon polyprotein translation (16, 37). Open boxes show in-frame deletions in NS1, NS3, and NS5 genes with the corresponding amino acid position of the deletion boundaries indicated. Abbreviations: C20, first 20 codons of KUN C protein; E22, last 22 codons of KUN E protein; SP6, SP6 RNA polymerase promoter for in vitro RNA transcription. (B) Deletions in the NS3 gene. Filled boxes represent KUN NS3 protein, with the numbers indicating the first and the last NS3 amino acids. Open boxes show in-frame deletions as in panel A but with the numbers representing NS3 codons. QRR{downarrow}GR in the repdNS1.1/3.8 construct shows a putative cleavage site releasing a truncated NS3' protein product. Note that all these NS3 deletions were accompanied by the deletion in NS1 protein (dNS1.1) (21). Relative complementation efficiencies shown on the right are approximate estimates from the results of ß-Gal expression (see Fig. 5A) and Northern blotting (see Fig. 5B). Symbols: ++, ~20 to 30% complentation; +++, ~40 to 50% complementation; ++++, ~80 to 100% complementation; —, no complementation.

Detection of replication and expression of complemented defective KUN replicon RNAs in transfected and puromycin-selected repBHK cells was performed by in situ staining with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal), or by ß-Gal assay of cell lysates using the ß-Gal enzyme assay system (Promega, Madison, Wis.). The amount of produced ß-Gal was expressed in milliunits (enzymatic activity) per 105 puromycin-resistant cells, which was calculated as recommended by the manufacturer using purified ß-Gal protein of known specific activity as a standard. The replication and accumulation of helper and complemented defective replicon RNAs were also analyzed by Northern blot with 32P-labeled cDNA probes representing the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence and a fragment from ß-Gal gene, respectively.


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RESULTS
 
The first 316 amino acids of NS5 protein are required for efficient trans-complementation. To examine whether the retention of one or more of the conserved domains a, b, or c in the N-terminal region of KUN NS5 protein was required for efficient complementation of replication of the defective KUN genomic RNA (20), we prepared a first series of deletion constructs, ns5c, ns5dc, ns5dc2, ns5dbc, and ns5dabc, where each of these domains was either retained or precisely deleted (Fig. 1). The deletion constructs were prepared on a background of the KUN full-length cDNA clone FLSDXHDVr, which was constructed from FLSDX plasmid (19) by inserting the hepatitis delta virus antigenomic ribozyme sequence (HDVr) immediately downstream of the last nucleotide of KUN cDNA. This modification was shown previously to improve efficiency of KUN replicon RNA transfection and replication (37). All these and the rest of the NS5 deletion constructs also contained a GST gene inserted in place of the deletions (not shown in Fig. 1) for a purpose not relevant to the scope of this study. The constructs ns5c and ns5dc2 are similar to our previous deletion constructs ns5dNB and ns5dBsB (20), respectively, except that they now have precise boundaries of the retained domains c and b, respectively. Transfection of RNAs produced from the first series of deleted constructs into repBHK cells showed efficient complementation of replication of ns5c and ns5dc constructs by day 3 after transfection, as detected by 100% of E-positive cells in IF analysis (data not shown) and strong signals in Northern blot analysis with a prM-specific probe (Fig. 2A, lanes 2 and 3 for ns5c and lanes 5 and 6 for ns5dc). Only a few E-positive cells were detected at 3 days after electroporation of ns5dc2 and ns5dbc RNAs, and longer exposure of the Northern blot to X-ray film resulted in detection of a weak signal in the 6-day sample from ns5dc2-transfected cells (Fig. 2A, lanes 7 to 9). No complementation was observed in repBHK cells transfected with ns5dbc and ns5dabc RNAs by 6 days after transfection (Fig. 2A, lanes 10 to 15). Titration of the defective viruses recovered in the 3-day CFs of cells transfected with ns5c and ns5dc RNAs on repBHK cells showed the presence of 4 x 106 and 1.2 x 106 infectious particles per ml, respectively (Fig. 1). No infectious particles were detected in undiluted 3-day CF collected from ns5dc2- and ns3dbc-transfected cells (not shown). However, titration of corresponding 6-day CF did result in detection of very low titers of infectious particles, ~100 and ~10 IU/ml, respectively (Fig. 1). No E-positive cells at all were detected in 6-day CF from ns5dabc-transfected cells. Thus, it appears from these results that retention of the region between domains b and c, but not of the domain c was essential for efficient trans-complementation.



