Functional non-coding RNAs derived from the flavivirus 3' untranslated region.

Flaviviruses are single-stranded positive sense RNA enveloped viruses. The flavivirus genus includes important human pathogens such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and Murray Valley encephalitis virus (MVEV). In addition to the viral proteins and viral genomic RNA, flaviviruses produce at least two functional non-coding RNAs derived from the 3' untranslated region (3'UTR), the subgenomic flavivirus RNA (sfRNA) and a putative WNV miRNA (KUN-miR-1). In this review we summarize published data from studies with WNV, YFV, DENV, JEV, and MVEV on sfRNA production following incomplete degradation of the viral genomic RNA by the cellular 5'-3' exoribonuclease 1 (XRN1), RNA structural elements involved in stalling XRN1 to generate sfRNA, and functions of sfRNA in modulating cellular mRNA decay and RNAi pathways as well as in modulating anti-viral type I interferon response. In addition, we also summarize data on the mechanisms of biogenesis of 3'UTR-derived KUN-miR-1 and its function in WNV replication in mosquito host, along with recent findings on a discovery of a second potential flaviviral miRNA vsRNA5, derived from the 3'UTR of DENV. This review thus summarizes the known mechanisms of generation and the functions of flaviviral 3'UTR-derived non-coding RNAs.

Identification of residues in West Nile virus pre-membrane protein that influence viral particle secretion and virulence.

The pre-membrane protein (prM) of West Nile virus (WNV) functions as a chaperone for correct folding of the envelope (E) protein, and prevents premature fusion during virus egress. However, little is known about its role in virulence. To investigate this, we compared the amino acid sequences of prM between a highly virulent North American strain (WNV(NY99)) and a weakly virulent Australian subtype (WNV(KUN)). Five amino acid differences occur in WNV(NY99) compared with WNV(KUN) (I22V, H43Y, L72S, S105A and A156V). When expressed in mammalian cells, recombinant WNV(NY99) prM retained native antigenic structure, and was partially exported to the cell surface. In contrast, WNV(KUN) prM (in the absence of the E protein) failed to express a conserved conformational epitope and was mostly retained at the pre-Golgi stage. Substitutions in residues 22 (Ile to Val) and 72 (Leu to Ser) restored the antigenic structure and cell surface expression of WNV(KUN) prM to the same level as that of WNV(NY99), and enhanced the secretion of WNV(KUN) prME particles when expressed in the presence of E. Introduction of the prM substitutions into a WNV(KUN) infectious clone (FLSDX) enhanced the secretion of infectious particles in Vero cells, and enhanced virulence in mice. These findings highlight the role of prM in viral particle secretion and virulence, and suggest the involvement of the L72S and I22V substitutions in modulating these activities.

Kunjin virus replicon-based vaccines expressing Ebola virus glycoprotein GP protect the guinea pig against lethal Ebola virus infection.

Pre- or postexposure treatments against the filoviral hemorrhagic fevers are currently not available for human use. We evaluated, in a guinea pig model, the immunogenic potential of Kunjin virus (KUN)-derived replicons as a vaccine candidate against Ebola virus (EBOV). Virus like particles (VLPs) containing KUN replicons expressing EBOV wild-type glycoprotein GP, membrane anchor-truncated GP (GP/Ctr), and mutated GP (D637L) with enhanced shedding capacity were generated and assayed for their protective efficacy. Immunization with KUN VLPs expressing full-length wild-type and D637L-mutated GPs but not membrane anchor-truncated GP induced dose-dependent protection against a challenge of a lethal dose of recombinant guinea pig-adapted EBOV. The surviving animals showed complete clearance of the virus. Our results demonstrate the potential for KUN replicon vectors as vaccine candidates against EBOV infection.

A Kunjin replicon vector encoding granulocyte macrophage colony-stimulating factor for intra-tumoral gene therapy.

We have recently developed a non-cytopathic RNA replicon-based viral vector system based on the flavivirus Kunjin. Here, we illustrate the utility of the Kunjin replicon system for gene therapy. Intra-tumoral injections of Kunjin replicon virus-like particles encoding granulocyte colony-stimulating factor were able to cure >50% of established subcutaneous CT26 colon carcinoma and B16-OVA melanomas. Regression of CT26 tumours correlated with the induction of anti-cancer CD8 T cells, and treatment of subcutaneous CT26 tumours also resulted in the regression of CT26 lung metastases. Only a few immune-based strategies are able to cure these aggressive tumours once they are of a reasonable size, illustrating the potential of this vector system for intra-tumoral gene therapy applications.

