Abstract
Double subgenomic Sindbis virus (dsSINV) vectors are widely used for the expression of proteins, peptides, and RNA sequences. These recombinant RNA viruses permit high level expression of a heterologous sequence in a wide range of animals, tissues, and cells. However, the alphavirus genome structure and replication strategy is not readily amenable to the expression of more than one heterologous sequence. The Rhopalosiphum padi virus (RhPV) genome contains two internal ribosome entry site (IRES) elements that mediate cap-independent translation of the virus nonstructural and structural proteins. Most IRES elements that have been characterized function only in mammalian cells but previous work has shown that the IRES element present in the 5′ untranslated region (UTR) of the RhPV genome functions efficiently in mammalian, insect, and plant 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/3′2J/mcs. While mammalian and insect cells infected with recombinant viruses containing 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only single marker proteins were routinely observed in cells infected with dsSINV vectors in which the RhPV IRES had been replaced by a luciferase fragment, an antisense RhPV IRES, or no intergenic sequence.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.
Highlights
Alphaviruses have a positive strand, nonsegmented, RNA genome,12 kilobases in length
The complete 59 untranslated region (UTR) of the Rhopalosiphum padi virus (RhPV) genome containing the internal ribosome entry site (IRES) element, (A) D1, or a truncated 59 UTR, (B) D200, was inserted between the Aequorea coerulescens green fluorescent protein (GFP) or Discosoma red fluorescent protein (DsRed) open reading frames (ORFs), downstream from the second subgenomic promoter of TE/392J. (C) A third construct reversed the order of GFP and DsRed in relation to the D1 sequence
Because it has been postulated that the stability of heterologous sequences in double subgenomic alphavirus vectors is inversely related to size [2,3], a second Double subgenomic Sindbis virus (dsSINV) construct was generated that contained a fragment of the RhPV 59 UTR lacking the 59 200 nt (RhPVD200, Fig. 1B)
Summary
Alphaviruses (family Togaviridae) have a positive strand, nonsegmented, RNA genome ,12 kilobases (kb) in length. The structural proteins are translated from the subgenomic 26S mRNA, which is transcribed from an internal promoter present in the minus strand RNA [1]. This genome structure and replication strategy is amenable to the construction of expression vectors. The utility of alphavirus vectors is 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]. 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