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  		FIG. 2. Detection of complementation of genomic RNAs with deletions in the NS5 gene. Seven and a half µg (A) and 10 µg (B) of total RNA isolated from repBHK cells at indicated times after transfection with NS5-deleted genomic RNAs identified as in Fig. 1 were used in Northern blot analysis with a 32P-labeled cDNA probe representing the KUN 3'UTR sequence. Lanes 1 to 6 in (A) and lanes 1 to 13 in (B) were exposed to X-ray film for 7 h, lanes 7 to 16 in (A) were exposed for 2d. The samples in corresponding gels in (A) and (B) contained similar amounts of total cell RNA as determined by visualization of 28S and 16S rRNA bands by staining of the gel with ethidium bromide. The arrow indicates the position in the gel of RNA of about 11kb determined relative to migration in the same gel of a 1Kb plus DNA ladder (Invitrogen) stained with ethidium bromide. The control lane contains ~10 ng of in vitro-transcribed full-length KUN RNA. (C) Reverse transcription (RT) and PCR amplification of complemented genomic RNAs isolated from secreted complemented viruses. RNAs were recovered from 4-day CF of repBHK cells transfected with the indicated genomic RNAs with NS5 deletions using a Nucleospin RNA virus kit (Macherey-Nagel, Duren, Germany). The presence of NS5 deletions in isolated viral RNAs was then detected by RT-PCR amplification using a Superscript one-step RT-PCR kit (Gibco BRL, Life Technologies, Carlsbad, Calif.) and primers ns5dSAM-F (KUN nucleotides 7935 to 7952) and GST-BamR (complementary to the C terminus of GST). The expected sizes of the RT-PCR fragments are 1,509 bp for ns5c (lane 2), 1,440 bp for ns5dc (lane 3), 1,351 bp for ns5dc1.1 (lane 4), and 1,288 bp for ns5dc1.2 (lane 5).

To define further the minimal complementable amino acid sequence in NS5 located in the region between domains b and c (amino acids 224 and 343), we prepared two deletion constructs ns5dc1.1 and ns5dc1.2 which retained the first 316 and 293 amino acids, respectively (Fig. 1). Northern blot analysis of repBHK cells transfected with these two RNAs and ns5dc RNA showed that ns5dc1.1 RNA was complemented with efficiency similar to ns5dc RNA, while ns5dc1.2. RNA was complemented inefficiently (Fig. 2B). The titers of complemented secreted viruses in 4-day CFs from ns5dc- and ns5dc1.1- transfected cells were also similar (9 x 105 and 5 x 105 infectious particles per ml, respectively), while only ~103 infectious particles per ml were detected in 6-day CF from ns5dc1.2-transfected cells, suggesting that the NS5 amino acids 294 to 316 were required for efficient complementation. Reverse transcription-PCR analysis of RNAs isolated from secreted complemented viruses confirmed retention of introduced deletions (Fig. 2C). No E-positive cells were detected after transfection with all the deletion constructs into normal BHK cells. Also, only a few E-positive normal BHK cells were detected after infection with the lower dilutions or undiluted CFs collected from repBHK cells transfected with most of the deleted RNAs (data not shown). As we previously demonstrated, this apparently resulted from complementation of replication of defective full-length RNA by helper replicon RNA both of which were delivered into the same cells by coinfection with two types of particles, one containing encapsidated defective full-length RNA and another one containing encapsidated helper replicon RNA (19). We concluded from these results that retention of the first 316 amino acids of KUN NS5 protein, which included conserved domains a and b, was required for efficient trans-complementation of viral RNA replication by the helper replication complex and the assembly and release of defective infectious virions.

