Heterologous gene expression by infectious and replicon vectors derived from tick-borne encephalitis virus and direct comparison of this flavivirus system with an alphavirus replicon

Abstract

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 ...

Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct

“…The ability to engineer infectious flavivirus clones to accommodate expression from two separate cistrons has a number of potentially useful applications, including their use as vehicles for the expression of foreign genes as well as tools for investigating the mechanisms of virus replication and assembly (7,10,16,18,24,32,34,36,37,39,40,44), but many of these systems have been hampered by poor efficiency and stability (7,16,18,32,37). We have recently shown that the ability to produce infectious, self-propagating bicistronic flaviviruses can be helpful in elucidating the details of the flavivirus replication and assembly processes by uncoupling the expression of individual proteins from the normal polyprotein processing mechanism (32), but the efficiency of this system also still requires improvement.…”

“…RNA was quantitated spectrophotometrically (19), and equal amounts of RNA were used for all transfections. BHK-21 cells were transfected by electroporation using a Bio-Rad GenePulser apparatus as described previously (7,27).…”

“…This RNA is approximately 11 kilobases in length and has a 5Ј cap structure, but it lacks a poly(A) tail at the 3Ј end (42, 43). All of the viral structural and nonstructural proteins are encoded within a single open reading frame and are processed co-and posttranslationally to form the mature products.Despite the fact that the flavivirus genome contains only a single cap-dependent translation initiation site, several studies have demonstrated that it is possible, by introducing a heterologous viral internal ribosome entry site (IRES) into the 3Ј noncoding region (3ЈNCR), to achieve expression from a separate, cap-independent cistron, thus making it feasible to use modified flaviviruses as bicistronic expression systems for foreign genes (7,10,16,18,24,32,34,36,37,39,40,44). However, many of these have been found to be unstable or inefficient and therefore need to be further developed and optimized (7,16,18,32,37).…”

“…However, many of these have been found to be unstable or inefficient and therefore need to be further developed and optimized (7,16,18,32,37).…”

Construction and Mutagenesis of an Artificial Bicistronic Tick-Borne Encephalitis Virus Genome Reveals an Essential Function of the Second Transmembrane Region of Protein E in Flavivirus Assembly

“…One such derivative, plasmid pTNd/ ⌬ME, encodes a TBEV replicon RNA lacking the structural proteins (pr)M and E and was described previously (11). The same replicon, but with an IRES-EGFP cassette replacing the variable region of the 3Ј noncoding region, can be transcribed from plasmid pTNd/⌬ME-EGFP, which was characterized in a previous publication (12). The bc mutants analyzed in this study were constructed by replacing the EGFP gene of plasmid pTNd/⌬ME-EGFP by the genes coding for proteins prM and E.…”