Essential role of cyclization sequences in flavivirus RNA replication.

A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first ~160 nucleotides) and the 3' untranslated region (last ~115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.

Coupling between replication and packaging of flavivirus RNA: evidence derived from the use of DNA-based full-length cDNA clones of Kunjin virus.

In order to study whether flavivirus RNA packaging is dependent on RNA replication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN1dGDD, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-length Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. As expected, transfection of pKUN1 plasmid DNA into BHK cells resulted in the recovery of secreted infectious Kunjin virions. Transfection of pKUN1dGDD DNA into BHK cells, however, did not result in the recovery of any secreted virus particles containing encapsidated dGDD RNA, despite an apparent accumulation of this RNA in cells demonstrated by Northern blot analysis and its efficient translation demonstrated by detection of correctly processed labeled structural proteins (at least prM and E) both in cells and in the culture fluid using coimmunoprecipitation analysis with anti-E antibodies. In contrast, when dGDD RNA was produced even in much smaller amounts in pKUN1dGDD DNA-transfected repBHK cells (where it was replicated via complementation), it was packaged into secreted virus particles. Thus, packaging of defective Kunjin virus RNA could occur only when it was replicated. Our results with genome-length Kunjin virus RNA and the results with poliovirus replicon RNA (C. I. Nugent et al., J. Virol. 73:427-435, 1999), both demonstrating the necessity for the RNA to be replicated before it can be packaged, strongly suggest the existence of a common mechanism for minimizing amplification and transmission of defective RNAs among the quasispecies in positive-strand RNA viruses. This mechanism may thus help alleviate the high-copy error rate of RNA-dependent RNA polymerases.

Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin.

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD-->GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity.

Stable expression of noncytopathic Kunjin replicons simulates both ultrastructural and biochemical characteristics observed during replication of Kunjin virus.

This report focuses mainly on the characterization of a Vero cell line stably expressing the flavivirus Kunjin (KUN) replicon C20SDrep (C20SDrepVero). We showed by immunofluorescence and cryoimmunoelectron microscopy that unique flavivirus-induced membrane structures, termed convoluted membranes/paracrystalline structures, were induced in the C20SDrepVero cells. These induced cytoplasmic foci were immunolabeled with KUN virus anti-NS3 antibodies and with antibodies to the cellular markers ERGIC53 (for the intermediate compartment) and protein disulfide isomerase (for the rough endoplasmic reticulum). However, in contrast to the large perinuclear inclusions observed by immunofluorescence with anti-double-stranded (ds)RNA antibodies in KUN virus-infected cells, the dsRNA in C20SDrepVero cells was localized to small isolated foci scattered throughout the cytoplasm, which were coincident with small foci dual-labeled with the trans-Golgi specific marker GalT. Importantly, persistent expression of the KUN replicons in cells did not produce cytopathic effects, and the morphology of major host organelles (including Golgi, mitochondria, endoplasmic reticulum, and nucleus) was apparently unaffected. The amounts of plus- and minus-sense RNA synthesis in replicon cells were similar to those in KUN virus-infected cells until near the end of the latent period, but subsequently increases of about 10- and fourfold, respectively, occurred in infected cells. Virus-specified protein synthesis in C20SDrepVero cells was also about 10-fold greater than that in infected cells. When several KUN replicon cell lines were compared with respect to membrane induction, the relative efficiencies increased in parallel with increases in viral RNA and protein synthesis, consistent with the increases observed during the virus infectious cycle. Based on these observations, cell lines expressing less-efficient replicons may provide a useful tool to study early events in flavivirus RNA replication, which are difficult to assess in virus infections.

cis- and trans-acting elements in flavivirus RNA replication.