Dual replicon complementation system. The dual replicon complementation system employs the use of repBHK cells persistently producing KUN replicon RNA which also encodes the neomycin resistance gene (Fig. 3) (19) as a helper for complementation of a second replicon RNA containing deletions in the nonstructural region of the genome (Fig. 3). This second (defective) replicon RNA also encodes the PAC gene and the reporter gene for ß-Gal. Complementation of replication of the defective replicon RNA results in expression of PAC and allows establishment of cell lines stably producing complemented defective RNA by selection with puromycin. Since defective replicon RNA can produce PAC only if it is replicating, and it can replicate only if it is complemented by the helper RC produced from the replicating helper replicon RNA, selection with only one antibiotic, puromycin, encoded by the defective replicon RNA is sufficient to establish a cell line stably producing both defective and helper replicon RNAs. Expression of ß-Gal by the complemented defective RNAs should allow detection of positive cells by X-Gal staining as well as comparative quantitative analysis of complementation via ß-Gal assays in cell lysates. The replication and accumulation of the defective replicon RNA in established stable cell lines can be confirmed and distinguished from the helper replicon RNA by Northern blot analysis with ß-Gal-specific radiolabeled probe. The advantage of the dual replicon complementation system over the complementation system using defective genomic RNAs is that the former focuses on the effects of complementable deletions or mutations on RNA replication only and virus assembly is not involved.



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  		FIG. 3. Dual replicon complementation system. Helper repBHK cells stably producing KUN replicon RNA with its functional replication complex (19) were used to complement replication of transfected defective KUN replicon RNAs containing deletions in the nonstructural genes (marked by asterisks). The defective replicon RNA also encodes the puromycin resistance gene (PAC) and the ß-Gal gene (see Fig. 4 for details), which are produced and released as a result of translation from complemented replicon RNA. Expression of PAC allows selection of stable cell line continuously producing complemented defective replicon RNA and expression of ß-Gal allows easy detection of positive cells by in situ X-Gal staining, as well as quantitative analysis of efficiencies of complementation via ß-Gal assay of the cell lysates. Accumulation of complemented defective replicon RNAs in puromycin-resistant cells can also be determined and distinguished from the helper replicon RNA by Northern blot analysis with a ß-Gal-specific radioactive probe.

Comparison of complementation efficiency of replicon RNAs with single and combined deletions in the NS1, NS3, and NS5 genes. We have previously demonstrated that replication of full-length KUN RNAs with a large deletion in the NS5 gene; with combined double deletions in the NS1 and NS5 genes; or with combined triple deletions in the NS1, NS3, and NS5 genes were complemented in repBHK cells (21). The complementation efficiencies for defective RNAs with a single deletion in NS5 (ns5dAB) (20) and combined double deletions in NS1 and NS5 (dNS1.1/5AB) (21), determined by the titers of the recovered defective viruses at 4 days after RNA transfection, were ~5 x 106 and 106 IU/ml, respectively, when assayed on repBHK cells (see Table 1 in reference 21). However, we could not compare the complementation efficiencies of these two RNAs with that of the RNA containing combined triple deletions in NS1, NS3, and NS5 genes due to the inability of the latter RNA to produce defective virus during complementation. In order to be able to compare the complementation efficiencies of RNAs with these deletions, we transferred the deletions into the replicon constructs encoding PAC and ß-Gal genes (Fig. 4A) and transfected the resulting replicon RNAs, repdNS5AB, repdNS1.1/5AB, and repdNS1.1/3.3/5AB, into repBHK cells. Propagation of cells in medium containing puromycin at 5 µg per ml for 7 to 11 days after transfection resulted in selection of puromycin-resistant cell colonies with all the cells expressing ß-Gal, as detected by X-Gal staining (data not shown). Complementation efficiencies of defective RNAs in these puromycin-resistant cells were compared by quantitation of ß-Gal expression in the corresponding cell lysates (Fig. 5A). Judging by the levels of ß-Gal expression, the complementation efficiency of RNA with a single deletion in NS5 gene (repdNS5AB, Fig. 4A) was slightly higher than that of the RNA with combined double deletions in NS1 and NS5 genes (repdNS1.1/5AB, Fig. 4A). These were 218.5 ± 12 mU/105cells and 186.9 ± 7.2 mU/105cells, respectively (values are presented as means ± standard deviations throughout) (Fig. 5A), which correlated well with the CF infectious titers in our previous complementation results with corresponding full-length RNAs (see Table 1 in reference 21). The complementation efficiencies of replicon RNAs with combined double deletions in NS1 and NS5 (repdNS1.1/5AB [Fig. 4A]) and combined triple deletions in NS1, NS3, and NS5 (repdNS1.1/3.3/5AB [Fig. 4A]) were similar, 186.9 ± 7.2 mU/105cells and 169.7 ± 5.3 mU/105cells, respectively (Fig. 5A), demonstrating that addition of a large deletion in NS3 to the deletions in NS1 and NS5 apparently did not significantly reduce the efficiency of complementation of the resulting RNA. Note that although repdNS1.1/3.3/5AB-transfected repBHK cells were harvested later (11 days after transfection) than repdNS5AB- and repdNS1.1/5AB-transfected repBHK cells (7 days after transfection) due the lower growth rate of the former, ß-Gal expression levels were calculated per the same amount of puromycin-resistant cells. The ß-Gal expression results were confirmed by the results of Northern blots with a ß-Gal-specific probe showing the total amount of accumulated complemented RNAs at the time of RNA isolation (Fig. 5B; top panel). The amounts of helper replicon RNA as well as host-keeping gene (ß-actin) mRNA in all three established cell lines were similar, as confirmed by Northern blotting with the probes to EMCV IRES (present in the helper replicon RNA) or ß-actin, respectively (Fig. 5A; middle and bottom panels, respectively).



