Viruses | Behind the Paper: “Picornavirus Evolution: Genomes Encoding Multiple 2ANPGP Sequences—Biomedical and Biotechnological Utility”

“Picornavirus Evolution: Genomes Encoding Multiple 2ANPGP Sequences—Biomedical and Biotechnological Utility”
by Garry A. Luke, Lauren S. Ross, Yi-Ting Lo, Hsing-Chieh Wu and Martin D. Ryan
Viruses 2024, 16(10), 1587; https://doi.org/10.3390/v16101587
Available online: https://www.mdpi.com/1999-4915/16/10/1587

This accompaniment to the publication proper tells the story behind the paper, detailing the highs and lows along the way.

First identified in foot-and-mouth disease virus (FMDV), the 2A oligopeptide sequence allows multiple discrete proteins to be synthesized from a single strand of virus RNA, which also functions as a messenger RNA (mRNA). The short 2A peptide, 18 amino acids (aa) in length, comprises two parts, an N-terminal region (without high sequence conservation) and a conserved motif comprising the seven C-terminal residues of 2A and the N-terminal proline of the downstream protein 2B (-D[V/I]ExNPG^↓P, underlined proline comprises the N-terminal residue of 2B). Briefly, the 2A region of the polyprotein manipulates the ribosome to “skip” the synthesis of the glycyl-prolyl peptide bond at its own carboxyl terminus leading to the release of the nascent protein followed by translation of the remaining downstream sequence ⁽¹⁾.

Probing databases with this “signature” motif, 2A^NPGP sequences have been reported in several viral genomes within different genera of the Picornaviridae, other positive strand viruses such as the Dicistroviridae and Iflaviridae^(2-3), double-strand RNA viruses belonging to the Totiviridae/Reoviridae  ⁽⁴⁾ families, and in a tentatively assigned negative-sense single-stranded RNA virus of the Bunyaviridae family ⁽³⁾. From bioinformatics analysis, 2A-like cellular sequences have also been identified in the open reading frames (ORFs) of CATERPILLER proteins that are involved in innate immunity⁽⁵⁾, non-LTR retrotransposons (non-LTRs) within the genome of trypanosomes⁽⁶⁾, and a wide range of primitive marine organisms, including sponges, sea slugs, purple sea urchins, acorn worms and amphioxi ⁽⁷⁾. Linking proteins with 2A or 2A-like peptide sequences results in cellular expression of multiple, discrete proteins derived from a single ORF. Of the many 2A peptides identified to date, four viral 2As have been widely used in biotechnology and biomedicine: FMDV (F2A; -QLLNFDLLKLAGDVESNPGP-), equine rhinitis A virus (E2A; -QCTNYALLKLAGDVESNPGP-), porcine teschovirus-1 (P2A; -ATNFSLLKQAGDVEENPGP-), and Thosea asigna virus (T2A; -EGRGSLLTCGDVESNPGP-) ⁽⁸⁾. These sequences are now being used to treat cancer, to produce antibodies and antigens that can be used in vaccine production, genome (DNA and RNA) editing in cell/gene therapies, and engineering multi-gene biosynthetic pathways amongst a host of other biomedical/biotechnological applications (https://www.st-andrews.ac.uk/ryanlab/Index.html). The positive strand RNA viruses typically possess one 2A/2A-like sequence, but some viruses have two, three, or more motifs. The primary aims of our recent paper were to identify genomes containing more than a single 2A^NPGP sequence in the family Picornaviridae, describe their occurrence in different viral genera, and determine their cleavage activity vis à vis potential biotechnology applications.

As a first step, in silico searches of picornavirus proteinase/polymerase and classical 2A^NPGP sequences (-D(V/I)ExNPGP-; -G(V/I)ExNPGP-; “x” is any amino acid which is not conserved) were made by screening the sequences currently available in the NCBI’s (National Centre for Biotechnology Information) growing database. Alignment of 3CD amino acid sequences revealed this family evolved into five “supergroups” (SG1-5; Caphthovirinae, Kodimesavirinae, Ensavirinae, Paavivirinae, and Heptrevirinae) with 2A^NPGP sequences found only within supergroups 1 and 4. Interestingly, the nature of the 2A region of the picornavirus polyprotein was highly correlated with this division of the family ⁽⁹⁾. In the case of the Caphthovirinae (SG1), most if not all viruses encoded a single copy of 2A^NPGP whilst members of the Paavivirinae (SG4) exhibited the highest variability in their 2A region with many viruses encoding multiple 2A^NPGP sequences (e.g., aali-, avisi-, grusopi-, kunsagi-, limnipi-, parecho-, and potamipiviruses) (Figure 1). For instance, the Duck Picornavirus GL/12 (aalivirus A1) has six 2A proteins (Figure 2, Panel A) — the first four all have C-terminal region (~25aa) similarity with the aphthovirus-like 2A^NPGP sequences whilst 2A5 and 2A6 closely resemble avihepatovirus 2A2 and 2A3, respectively ⁽¹⁰⁾.

