Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

Abstract

Insertion of picornaviral 2A sequences into mRNAs causes ribosomes to skip formation of a peptide bond at the junction of the 2A and downstream sequences, leading to the production of two proteins from a single open reading frame. Adenoviral protein IX is a minor capsid protein that has been used to display foreign peptides on the surface of the capsid. We have used 2A sequences from the foot-and-mouth disease virus (FMDV) and porcine teschovirus 1 (PTV-1) to express protein IX (pIX) and green fluorescent protein (GFP) from pIX-2A-GFP fusion genes in an oncolytic virus derived from human adenovirus 5. GFP was efficiently expressed by constructs containing either 2A sequence. Peptide bond skipping was more efficient with the 58 aa FMDV sequence than with the 22 aa PTV-1 2A sequence, but the virus with the FMDV 2A sequence showed a reduction in plaque size, cytopathic effect, viral burst size and capsid stability. We conclude that ribosome skipping induced by 2A sequences is an effective strategy to express heterologous genes in adenoviruses; however, careful selection or optimization of the 2A sequence may be required if protein IX is used as the fusion partner. INTRODUCTIONReplication-competent oncolytic adenoviruses have been extensively tested in animals, and a virus lacking the E1B 55K gene was recently approved for cancer therapy in China (reviewed by Alemany, 2007). The main factor limiting widespread application of oncolytic adenoviruses is their low efficacy. Many groups have attempted to develop more active viruses, for example by inserting genes for toxic proteins or prodrug-activating enzymes (reviewed by Alemany, 2007). We and others have previously used internal ribosome entry sites (IRESs) to initiate the translation of prodrug-activating enzymes in adenoviruses but the results were disappointing (reviewed by de Felipe, 2002;Fuerer & Iggo, 2004;Lukashev et al., 2005). Ribosome skipping is a recently described mechanism that allows translation of multiple proteins from a single mRNA. It is based on the use of a picornaviral 2A sequence that causes the ribosome to continue translation after skipping the formation of one peptide bond. This process was first characterized in foot-and-mouth disease virus (FMDV) (Ryan & Drew, 1994). The process was originally termed 'cleavage' by analogy with the proteasemediated cleavages occurring at other sites in the FMDV polyprotein but this is misleading because the 'cleavage' results from failure to form a peptide bond during translation. Unlike reinitiation of translation by IRESs, skipping induced by 2A sequences gives approximately equal expression of the proteins upstream and downstream of the 2A site (de Felipe et al., 2006). 2A and 2A-like sequences have previously been used in biotechnology applications, but not in adenoviruses (reviewed by de Felipe et al., 2006). Protein IX is a small cement protein located between the hexons in the capsid of the adenovirus (Furcinitti et al., 1989). It is essential for the packaging of full-length viral genomes (Ghosh-Cho...

Signed outside: a surface marker system for transgenic cytoplasmic proteins

“…[7][8][9] The cleavage efficiency of the present construct containing 31aa of 2A is consistent with efficiencies previously reported. Owing to the high cleavage efficiency of our marker system, the detection of the 'DLNGFR-2A' surface marker provides a direct measure for the presence of the cytoplasmic p47phox transgene product.…”

A bicistronic lentiviral vector based on the 1D/2A sequence of foot-and-mouth disease virus expresses proteins stoichiometrically

“…Longer versions of 2A with extra sequences (total ∼30aa) derived from capsid protein 1D produce higher levels of cleavage (Donnelly et al, 2001;Klump et al, 2001;Luke et al, 2008;Ryan et al, 1991). Of note, the 1D/2A sequence has been successfully used in a number of in vitro and in vivo heterologous systems to achieve efficient bicistronic production of various combinations of reporter proteins and proteins requiring discrete-cotranslational or post-translational-subcellular localization (Donnelly et al, 1997(Donnelly et al, , 2001Funston et al, 2008;Furler et al, 2001;Halpin et al, 1999;Trichas et al, 2008). In contrast to IRESes, incorporation of these sites into viral vectors generally leads to coordinated expression of both upstream and downstream genes as measured by: (1) correlated enzymatic activity between chloramphenicol acetyl transferase and ␤-glucuronidase (Halpin et al, 1999); (2) GFP-FACS paired with antibiotic resistance (Lorens et al, 2004); (3) second gene expression and Western blot (Chinnasamy et al, 2006;Furler et al, 2001); (4) co-fluorescence reporting Samalova et al, 2006); (5) protein segregation (El Amrani et al, 2004;Trichas et al, 2008); and (6) FRET analysis (Szymczak et al, 2004).…”

Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

“…For reasons that remain to be determined, the 2A strategy also resulted in reduced E1A expression, replication, and transgene expression for the Ad5TL virus, but not the Ad5TyrTL virus. Iggo and co-workers recently reported feasibility of 2A sequences derived from the footand-mouth disease virus (F2A) and from the porcine teschovirus-1 (P2A) for co-expression of green fluorescent protein with Ad pIX (Funston et al, 2008). They also observed reduced replication and spread for the 2A viruses, FIG.…”