Double Subgenomic Alphaviruses Expressing Multiple Fluorescent Proteins Using a Rhopalosiphum padi Virus Internal Ribosome Entry Site Element
| Autor: | Michael R. Wiley, Lisa O. Roberts, Kevin M. Myles, Zach N. Adelman |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
Untranslated region
Sindbis virus Five prime untranslated region Recombinant Fusion Proteins Blotting Western Genetic Vectors Green Fluorescent Proteins lcsh:Medicine Alphavirus Genome Viral Picornaviridae Cell Line Aedes Virology Chlorocebus aethiops Animals lcsh:Science Vero Cells Gene Cells Cultured Subgenomic mRNA Rhopalosiphum padi virus Binding Sites Multidisciplinary biology lcsh:R fungi biology.organism_classification Luminescent Proteins Internal ribosome entry site Microscopy Fluorescence Virology/Viral Replication and Gene Regulation lcsh:Q Sindbis Virus Ribosomes Research Article Molecular Biology/Translation Mechanisms |
| Zdroj: | PLoS ONE PLoS ONE, Vol 5, Iss 11, p e13924 (2010) |
| Popis: | 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only single marker proteins were routinely observed in cells infected with dsSINV vectors in which the RhPV IRES had been replaced by a luciferase fragment, an antisense RhPV IRES, or no intergenic sequence.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. |
| Databáze: | OpenAIRE |
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