Figure 1

 

Figure 2

These multiple 2A^NPGP sequences were then tested for translational recoding activity using our pSTA1 dual reporter system [GFP-2A-GUS] ⁽²⁾. Plasmids containing 2A-like sequences were used to programme a Wheat Germ Extract coupled transcription/translation system and proteins resolved by SDS-PAGE separation (Figure 2, Panel B). Constructs were evaluated for recoding activity relative to the positive FMDV 2A control by visual inspection: [GFP2A] + GUS for high activity, [GFP-2A-GUS] + GUS + [GFP2A] for high/moderate activity, and [GFP-2A-GUS] for no activity. Careful analysis of the translational profiles was both surprising and bewildering. Several sequences displayed higher activity than FMDV 2A and could prove useful for future biotechnological applications: aalivirus A1/B1 2A1-2A4; Grusopivirus A1/C 2A1-2A3; Limnipivirus A1 2A1, B1 2A2, C1 2A1, D1 2A1-3; and Mosavirus B1 2A1 and 2A2 (Table 1). In a number of cases, however, only [GFP2A] could be detected: kunsagivirus C1 2A1, limnipivirus A1 2A2, limnipivirus B1 2A1, potamipivirus B1 2A2, Wuhan carp picornavirus (WCP) 2A1/2A2/2A3, and Wenzhou picorna-like virus 48 (WP-LV48) 2A1/2A2 and 2A3. In terms of our current model of ribosome skipping and virus functionality, an enigma wrapped in a conundrum. In our study, we draw a parallel with no-go decay (NGD), a eukaryote translation-coupled mRNA quality control system ⁽¹¹⁾. NGD, one of at least three mRNA surveillance pathways that include nonsense-mediated decay (NMD) and non-stop decay (NSD), is triggered by the presence of mRNA secondary structures leading to ribosome stalling. Persistent stalling sets in motion a cascade of events that aim to inhibit translation re-initiation on the defective mRNA and simultaneously remove faulty mRNAs and recycle the ribosomes in a process mediated by the Pelota/Hbs1/ABCE1 complex ⁽¹²⁾. Consistent with our observations, this would produce [GFP2A] alone, although the NGD pathway implies degradation of the virus RNA, which is perhaps the most important question yet to be addressed.

Table 1. 2A/2A-like sequences used for protein co-expression

Genus	Species	2ANPGP	Amino Acid Sequence	Accession no
Positive-stranded RNA viruses: Picornaviridae
Aalivirus	AalV-A1	2A1	LLTSEGATNSSLLKLAGDVEENPGP	KJ000606
		2A2	FEMPYDDPEWDRLLQAGDIEQNPGP
		2A3	PIPARPDPQWNNLQQAGDVEMNPGP
		2A4	EHFNQTGGWVPDLTQCGDVESNPGP
	AalV-B1	2A1	ATTLQVSEYLKDLTIDGDVESNPGP	MH453803
		2A2	LKVKKLEGDYVRDLTQEGVEPNPGP
		2A3	SVRVTDAGWVRDLTVDGDVESNPGP
		2A4	VFKCHDKCWVDDLTNCGDVESNPGP
Grusopivirus	GrV-A1	2A1	FEKHVKPWRSQEDLSKEGIEPNPGP	KY312544
		2A2	ITDNRYKETDAKWLSRYGVEMNPGP
		2A3	VTQDLYAATNQDQLSNQGIESNPGP
	GrV-C	2A1	YFEERSPHPTQKELGQFGVETNPGP	MK443503
		2A2	ENNSNYDERDAKHLSRYGIEMNPGP
		2A3	CVCTRWSPTMQSELGKYGIEKNPGP
Limnipivirus	A1	2A1	CKEFVRESDNQELLKCGDVESNPGP	JX134222
	B1	2A2	IDLVQAAYSRMRLLLSGDVEQNPGP	KF306267
	C1	2A1	KLLEQILAYKRDLTACGDVESNPGP	KF874490
	D1	2A1	EEEVDWGVGRMRLKMSGDVEENPGP	MG600094
		2A2	AVHLLVTWMRRRLTLSGDIESNPGP
		2A3	DLRAVKSFIESQLMRAGDVERNPGP
Mosavirus	B1	2A1	ESRGTGNCDATTISQCGDVETNPGP	KY855435
		2A2	YVRRSANRTAADISQDGDVETNPGP

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Figure Legends

Figure 1. Supergroup 4 – ‘Paavivirinae’ Viruses not encoding a 2A^NPGP are shown in blue text, those encoding a single copy of 2A^NPGP are shown in black text, whilst those encoding multiple instances are shown in red text.

Figure 2. Schematic (drawn to scale) showing the positions of 2A^NPGP sequences (25aa) within the genome of Aalivirus A1 polyprotein (Panel A). Artificial reporter polyprotein (boxed area) used to programme in vitro Wheat Germ Extract coupled transcription/translation systems together with translation profiles (below). The translation profile from the control FMDV constructs shows three major products: uncleaved [GFP2AGUS] and the cleavage products [GFP2A] and [GUS] whilst the Aalivirus A1 construct shows the two major cleavage products [GFP2A] and [GUS] (Panel B).