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  		FIG. 5. Complementation of KUN RNAs with single and combined deletions in the NS1, NS3, and NS5 genes. repBHK cells were electroporated with the indicated replicon RNAs with deletions, and cells producing complemented RNAs were selected by incubation with the medium containing puromycin at 5 µg/ml. At 7 to 11 days after commencement of selection, puromycin-resistant cells were trypsinized, counted, lysed, and assayed for ß-Gal expression by ß-Gal assay (A) and for accumulation of complemented RNA by Northern blot with a 32P-labeled ß-Gal-specific cDNA probe (B, top panel). (A) Results show ß-Gal enzymatic activity produced in 105 puromycin-resistant cells determined as described in Materials and Methods. Error bars, standard deviations. (B) Approximately 10 µg of total RNA was used, and the presence of similar amounts of the helper replicon RNA and total cellular RNA in different samples was confirmed by using 32P-labeled cDNA probes detecting EMCV IRES (middle panel) and ß-actin (bottom panel), respectively. The arrow indicates the position in the gel of RNA of about 11.6 kb (top panel), 11 kb (middle panel), and 1.1 kb (bottom panel), determined relative to migration in the same gel of the ethidium bromide-stained 1Kb plus DNA ladder (Invitrogen). The control lane contains ~10 ng of in vitro-transcribed full-length KUN RNA.

The N-terminal 178 codons of NS3 gene are essential for complementation of KUN RNA replication. Encouraged by the successful complementation of replicon RNAs with large deletions of the helicase region of NS3 gene (Fig. 4 and 5), we decided to define the maximum extent of deletions in the NS3 gene which can be complemented in trans. We prepared a series of progressive deletions towards the N terminus and the C terminus of NS3 gene (Fig. 4B). All these constructs also had a deletion in the NS1 gene (dNS1.1) (21). The resulting RNAs were electroporated into repBHK cells, and cells producing complemented defective RNAs were selected by incubation in the medium with puromycin. X-Gal staining of cells at 9 days after commencement of selection (11 days after transfection) showed that replicon RNA with a deletion of the region between amino acids 178 and 567 was complemented efficiently, as indicated by the detection of a significant number (n = 169) of ß-Gal-expressing cell colonies (repdNS1.1/3.3 in Fig. 6A). ß-Gal expression analysis of the cell lysates also showed production of high levels of ß-Gal (175 ± 5.6 mU/105 cells, Fig. 6B). Replicon RNA with an additional deletion of 44 codons extending further into the NS3 C terminus and retaining only the last eight codons of the KUN NS3 gene (repdNS1.1/3.7 [Fig. 4B]) was also complemented as judged by X-Gal staining and ß-Gal assay, albeit with a lower efficiency (59 ß-Gal-expressing cell colonies and 69.7 ± 5.1 mU/105 cells, respectively; Fig. 6). The replication and accumulation of complemented defective RNAs in these puromycin-resistant cells were confirmed by Northern blot analysis of total cellular RNA with ß-Gal-specific probe (Fig. 7A, top panel). In addition, the retention of introduced deletions in NS3 in complemented RNAs isolated from these puromycin-resistant cells was confirmed by RT-PCR analysis, using RT with a primer selectively recognizing complemented replicon RNAs and PCR with a pair of primers surrounding the deleted regions (Fig. 7B).



