Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps

Virol J. 2007 Jun 5:4:51. doi: 10.1186/1743-422X-4-51.

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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Codon / genetics
  • Cytoplasm / chemistry
  • Exons
  • Gene Expression*
  • Genetic Techniques*
  • Genetic Vectors
  • Humans
  • Introns
  • Membrane Glycoproteins / biosynthesis*
  • Membrane Glycoproteins / genetics
  • Plasmids
  • RNA 3' Polyadenylation Signals / genetics
  • RNA Polymerase II / metabolism*
  • Respiratory Syncytial Viruses / genetics*
  • Vesicular stomatitis Indiana virus / genetics*
  • Viral Envelope Proteins / biosynthesis*
  • Viral Envelope Proteins / genetics
  • Viral Fusion Proteins / biosynthesis*
  • Viral Fusion Proteins / genetics

Substances

  • Codon
  • G protein, vesicular stomatitis virus
  • Membrane Glycoproteins
  • Viral Envelope Proteins
  • Viral Fusion Proteins
  • RNA Polymerase II