2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology

Abstract

2A is an oligopeptide sequence that mediates a ribosome “skipping” effect and can mediate a co-translation cleavage of polyproteins. These sequences are widely distributed from insect to mammalian viruses and could act by accelerating adaptive capacity. These sequences have been used in many heterologous co-expression systems because they are versatile tools for cleaving proteins of biotechnological interest. In this work, we review and update the occurrence of 2A/2A-like sequences in different groups of viruses by screening the sequences available in the National Center for Biotechnology Information database. Interestingly, we reported the occurrence of 2A-like for the first time in 69 sequences. Among these, 62 corresponded to positive single-stranded RNA species, six to double stranded RNA viruses, and one to a negative-sense single-stranded RNA virus. The importance of these sequences for viral evolution and their potential in biotechnological applications are also discussed.

Ubiquiton—An inducible, linkage-specific polyubiquitylation tool

“…EGFR ∗CUbo-GFP was combined with NUbo E3s or—as a monoubiquitin mimic—a non-catalytic NUbo FLAG construct in HeLa cells deleted for endogenous EGFR. For the K48-Ubiquiton, we fused yeast Ubc7 to the C terminus of NUbo E3(48) via an in vivo -cleavable viral P2A peptide 48 in order to supply E3 and E2 via the same construct ( Figure S8 A). Polyubiquitylation of EGFR ∗CUbo-GFP by NUbo E3(48) and NUbo E3(63) upon Rapa treatment was confirmed by immunoprecipitation of the receptor and blotting for ubiquitin ( Figure 6 A).…”

WIV, a protein domain found in a wide number of arthropod viruses, which probably facilitates infection

“…For example, in the ORF1 of the virus wpk049shi07 [75] (GenBank accession QKE55054.1), related to Sinhaliviridae , the WIV domain, located between aa 1–113, is immediately followed by a 2A ‘StopGo’ sequence (aa 127–139) (our observations; see , top). Such sequences (also called ‘Stop-Carryon’) mediate ribosome skipping during translation, which separates two proteins, akin to a cleavage, but without requiring a protease [76]. Their core motif is DxExNPGP, and the proteins are separated between the penultimate G and the final P (respectively G134 and P135 in the sequence of ORF1).…”

“…2a, top). Such sequences (also called 'Stop-Carryon') mediate ribosome skipping during translation, which separates two proteins, akin to a cleavage, but without requiring a protease [76]. Their core motif is DxExNPGP, and the proteins are separated between the penultimate G and the final P (respectively G134 and P135 in the sequence of ORF1).…”

The 2A Story: The End of the Beginning

“…The presence of 2A-like sequences has been reported in a number of viral genomes within different genera of the Picornaviridae, other positive strand viruses such as the Dicistroviridae and Iflaviridae [16,72], double-strand RNA viruses belonging to the Totiviridae/Reoviridae families [73], and surprisingly in a tentatively assigned negative-sense single-stranded RNA virus of the Bunyaviridae family [72]. The positivestrand RNA viruses typically possess one 2A/2A-like sequence, but some viruses have two, three or more motifs-the picornavirus duck egg-reducing syndrome virus (DERSV) was shown to have a total of seven 2A proteins, the first six separated by the "DxExNPGP" sequence motif (Table 1) [74].…”