Virus constructs containing a fragment of the (D) firefly luciferase (LUC) gene, (E) the 1 sequence in the antisense orientation, or (F) a construct lacking any intergenic sequence between GFP and DsRed served as negative controls. region (UTR) of the RhPV genome functions efficiently in mammalian, insect, and herb systems. To determine if the 5 RhPV IRES element could be used to express more than one heterologous sequence from a dsSINV vector, RhPV 5 IRES sequences were placed between genes for two different fluorescent marker proteins in the dsSINV, TE/32J/mcs. While mammalian and insect cells infected with recombinant viruses made up of the RhPV sequences expressed both fluorescent marker proteins, only single marker proteins were routinely observed in cells infected with dsSINV vectors in which the RhPV IRES had been replaced by a luciferase fragment, an antisense RhPV IRES, or no intergenic sequence. Thus, we report development of a versatile tool for the expression of multiple sequences in diverse cell types. == Introduction == Alphaviruses (familyTogaviridae) have a positive strand, non-segmented, RNA genome 12 kilobases (kb) in length. The first two-thirds of the genome encode the nonstructural or replicase proteins, while the 3 one-third encodes the structural proteins. In the infected cell, the 49S genomic RNA serves both as mRNA for the translation of the nonstructural proteins and as a template for synthesis of full-length minus strand RNA copies[1]. The structural proteins are translated from the subgenomic 26S mRNA, which is usually transcribed from an internal promoter present in the minus strand RNA[1]. This genome structure and replication strategy is usually amenable to the construction of expression vectors. Replication and packaging qualified alphavirus vectors have been developed by duplicating the subgenomic RNA promoter element in the genome[2],[3]. Heterologous sequences can be expressed as an additional subgenomic RNA transcribed from the duplicated promoter. Double subgenomic alphavirus vectors have several advantages as transient expression systems. These include a tremendously broad host range (e.g. vertebrates and invertebrates), routine construction and manipulation with standard recombinant Lenalidomide-C5-NH2 DNA techniques, and high level expression of proteins, peptides, and RNA sequences[2]. However, expression levels typically diminish with virus passage because of instability in the region Lenalidomide-C5-NH2 of the genome made up of the duplicated Lenalidomide-C5-NH2 promoter and heterologous sequence[3],[4]. The utility of alphavirus vectors is also limited by an inability to express more than a single exogenous gene or sequence from the subgenomic promoter. This has previously been addressed by inserting the foot-and-mouth disease virus (FMDV) 2A protein between the N-terminal capsid and PE2 glycoprotein encoded in the 26S mRNA[5]. The alphavirus capsid protein autoproteolytically cleaves itself from the structural polyprotein[6]. The 20 amino acid FMDV 2A sequence mediates self-processing through a proposed ribosomal-skip mechanism[7]. Thus, a 2A fusion protein located at this position in the viral genome can be expressed as a discrete product from the structural polyprotein[5]. Although a protein expressed from the duplicated subgenomic promoter will be in native form, the other protein is usually always expressed in conjunction with the FMDV 2A peptide sequence[5]. This limits the usefulness of these vectors for some applications, not least of which is the expression of proteins that exhibit reduced bioactivity as fusion products. An alternative mechanism for achieving the expression of more than one protein from a single mRNA is the insertion of a viral internal ribosome entry site (IRES) element between the two open reading frames (ORFs). An IRES directs a cap-independent mechanism of protein synthesis and therefore efficient expression of both ORFs can be achieved. IRES elements found within the 5 untranslated regions (UTRs) of picornavirus genomes have been extensively studied for this purpose and are able to direct efficient translation of a Rabbit polyclonal to NOTCH1 downstream ORF within a discistronic mRNA within mammalian cells[8]. As such, much interest has been focused on the use of picornavirus IRES elements in protein expression systems. While these elements have been effectively used in alphavirus expression systems, the mammalian picornavirus IRES elements do not function efficiently in insect cell systems[9],[10],[11]. This limits the usefulness of alphavirus expression systems in dipterans (fruit flies and mosquitoes) and lepidopterans[12],[13],[14]. Here, we have employed an IRES element found within the genome ofRhopalosiphum padivirus (RhPV), a virus belonging to theDicistroviridaefamily..