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  		FIG. 6. Complementation of replicon RNAs with deletions in the NS3 gene. Cells producing complemented replicon RNAs were selected and characterized as described in the legend to Fig. 5, except that at 9 days after commencement of puromycin selection they were either stained in situ with X-Gal to count the numbers of puromycin-resistant and ß-Gal-expressing cell colonies (indicated under the panels [A]) or lysed and analyzed for ß-Gal expression (B).



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  		FIG. 7. Northern blot (A) and RT-PCR (B) analyses of complemented RNAs with NS3 deletions. (A) Approximately 10 µg of total RNA isolated from repBHK cells transfected with RNAs with deletions and selected with 5-µg/ml puromycin for 9 days (passage 2) were subjected to Northern blotting with 32P-labeled cDNA probes detecting ß-Gal cDNA (top panel, complemented RNAs), EMCV IRES (middle panel, helper replicon RNA), and ß-actin (bottom panel, cellular RNA). The arrow indicates the position in the gel of RNA of about 11.6 kb (top panel), 11 kb (middle panel), and 1.1 kb (bottom panel), determined relative to the migration in the same gel of the ethidium bromide-stained 1Kb plus DNA ladder (Invitrogen). The control lane contains ~10 ng of in vitro-transcribed full-length KUN RNA. (B) One microgram of RNA isolated (as in panel A) was reverse transcribed using SuperScript II reverse transcriptase (Gibco BRL, Life Technologies) and primer 3'UTRdxR. Primer 3'UTRdxR is complementary to the beginning of 3'UTR sequence (KUN nucleotides 10443 to 10469), which is not present in the helper replicon RNA in repBHK cells (20). Obtained cDNA was then PCR amplified with primers FLdNS3NdeF (KUN nucleotides 4612 to 4629) and NS5dSAM-R (complementary to KUN nucleotides 7888 to 7903). The expected sizes of the RT-PCR fragments are 2,127 bp for redNS1.1/3.3 RNA (lane 2), 1,995 bp for repdNS1.1/3.7 RNA (lane 3), and 1,269 bp for repdNS1.1/3.8 RNA (lane 4).

None of the deletions extending further into the NS3 N terminus (upstream of codon 178) were complemented for RNA replication, as judged by the absence of ß-Gal-expressing cell colonies after selection with puromycin (see for example results for repdNS1.1/3.9 and repdNS1.1/3.4 in Fig. 6A; data not shown for other constructs). The results in this section demonstrated that deletion of as many as 434 codons in the C terminus of NS3 gene (codons 179 to 611) can be complemented in trans, while the first 178 codons of NS3 are required in cis for complementation of RNA replication.

