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.

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.

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.

Expression and purification of the seven nonstructural proteins of the flavivirus Kunjin in the E. coli and the baculovirus expression systems.

All seven nonstructural (ns) proteins of the flavivirus Kunjin (KUN) ranging from NS1 to NS5 were expressed either alone or as fusion proteins with Glutathione-S-transferase (GST). High level expression of recombinant proteins was achieved in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system in contrast to the low level of expression in E. coli. The order of the level of expression of the recombinant fusion proteins per 4 x 10(7) Sf9 cells was: GST-NS5 (yields approximately 4-5 mg) > GST-delta NS3 (approximately 1-2 mg) > GST-4A (approximately 1 mg) > GST-2B (approximately 0.5-1 mg) > GST-2A (approximately 0.5 mg) > GST-4B (approximately 0.1-0.2 mg). NS1 protein was expressed in a native form at the level of approximately 2-4 mg per 4 x 10(7) Sf9 cells. All the GST-fusion proteins were purified by adsorption on Glutathione Sepharose (GS) beads from solubilized lysates of Sf9 cells infected with the recombinant baculoviruses, or of E. coli cultures transformed with the expression plasmid and induced with IPTG. Only delta NS3 protein was recovered intact by removing GST from the fusion protein by digestion with Factor Xa protease. Attempts to cleave off the GST moiety from all the other purified recombinant proteins resulted either in inefficient cleavage or in degradation of the proteins. No GST-NS5 but from 20 to 50% of the purified GST-NS2A, GST-NS2B, GST-delta NS3, GST-NS4A, and GST-NS4B was eluted off the GS beads by adding glutathione. Thus, KUN purified recombinant proteins, either in eluted form or while immobilized on GS beads, could be used to raise monospecific antibodies, to perform functional assays or to participate in protein-protein or RNA-protein binding reactions.

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.

Immunization with recombinant vaccinia viruses expressing structural and part of the nonstructural region of tick-borne encephalitis virus cDNA protect mice against lethal encephalitis.

Three recombinant vaccinia viruses containing different fragments of tick-borne encephalitis virus (TBEV) cDNA representing the 5'-noncoding region (5'NCR), all structural and part of the nonstructural regions were constructed. Western blot analysis showed that E and NS1 proteins were expressed and processed correctly in cells infected with recombinant viruses vC-NS1 (coding for C-prM-E-NS1 region) and vC-NS3 (coding for C-prM-E-NS1-NS2A-NS2B-NS3 region). In contrast, in cells infected with recombinant virus v5'C-NS2A (coding for 5'NCR and C-prM-E-NS1-NS2A regions) expression of NS1 protein was greatly reduced and no E protein was detected. Immunization of mice with vC-NS3 induced high levels of TBEV-specific antibodies and protected them against intraperitoneal challenge with 10(7) LD50 of TBEV. The level of protection was very similar to the level of protection achieved by immunization with commercially available inactivated TBEV vaccine. Although the immunization of mice with recombinants vC-NS1 and v5'C-NS2A induced much lower levels of TBEV-specific antibodies, they were still protected against intraperitoneal challenge with 10(4) and 10(3.6) LD50 of TBEV, respectively. The high level of protection against TBEV infection achieved by the immunization of mice with the recombinant vaccinia virus vC-NS3 makes this virus a very attractive candidate for development of a live recombinant vaccine against TBEV.

Subgenomic replicons of the flavivirus Kunjin: construction and applications.

Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA deltaME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon deltaME/76 with a deletion of 76 nucleotides in the 3'UTR of deltaME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon deltaME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of deltaME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (deltaME/76CAT[+])- and minus (deltaME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental deltaME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of deltaME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the deltaME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system.

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.

RNA binding properties of core protein of the flavivirus Kunjin.

Kunjin virus (KUN) C is a typical flavivirus core protein which is truncated in vivo to a mature form of 105 residues enriched in lysine and arginine. In order to study the possible association of KUN C with RNA in vitro, we prepared several recombinant C proteins with specific deletions, each fused at the amino-terminus to glutathione-S-transferase (GST) and expressed in E. coli. They were reacted with KUN RNA probes transcribed in vitro from cDNA representing the 5' untranslated region (5' UTR, 93 to 96 nucleotides), the 3' UTR (624 nucleotides), and the 5' UTR plus most of the C coding region (5' core, 440 nucleotides). Fusion protein C107 (incorporating mature C) bound strongly to all KUN RNA probes with apparent specificity, being completely resistant to inhibition by 800 mM NaCl, and to competition by a large excess of tRNA. In reactions with labelled KUN RNA probes putative binding sites were identified in the isolated amino-terminal (32 residues) and carboxy-terminal (26 residues) basic amino acid domains; this binding was strongly competed by unlabelled KUN UTR probes but weakly or not at all by tRNA. These small domains probably acted co-operatively in binding of mature C to KUN RNA probes. The KUN RNA-core protein binding reactions are similar to those reported with other viral coat or capsid proteins and viral RNAs.

Completion of Kunjin virus RNA sequence and recovery of an infectious RNA transcribed from stably cloned full-length cDNA.

Completion of the Kunjin virus (KUN) RNA sequence showed that it is the longest flavivirus sequence reported (11,022 bases), commencing with a 5' noncoding region of 96 bases. The 3' noncoding sequence of 624 nucleotides included a unique insertion sequence of 46 bases adjacent to the stop codon, but otherwise it had properties similar to those of RNAs of closely related flaviviruses. A full-length KUN cDNA clone which could be stably propagated in Escherichia coli DH5 alpha was constructed; SP6 polymerase RNA transcripts from amplified cDNA were infectious when transfected into BHK-21 cells. A mutational change abolishing the BamHI restriction site at position 4049, leading to a conservative amino acid change of Arg-175 to Lys in the NS2A protein, was introduced into the cDNA during construction and was retained in the recovered virus. Extra terminal nucleotides introduced during cloning of the cDNA were shown to be present in the in vitro RNA transcripts but absent in the RNA of recovered virus. Although recovered virus differed from the parental KUN by a smaller plaque phenotype and delayed growth rate in BHK-21 cells and mice, it was very similar as assessed by several other criteria, such as peak titer during growth in cells, infectivity titer in cells and in mice, rate of adsorption and penetration in cells, replication at 39 degrees C, and neurovirulence after intraperitoneal injection in mice. The KUN stably cloned cDNA will provide a useful basis for future studies in defining and characterizing functional roles of all the gene products.

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.

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.

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.

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.