title: Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps.
abstract: BACKGROUND
  Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV) and the F protein of respiratory syncytial virus (RSV) by eukaryotic promoters revealed restrictions at several steps of gene expression.
  
  
  RESULTS
  Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF) of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation.
  
  
  CONCLUSIONS
  Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.
authors.affiliation: Department of Molecular and Medical Virology, Ruhr-Universität Bochum, Bochum, Germany. nicola.ternette@rub.de
authors.forename: Nicola
authors.initials: N
authors.lastname: Ternette
authors.forename: Daniela
authors.initials: D
authors.lastname: Stefanou
authors.forename: Seraphin
authors.initials: S
authors.lastname: Kuate
authors.forename: Klaus
authors.initials: K
authors.lastname: Uberla
authors.forename: Thomas
authors.initials: T
authors.lastname: Grunwald
mesh_terms: D002460:Cell Line; D003062:Codon / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry; D005091:Exons; D015870:Gene Expression; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D007438:Introns; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D039104:RNA 3' Polyadenylation Signals / Q000235:genetics; D012319:RNA Polymerase II / Q000378:metabolism*; D012136:Respiratory Syncytial Viruses / Q000235:genetics*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics; D014760:Viral Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003062:Codon; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins; D012319:RNA Polymerase II