Most of the seven flavivirus nonstructural proteins (NS1 to NS5) encoded in the distal two-thirds of the RNA positive-sense genome are believed to be essential components of RNA replication complexes. To explore the functional relationships of these components in RNA replication, we used trans-complementation analysis of full-length infectious RNAs of Kunjin (KUN) virus with a range of lethal in-frame deletions in the nonstructural coding region, using as helper a repBHK cell line stably producing functional replication complexes from KUN replicon RNA. Recently we showed that replication of KUN RNAs with large carboxy-terminal deletions including the entire RNA polymerase region in the NS5 gene, representing 34 to 75% of the NS5 coding content, could be complemented after transfection into repBHK cells. In this study we have demonstrated that KUN RNAs with deletions of 84 to 97% of the NS1 gene, or of 13 to 63% of the NS3 gene including the entire helicase region, were also complemented in repBHK cells with variable efficiencies. In contrast, KUN RNAs with deletions in any of the other four nonstructural genes NS2A, NS2B, NS4A, and NS4B were not complemented. We have also demonstrated successful trans complementation of KUN RNAs containing either combined double deletions in the NS1 and NS5 genes or triple deletions in the NS1, NS3, and NS5 genes comprising as much as 38% of the entire nonstructural coding content. Based on these and our previous complementation results, we have generated a map of cis- and trans-acting elements in RNA replication for the nonstructural coding region of the flavivirus genome. These results are discussed in the context of our model on formation and composition of the flavivirus replication complex, and we suggest molecular mechanisms by which functions of some defective components of the replication complex can be complemented by their wild-type counterparts expressed from another (helper) RNA molecule.

Replicon-based vectors of positive strand RNA viruses.

Vectors based on self-replicating RNAs (replicons) of positive strand RNA viruses are becoming powerful tools for gene expression in mammalian cells and for the development of novel antiviral and anticancer vaccines. A relatively small genome size and simple procedure allow rapid generation of recombinants. Cytoplasmic RNA amplification eliminates nuclear involvement and leads to extremely high levels of gene expression, and continuous synthesis of double stranded RNA results in induction of enhanced immune responses, making these vectors unique among other gene expression systems. Both cytopathic replicon vectors allowing short-term transient expression, and non-cytopathic replicon vectors allowing long-term stable expression, are now available with the choice of vector depending on particular applications.

Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors.

Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366-375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of beta-galactosidase (beta-Gal) in several mammalian cell lines, and expression remained high ( approximately 150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by beta-Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-beta-Gal antibody response after intramuscular inoculation of the beta-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo.

Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase.

Successful trans-complementation of the defective Kunjin virus (KUN) RNA FLdGDD with a deletion of the RNA polymerase motif GDD in the NS5 gene by using a BHK cell line, repBHK, that continuously produced a functionally active KUN replication complex (RC) from replicon RNA was recently reported (A. A. Khromykh, M. T. Kenney, and E. G. Westaway, J. Virol. 72:7270-7279, 1998). In order to identify whether this complementation of FLdGDD RNA was provided by the wild-type NS5 protein alone or with the help of other nonstructural (NS) proteins also expressed in repBHK cells, we generated BHK cell lines stably producing the individual NS5 protein (SRns5BHK) or the NS1-NS5 polyprotein (SRns1-5BHK) by using a heterologous expression vector based on a modified noncytopathic Sindbis replicon. Western blot analysis with anti-NS5 antibodies showed that the level of production of NS5 was significantly higher in SRns5BHK cells than in SRns1-5BHK cells. Despite the higher level of expressed NS5, trans-complementation of FLdGDD RNA was much less efficient in SRns5BHK cells than in SRns1-5BHK cells and produced at least 100-fold less of the secreted complemented virus. In contrast, efficient complementation of KUN RNA with lethal cysteine-to-alanine mutations in the NS1 gene was achieved both in BHK cells producing the individual KUN NS1 protein from the Sindbis replicon vector and in repBHK cells, with both cell lines expressing similar amounts of NS1 protein. These results clearly demonstrate that flavivirus NS5 coexpressed with other components of the viral replicase possesses much higher functional (trans-complementing) activity than individually expressed NS5 and that efficient trans-complementation of mutated flavivirus NS1 and NS5 proteins occurs by different mechanisms. The results are interpreted and discussed in relation to our proposed model of formation of the flavivirus RC largely based on previous ultrastructural and biochemical analyses of KUN replication.

Loss of dimerisation of the nonstructural protein NS1 of Kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of NS1.