Full-length NS3 is required in cis for virus assembly. Our previous complementation experiments with genomic KUN RNAs containing different deletions in the NS3 gene showed that none of these RNAs could be assembled into secreted defective virions during complementation in repBHK cells (21). One of the suggested explanations for the lack of virus assembly and/or release was possible involvement of C-terminally truncated NS3 product, NS3' or p50, resulting from an alternative cleavage at QRR{downarrow}GR site (Fig. 4B) and shown to be present in tick-borne encephalitis virus- and dengue virus-infected cells (1, 30, 35) and in KUN-infected cells (W. J. Liu and A. A. Khromykh, unpublished results) in virus assembly. To examine a possible role of truncated NS3' product in the production of the secreted defective virions, we first prepared the replicon deletion construct repdNS1.1/3.8 in which the proposed cleavage site QRR{downarrow}GR was retained (Fig. 4B) and tested whether this deletion can be complemented for RNA replication using the dual replicon complementation system. The results of X-Gal staining, ß-Gal assay, and Northern blotting clearly demonstrated that replication of repdNS1.1/3.8 RNA was complemented in repBHK cells, albeit with lower efficiency than that of repdNS1.1/3.3 RNA (Fig. 6 and 7). We then prepared genome-length RNA dns1.1/3.8/5AB containing the deletion NS3.8 in addition to deletions in NS1 and NS5 by replacing a large NS3.3 deletion (codons 178 to 567 [Fig. 4B]) in the ns1.1/3.3/5AB construct (21) with the smaller NS3.8 deletion (codons 465 to 567 [Fig. 4B]) in order to examine whether restoration of the NS3' cleavage site would allow production of secreted defective virus. Similar to our previous results with NS3 deletions, transfection of repBHK cells with dns1.1/3.8/5AB RNA resulted in detection of a low number (~2 to 5%) of E-positive cells 2 days after transfection (data not shown), demonstrating complementation of RNA replication in positive cells. However, no increase in the proportion of E-positive cells or appearance of multicellular E-positive foci was observed at day 4 and day 6 after transfection with dns1.1/3.8/5AB RNA (data not shown). When undiluted CFs from these repBHK cells collected at 2, 4, and 6 days after transfection were assayed for infectivity on fresh repBHK cells, no E-positive cells were detected 2 days later (data not shown), clearly demonstrating that restoration of the NS3' cleavage site in NS3-deleted RNA did not lead to the assembly of secreted defective virions.

Another suggestion for the inability of RNAs with deletions in the helicase region of NS3 to be packaged into secreted virus particles was the possible negative effect of these deletions on protease activity of the NS2B-(deleted)NS3 complex in cis for the cleavage between C and prM proteins. If this is true, then structural proteins produced from the complemented NS3-deleted genomic RNA should be also deficient in packaging of the helper replicon RNA. Indeed, our previous preliminary attempts to find infectious virus particles with encapsidated helper replicon RNA in 6 days CF of dns1.1/3.3/5AB genomic RNA-transfected repBHK cells failed (21). However, we also showed that the amount of complemented RNA (and obviously structural proteins produced from it) later in transfection (6 days) was significantly lower (almost undetectable) than that earlier in transfection (2 and 4 days) (21). This decrease in the level of accumulated complemented defective RNA was attributed to the apparent loss of positive cells during their harvest for RNA isolation later in transfection due to the cytopathic affect of accumulated structural proteins (21). To examine whether the structural proteins produced from the complemented NS3-deleted RNAs earlier in transfection and complementation (4 days) were functionally capable of packaging helper replicon RNA and producing secreted virus particles, 100 µl of each undiluted 4 days CF from dns1.1/3.3/5AB RNA- or dns1.1/3.8/5AB RNA-transfected repBHK cells were used to infect normal BHK cells which were then analyzed for NS3 expression by IF analysis 2 days later. A small number of single NS3-positive cells were detected in both CFs (23 and 39 cells, respectively), thus demonstrating that structural proteins produced from complemented NS3-deleted RNAs earlier in transfection were in fact in sufficient amounts and capable of packaging RNA (at least helper replicon RNA) into secreted virus particles. These NS3-positive BHK cells were not positive for E expression when assayed by dual IF analysis with anti-NS3 and anti-E antibodies, showing that none of these NS3-positive cells was infected with particles containing defective NS3-deleted genomic RNAs.

