Functional expression of secreted proteins from a bicistronic retroviral cassette based on foot-and-mouth disease virus 2A can be position dependent.
The expression of two or more genes from a single viral vector has been widely used to label or select for cells containing the transgenic element. Identification of the foot-and-mouth disease virus (FMDV) 2A cleavage peptide as a polycistronic linker capable of producing equivalent levels of transgene expression has greatly improved this approach in the field of gene therapy. However, as a consequence of 2A posttranslational cleavage the upstream protein is left with a residual 19 amino acids from the 2A sequence on its carboxy terminus, and the downstream protein is left with an additional 2 to 5 amino acids on its amino terminus. Here we have assessed the functional consequences of the FMDV 2A cleavage motif on two secreted proteins (interleukin [IL]-2 and transforming growth factor [TGF]-β) when expressed from a retroviral bicistronic vector. Whereas IL-2 expression and function were found to be unaffected by the 2A motif in either orientation, functional expression of secreted TGF-β was significantly abrogated when the transgene was expressed upstream of the 2A sequence. We believe this is a consequence of aberrant cleavage and intracellular trafficking of the TGF-β polyprotein. These results highlight that to achieve functional expression of secreted proteins consideration must be taken of the transgenic protein's posttranslational modification and trafficking when using 2A-based bicistronic cassettes.
Cancer Research UK Department of Medical Oncology, School of Cancer and Imaging Sciences, University of Manchester, Manchester Academic Health Science Centre, Christie NHS Trust, Manchester M20 4BX, United Kingdom. drothwell@picr.man.ac.uk
Dominic G
DG
Rothwell
Rachel
R
Crossley
John S
JS
Bridgeman
Victoria
V
Sheard
Yining
Y
Zhang
Tyson V
TV
Sharp
Robert E
RE
Hawkins
David E
DE
Gilham
Tristan R
TR
McKay
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005536:Foot-and-Mouth Disease / Q000235:genetics; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011499:Protein Processing, Post-Translational; D012190:Retroviridae / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D007376:Interleukin-2; D016212:Transforming Growth Factor beta; D014764:Viral Proteins