The flavivirus nonstructural protein NS1 has been implicated in viral RNA replication, although its precise role has not been identified. In its native state NS1 exists as a heat labile homodimer that is thought to be required for NS1 function and secretion. However, we have recently identified a cDNA clone of KUN virus (FLSD) that replicates efficiently in cell culture but produces and secretes NS1 in monomeric form. Sequence analysis of the NS1 gene in FLSD revealed a single amino acid substitution (proline(250) to leucine) when compared with the parental KUN virus. When site-directed mutagenesis was used to substitute leucine(250) with proline in FLSD to produce the clone 250pro, dimerisation was fully restored. Furthermore, time course experiments revealed that 250pro replicated in Vero cells significantly faster than FLSD and produced 100-fold more infectious virus early (12-24 h) in infection. This correlated with our observations that FLSD required approximately 10-fold more infectious virus than 250pro to produce disease in weanling mice after intraperitoneal inoculation. Taken together our results indicate that mutation from proline to leucine at residue 250 in KUN NS1 ablates dimer formation, slows virus replication, and reduces virulence in mice.

trans-Complementation analysis of the flavivirus Kunjin ns5 gene reveals an essential role for translation of its N-terminal half in RNA replication.

Recently we described rescue of defective Kunjin virus (KUN) RNAs with small deletions in the methyltransferase and RNA polymerase motifs of the ns5 gene, using BHK cells stably expressing KUN replicon RNA (repBHK cells) as helper (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). We have now extended our previous observations and report successful trans-complementation of defective KUN RNAs with most of the ns5 gene deleted or substituted with a heterologous (dengue virus) ns5 sequence. Replication of full-length KUN RNAs with 3'-terminal deletions of 136 (5%), 933 (34%), and 1526 (56%) nucleotides in the ns5 gene was complemented efficiently in transfected repBHK cells. RNA with a larger deletion of 2,042 nucleotides (75%) was complemented less efficiently, and RNA with an even larger deletion of 2,279 nucleotides (84%) was not complemented at all. Chimeric KUN genomic RNA containing 87% of the KUN ns5 gene replaced by the corresponding sequence of the dengue virus type 2 ns5 gene was unable to replicate in normal BHK cells but was complemented in repBHK cells. These results demonstrate for the first time complementation of flavivirus RNAs with large deletions (as much as 75%) in the RNA polymerase gene and establish that translation of most of the N-terminal half of NS5 is essential for complementation in trans. A model of formation of the flavivirus replication complex implicating a possible role in RNA replication of conserved coding sequences in the N-terminal half of NS5 is proposed based on the complementation and earlier results with KUN and on reported data with other flaviviruses.

Nascent flavivirus RNA colocalized in situ with double-stranded RNA in stable replication complexes.

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.

Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes.

Noncytopathic replicons of the flavivirus Kunjin (KUN) were employed for expression and delivery of heterologous genes. Replicon vector C20DX2Arep, containing a unique cloning site followed by the sequence of 2A autoprotease of foot-and-mouth disease virus, was constructed and used for expression of a number of heterologous genes including chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), beta-galactosidase, glycoprotein G of vesicular stomatitis virus, and the Core and NS3 genes of hepatitis C virus. The expression and proper processing of these genes upon transfection of BHK21 cells with the recombinant replicon RNAs were demonstrated by immunofluorescence, radioimmunoprecipitation, and appropriate reporter gene assays. Most of these recombinant KUN replicon RNAs were also successfully packaged into secreted virus-like particles (VLPs) by subsequent transfection with Semliki Forest virus replicon RNA expressing KUN structural genes. Infection of BHK21 and Vero cells with these VLPs resulted in continuous replication of the recombinant replicon RNAs and prolonged expression of the cloned genes without any cytopathic effect. We also developed a replicon vector for generation of stable cell lines continuously expressing heterologous genes by inserting an encephalomyelocarditis virus internal ribosomal entry site-neomycin transferase gene cassette into the 3'-untranslated region of the C20DX2Arep vector. Using this vector (C20DX2ArepNeo), stable BHK cell lines persistently expressing GFP and CAT genes for up to 17 passages were established. Thus noncytopathic KUN replicon vectors with the ability to be packaged into VLPs should provide a useful tool for the development of noninfectious and noncytopathic vaccines as well as for gene therapy applications.

trans-Complementation of flavivirus RNA polymerase gene NS5 by using Kunjin virus replicon-expressing BHK cells.

