RNA replicons derived from flavivirus genomes show considerable potential as gene transfer and immunization vectors. A convenient and efficient encapsidation system is an important prerequisite for the practical application of such vectors. In this work, tick-borne encephalitis (TBE) virus replicons and an appropriate packaging cell line were constructed and characterized. A stable CHO cell line constitutively expressing the two surface proteins prM/M and E (named CHO-ME cells) was generated and shown to efficiently export mature recombinant subviral particles (RSPs). When replicon Nd⌬ME lacking the prM/M and E genes was introduced into CHO-ME cells, virus-like particles (VLPs) capable of initiating a single round of infection were released, yielding titers of up to 5 ؋ 10 7 /ml in the supernatant of these cells. Another replicon (Nd⌬CME) lacking the region encoding most of the capsid protein C in addition to proteins prM/M and E was not packaged by CHO-ME cells. As observed with other flavivirus replicons, both TBE virus replicons appeared to exert no cytopathic effect on their host cells. Sedimentation analysis revealed that the Nd⌬ME-containing VLPs were physically distinct from RSPs and similar to infectious virions. VLPs could be repeatedly passaged in CHO-ME cells but maintained the property of being able to initiate only a single round of infection in other cells during these passages. CHO-ME cells can thus be used both as a source for mature TBE virus RSPs and as a safe and convenient replicon packaging cell line, providing the TBE virus surface proteins prM/M and E in trans.Subgenomic replicons of positive-stranded RNA viruses contain all of the genetic elements needed to amplify themselves in susceptible host cells but lack some or all of the genes coding for structural proteins. Consequently, these RNAs are replicated in cells but are not packaged into viral particles. Replicons have proven to be valuable tools for studying replication independently of virion assembly and maturation (4,20,30). Moreover, they have great potential as molecular tools for gene expression and as vectors for therapeutic and prophylactic purposes (15,19,40). The delivery of replicon RNAs to host cells can be achieved in three different ways: (i) transfection with in vitro-transcribed replicon RNA, (ii) transfection with plasmid DNA encoding replicon sequences under the control of a cellular RNA polymerase II promoter, and (iii) infection with virus-like particles (VLPs) generated by encapsidation of replicon RNA with viral structural proteins provided in trans. VLPs are capable of initiating a single round of infection, providing an efficient and easy way to deliver the replicon vector into specific host cells, and are therefore particularly useful tools in gene therapy or for immunization purposes (19).Flaviviruses, members of the genus Flavivirus (family Flaviviridae), are positive-stranded RNA viruses and include important human pathogens such as yellow fever virus, the four serotypes of dengue virus, Japanese encephaliti...

The flavivirus tick-borne encephaltis virus (TBEV) was established as a vector system for heterologous gene expression. The variable region of the genomic 39 non-coding region was replaced by an expression cassette consisting of the reporter gene enhanced green fluorescent protein (EGFP) under the translational control of an internal ribosomal entry site element, both in the context of an infectious virus genome and of a replicon lacking the genes of the surface proteins prM/M and E. The expression level and the stability of expression were measured by fluorescence-activated cell-sorting analysis and compared to an established alphavirus replicon vector derived from Venezuelan equine encephaltis virus (VEEV), expressing EGFP under the control of its natural subgenomic promoter. On the first day, the alphavirus replicon exhibited an approximately 180-fold higher expression level than the flavivirus replicon, but this difference decreased to about 20-and 10-fold on days 2 and 3, respectively. Four to six days post-transfection, foreign gene expression by the VEEV replicon vanished almost completely, due to extensive cell killing. In contrast, in the case of the TBEV replicon, the percentage of positive cells and the amount of EGFP expression exhibited only a moderate decline over a time period of almost 4 weeks. The infectious TBEV vector expressed less EGFP than the TBEV replicon at all times. Significant expression from the infectious vector was maintained for four cell-culture passages. The results indicate that the VEEV vector is superior with respect to achieving high expression levels, but the TBEV system may be advantageous for applications that require a moderate, but more enduring, gene expression. INTRODUCTIONPositive-stranded RNA viruses belonging to various viral families have been used to develop vectors for heterologous gene expression. In principle, such viral vectors can be generated in two ways: either in the form of infectious mutants (Dufresne et al., 2002;Jia et al., 2002;Yun et al., 2003) or non-infectious replicons (Hewson, 2000;Khromykh, 2000;Lundstrom, 2003). In the first case, the foreign gene is inserted into the viral genome in addition to all of the viral genes that are necessary for a productive viral life cycle. In this approach, the vector spreads autonomously and thus can carry the gene of interest to a large number of potential host cells. Clear limitations of this approach derive from the potential pathogenicity of the vector. In contrast, replicons contain only the genomic information that is necessary to establish RNA replication and translation, but are incapable of forming infectious virus progeny. Replicons are commonly generated by deleting part or all of the genetic information that encodes viral structural proteins from the genome. By providing the missing structural proteins in trans, replicons can be packaged into virus-like particles (VLPs) that are capable of establishing only a single round of infection. Due to their safety and simplicity, RNA replicons have recently ...