To examine whether any virus particles containing packaged defective genomic RNAs were produced in cells but could not be secreted into the CF, we prepared lysates from dns3.3 RNA (full-length RNA with a single NS3.3 deletion)- or dns1.1/3.8/5AB RNA-transfected repBHK cells by freeze-thawing and sonication and used these cell lysates to infect repBHK cells. We have used this approach previously to isolate intracellular virus-like particles containing encapsidated replicon RNA and showed that sonication did not adversely effect their infectivity (A. N. Varnavski and A. A. Khromykh, unpublished results). No anti-E-positive repBHK cells were detected at 2 days after infection with 2- and 4-day cell lysates (data not shown), demonstrating that no intracellular defective virus particles were produced during complementation of defective genomic RNAs in repBHK cells. These results showed that the defect caused by NS3 deletion was at the stage of assembly of virus particles, and not of their secretion.


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DISCUSSION
 
In this study we completed complementation mapping of the KUN NS5 gene by defining the minimal region (first 316 codons) required for efficient trans-complementation by the helper RC produced in repBHK cells. The minimal region contained the methyltransferase motifs and conserved domains a and b. We also developed a dual replicon complementation system allowing selection and propagation of repBHK cells, producing complemented defective replicon RNAs, and used this system to map the minimal complementable region in the NS3 gene to the first 178 codons (containing the protease domain).

Our previously proposed model of the formation of flavivirus RC suggested that the N-terminal region of NS5 protein was involved in binding to other components of the RC (i.e., NS3 and NS2A) via interactions of conserved domains a (KUN NS5 codons 141 to 151), b (KUN NS5 codons 203 to 223), and c (KUN NS5 codons 343 to 365) (20). In the previous complementation studies we showed that the first 397 codons of NS5 (containing all three conserved domains) were sufficient to allow formation of the defective RC which was able to exchange with the helper RC to allow complementation of RNA replication (20). The results obtained in this study eliminated NS5 domain c as a possible binding site in the RC.

The complementation experiments with deleted NS3 using the dual replicon complementation system showed that while the N-terminal 178 codons were required for assembly of the defective complementable RC, the C-terminal 434 codons were dispensable. Thus, our complementation experiments suggest that the translation products of the first 316 codons in NS5 protein and of the first 178 codons in NS3 protein may be involved in interactions with each other and/or with other components of RC during its assembly. Recent binding studies with dengue virus NS3 and NS5 proteins using the Saccharomyces cerevisiae two-hybrid system mapped domains in NS3 and NS5 proteins proposed to be involved in binding to each other at the C-terminal region expressed between codons 303 and 405 in NS3 protein and at the N-terminal region expressed between codons 320 and 368 in NS5 protein (14). These binding domains apparently differ from those proposed in our complementation studies described above. It is possible that binding of two individual proteins in the artificial environment such as the nucleus of a yeast cell may differ from binding of the same proteins in a natural environment, such as that present during formation of the RC in the cytoplasm of mammalian cells. Other factors, i.e., other components of RC and/or cellular proteins, may affect the binding. In addition, the presence of the helper RC in our complementation experiments may interfere with or block binding of the components of the complementable defective RC during its formation. Clearly, additional experimental data on binding of the truncated NS3 and NS5 proteins during formation of the defective RC in the cellular environment free of helper RC are required to draw the final conclusion on the exact location of the functional binding domains.

The results with complementation of genomic length RNAs with deletions in NS3 obtained in this study confirmed our previous observations (21) that deletions in NS3 abolished the ability of these RNAs to be packaged into virus particles. In this study we extended the range of deletions and showed that although the RNAs with these deletions could be complemented for RNA replication, they could not be assembled into secreted or intracellular virions. In one of the genomic length constructs with deletions (a derivative of repdNS1.1/3.8, Fig. 4) we retained the proposed cleavage site in the helicase domain QRR{downarrow}GR to allow the release of truncated NS3' protein (1, 30, 35) in order to examine whether NS3' may have a function in virus assembly. However, despite demonstrable complementation of RNA replication in repBHK cells, no secreted or intracellular virus particles were detected in this experiment, thus demonstrating that allowing production and release of NS3' did not rescue the inability of NS3-deleted RNA to assemble into virions.