A BHK cell line persistently expressing a Kunjin (KUN) virus replicon RNA (repBHK, similar to our recently described ME/76Neo BHK cell line [A. A. Khromykh and E. G. Westaway, J. Virol. 71:1497-1505, 1997]) was used for rescue and propagation of KUN viruses defective in the RNA polymerase gene (NS5). A new infectious full-length KUN virus cDNA clone, FLSDX, prepared from our previously described cDNA clone pAKUN (A. A. Khromykh and E. G. Westaway, J. Virol. 68:4580-4588, 1994) and possessing approximately 10(5)-fold higher specific infectivity than that of pAKUN, was used for preparation of defective mutants. Deletions of the predicted RNA polymerase motif GDD (producing FLdGDD) and of one of the predicted methyltransferase motifs (S-adenosylmethionine [SAM] binding site, producing FLdSAM) were introduced separately into FLSDX. Transcription and transfection of FLdGDD and FLdSAM RNAs into repBHK cells but not into normal BHK cells resulted in their replication and the recovery of defective viruses able to replicate only in repBHK cells. Reverse transcription-PCR and sequencing analyses showed retention of the introduced deletions in the genomes of the recovered viruses. Retention of these deletions, as well as our inability to recover viruses able to replicate in normal BHK cells after prolonged incubation (for 7 days) of FLdGDD- or FLdSAM-transfected repBHK cells, excluded the possibility that recombination had occurred between the deleted defective NS5 genes present in transfected RNAs and the functional NS5 gene present in the repBHK cells. An RNA with a point mutation in the GDD motif (FLGVD) was also complemented in transfected repBHK cells, and defective virus was recovered by day 3 after transfection. However, in contrast to the results with FLdGDD and FLdSAM RNAs, prolonged (4 days or more) incubation of FLGVD RNA in normal BHK cells allowed recovery of a virus in which the GVD mutation had reverted via a single base change to the wild-type GDD sequence. Overall, these results represent the first demonstration of trans-complementation of defective flavivirus RNAs with deleterious deletions in the flavivirus RNA polymerase gene NS5. The complementation system described here may prove to be useful for the in vivo complementation of deletions and mutations affecting functional domains or the essential secondary structure in any of the other flavivirus nonstructural proteins.

Encapsidation of the flavivirus kunjin replicon RNA by using a complementation system providing Kunjin virus structural proteins in trans.

Kunjin virus (KUN) replicon RNA was encapsidated by a procedure involving two consecutive electroporations of BHK-21 cells, first with KUN replicon RNA C20DXrep (with prME and most of C deleted) and about 24 h later with a recombinant Semliki Forest virus (SFV) replicon RNA(s) expressing KUN structural proteins. The presence of KUN replicon RNA in encapsidated particles was demonstrated by its amplification and expression in newly infected BHK-21 cells, detected by Northern blotting with a KUN-specific probe and by immunofluorescence analysis with anti-NS3 antibodies. No infectious particles were produced when C20DXrep RNA and recombinant SFV RNAs were electroporated simultaneously. When the second electroporation was performed with a single SFV replicon RNA expressing the KUN contiguous prME genes and the KUN C gene together but under control of two separate 26S subgenomic promoters (SFV-prME-C107), a 10-fold-higher titer of infectious particles was achieved than when two different SFV replicon RNAs expressing the KUN C gene (SFV-C107) and prME genes (SFV-prME) separately were used. No SFV replicon RNAs expressing KUN structural proteins were encapsidated in secreted particles. Infectious particles pelleted by ultracentrifugation of the culture fluid from cells sequentially transfected with C20DXrep and SFV-prME-C107 RNAs were neutralized by preincubation with monoclonal antibodies to KUN E protein. Radioimmunoprecipitation analysis with anti-E antibodies of the culture fluid of the doubly transfected cells showed the presence of C, prM/M, and E proteins in the immunoprecipitated particles. Reverse transcription-PCR analysis showed that the immunoprecipitated particles also contained KUN-specific RNA. The encapsidated replicon particles sedimented more slowly than KUN virions in a 5 to 25% sucrose density gradient and were uniformly spherical, with an approximately 35-nm diameter, compared with approximately 50 nm for KUN virions. The results of this study demonstrate for the first time packaging of flavivirus RNA in trans, and they exclude a role in packaging for virtually all of the structural region. Possible applications of the developed packaging system include the definition of the packaging signal(s) in flavivirus RNA as well as the amino acid motif(s) in the structural proteins involved in RNA encapsidation, virion assembly, and secretion. Furthermore, it could facilitate the development of a noninfectious vaccine delivery system based on encapsidation of a noncytopathic flavivirus replicon expressing heterologous genes.