We have also shown the functional capability of the structural proteins produced from the complemented NS3-deleted genomic RNAs to be assembled into secreted virions by detecting infectivity of the released virus particles containing encapsidated helper replicon RNA after passage on normal BHK cells. There are several possible explanations for the lack of detectable virus particles containing NS3-deleted genomic RNA during its complementation in repBHK cells. For example, the presence of a packaging signal in RNA within the NS3-deleted sequence, or a requirement for full-length NS3 protein to be translated from the same RNA molecule (in cis) destined to be packaged. As we speculated in our previous study (21), it is unlikely (but not impossible) in view of the ~270 nucleotides distance between some of the deletions (see deletions 3.1 and 3.2 in Fig. 1 of reference 21) that the packaging signal would be so long. It is also possible that the deletions could affect an overall conformational structure of the RNA molecule and render it unpackageable. The use of a construct retaining the intact RNA sequence in the complementable deleted region in NS3 gene, but eliminating translation of this region while still allowing translation of the downstream nonstructural region (say from an internal ribosomal entry site), would allow one to make a definite conclusion. Unfortunately, our numerous attempts to make such a construct failed.

One possible scenario for the requirement of NS3 in cis in virus assembly is that full-length NS3 protein may be involved in bringing together newly synthesized genomic RNA coming out of the RC and the structural proteins during assembly of virions in the endoplasmic reticulum near the sites of RNA replication in vesicle packets (28). Flavivirus NS3 was shown to bind to viral RNA and to NS5 protein in vitro and in vivo (7, 9, 14, 15, 33). KUN NS3 was shown to be strongly associated with the sites of RNA replication by immunostaining (41) and with the components of RC by biochemical assays (29). This proposed scenario could also explain the dependence of RNA packaging on RNA replication shown in our previous studies (22). Obviously, NS3 with deletion produced in cis or full-length NS3 produced in trans (from the helper replicon RNA) did not perform this packaging function.

During final preparation of this work, the data demonstrating involvement of yellow fever virus NS2A and possibly of NS3 in virus assembly were published (26). The authors showed that mutations in the putative alternative cleavage site QK{downarrow}T of NS2A (yellow fever virus NS2A codons 189 to 191) blocked virus assembly and that compensatory mutations in NS3 (at yellow fever virus NS3 codon 343 in the helicase region) restored virus assembly in some of the NS2A QK{downarrow}T mutants. The authors suggested that NS2A-NS3 interactions, or rather interactions in the NS2A-3 region, might modulate nucleocapsid assembly or budding. We have also recently identified a mutation in the KUN NS2A different from that reported for yellow fever virus which resulted in a block in virus assembly (W. J. Liu, H. Chen, and A. A. Khromykh, unpublished results). Our previous (21) and present complementation results demonstrating a role for KUN NS3 in virus assembly, and the results of identification of a mutation in KUN NS2A blocking virus assembly, are in agreement with those of the yellow fever studies, and together they show an unexpected role for the nonstructural proteins in virus assembly.

In summary, this study completes our complementation mapping of the KUN NS3 and NS5 proteins by defining the minimal regions required for efficient trans-complementation of RNA replication and demonstrates a role for NS3 in virus assembly. Further experimental data directly demonstrating the involvement of identified domains in NS3 and NS5 in their interactions with each other or with other components of RC during its assembly, and definition of interactions of NS3 with viral RNA and structural proteins during virus assembly, will significantly contribute to the elucidation of the poorly understood mechanisms of flavivirus RNA replication and virus assembly.


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ACKNOWLEDGMENTS
 
We are grateful to Ed Westaway for the critical reading of the manuscript and helpful comments.

This work was supported by grants 981442 and 142983 from the National Health and Medical Research Council of Australia.


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FOOTNOTES
 
* Corresponding author. Mailing address: Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Queensland 4029, Australia. Phone: 617 36361568. Fax: 617 36361401. E-mail: a.khromykh{at}mailbox.uq.edu.au. Back

{dagger} This is publication no. 145 from the Sir Albert Sakzewski Virus Research Centre. Back


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Journal of Virology, November 2002, p. 10766-10775, Vol. 76, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.21.10766-10775.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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