Garry A. Luke

Occurrence, function and evolutionary origins of ‘2A-like’ sequences in virus genomes

2A is an oligopeptide sequence mediating a ribosome ‘skipping’ effect, producing an apparent ‘cleavage’ of polyproteins. First identified and characterized in picornaviruses, ‘2A-like’ sequences are found in other mammalian viruses and a wide range of insect viruses. Databases were analysed using a motif conserved amongst 2A/2A-like sequences. The newly identified 2A-like sequences (30 aa) were inserted into a reporter polyprotein to determine their cleavage activity. Our analyses showed that these sequences fall into two categories. The majority mediated very high (complete) cleavage to separate proteins and a few sequences mediated cleavage with lower efficiency, generating appreciable levels of the uncleaved form. Phylogenetic analyses of 2A-like sequences and RNA-dependent RNA polymerases (RdRps) indicated multiple, independent, acquisitions of these sequences at different stages during virus evolution. Within a virus family, 2A sequences are (probably) homologous, but diverge due to other evolutionary pressures. Amongst different families, however, 2A/2A-like sequences appear to be homoplasic.

Inhibition of 2A-mediated 'cleavage' of certain artificial polyproteins bearing N-terminal signal sequences

Where 2A oligopeptide sequences occur within ORFs, the formation of the glycyl–prolyl peptide bond at the C‐terminus of (each) 2A does not occur. This property can be used to concatenate sequences encoding several proteins into a single ORF: each component of such an artificial polyprotein is generated as a discrete translation product. 2A and ‘2A‐like’ sequences have become widely utilised in biotechnology and biomedicine. Individual proteins may also be co‐ and post‐translationally targeted to a variety of sub‐cellular sites. In the case of polyproteins bearing N‐terminal signal sequences we observed, however, that the protein downstream of 2A (no signal) was translocated into the endoplasmic reticulum (ER). We interpreted these data as a form of ‘slipstream’ translocation: downstream proteins, without signals, were translocated through a translocon pore already formed by the signal sequence at the N‐terminus of the polyprotein. Here we show this effect is, in fact, due to inhibition of the 2A reaction (formation of fusion protein) by the C‐terminal region (immediately upstream of 2A) of some proteins when translocated into the ER. Solutions to this problem include the use of longer 2As (with a favourable upstream context) or modifying the order of proteins comprising polyproteins.

2A to the fore - research, technology and applications.

Abstract The 2A region of the foot-and-mouth disease virus (FMDV) encodes a short sequence that mediates self-processing by a novel translational effect. Translation elongation arrest leads to release of the nascent polypeptide and re-initiation at the next in-frame codon. In this way discrete translation products are derived from a single open read-size of 2A peptides compared to internal promoters or IRES sequences makes them ideal candidates for use in size-restricted viral and nonviral vectors. Additionally, the diversity of the 2A sequence minimizes the chances for homologous recombination which is an important consideration when using retroviral or lentiviral systems. One outstanding question is the effect of the 2A “tag” attached to the C-terminus of the upstream protein. This may interfere with function, or more importantly may present a new epitope that could be subject to immunological surveillance. However, the attachment of extra amino acids is a routine method for labelling transgene products while leaving their function intact (e.g. tags such as the His tag and Myc tag). To our knowledge, the 2A tag does not impair activity and expression - proteins that require authentic termini, or are N-/C- terminally modified, can be introduced as the first or final polyprotein domain, respectively. In any event, strategies have now been devised that allow removal of the 2A linker (see François et al., 2004; Fang et al., 2005). The “unwanted” tag may however stick – antibodies directed against 2A can be used to detect the gene cloned upstream (Ryan and Drew, 1994; de Felipe et al., 2003, 2006). Lastly, the presence of a proline residue at the N-terminus of the downstream protein, as a relict of the 2A self-cleaving process, does not normally interfere with function – it does, however, confer high protein stability (Varshavsky, 1992). Aware of the factors that influence expression levels it is important to empirically design any co-expression cassette to ensure the polyprotein is the most suitable arrangement in respect to desired function. As a form of control of protein biogenesis, 2A sequences are much more wide-spread than was first suspected. To appease different and opposing sensibilities, 2A variants that are not found in mammalian viruses can be used just as effectively for the production of multiple protein products. Although a relative new-kid-on-the-block in terms of co-expression studies, 2A can safely be considered an “established” player. It is clear that assorted 2A-derived proteins with diverse and distinct localized functions may be stably expressed in several different cell types demonstrating the applicability of this technology in biomedicine and biotechnology. The biotechnological applications of 2A are continually updated on www.st-andrews.ac.uk/ryanlab/Index.htm We envisage that 2A technology will become one of the predominant strategies for multigene delivery in the coming years.

Heat shock protein 40 (Hsp40) plays a key role in the virus life cycle

The heat shock proteins (Hsps) are a diverse subset of molecular chaperones that generally promote the proper folding of proteins after translation and also prevent their aggregation during cellular stress. Paradoxically, cellular chaperones might perform important antiviral functions for host cells, yet, at the same time, might be beneficial for virus replication. Among them, Hsp40 is a specialized co-chaperone that has recently received much attention for its crucial role in both constitutive cellular functions and virus pathogenicity. The aim of this review is to raise awareness of its importance in the life cycles of a wide range of viruses.

Baculovirus-based strategies for the management of insect pests: a focus on development and application in South Africa

There is growing concern among governments, scientists, agricultural practitioners and the general public regarding the negative implications of widespread synthetic chemical pesticide application for the control of crop pests. As a result, baculovirus biopesticides are gaining popularity as components of integrated pest management (IPM) programmes in many countries despite several disadvantages related to slow speed of kill, limited host range and complex large scale production. In South Africa, baculoviruses are incorporated into IPM programmes for the control of crop pests in the field, and recent bioprospecting has led to the characterisation of several novel isolates with the potential to be formulated as commercial products. This contribution will provide an overview of the use of baculoviruses against insect pests in South Africa, as well as research and development efforts aimed at broadening their application as biocontrol agents. Challenges faced by researchers in developmental projects as well as potential users of baculoviruses as biopesticides in the field are also discussed.

FMDV replicons encoding green fluorescent protein are replication competent

Highlights • FMDV replication can be studied outwith high disease secure facilities.• FMDV replicon genomes encoding GFP are replication competent.• These FMDV replicon systems can be used to study replication by live-cell imaging/image analyses.

2A-like signal sequences mediating translational recoding : a novel form of dual protein targeting

We report the initial characterization of an N‐terminal oligopeptide ‘2A‐like’ sequence that is able to function both as a signal sequence and as a translational recoding element. Owing to this translational recoding activity, two forms of nascent polypeptide are synthesized: (i) when 2A‐mediated translational recoding has not occurred: the nascent polypeptide is fused to the 2A‐like N‐terminal signal sequence and the fusion translation product is targeted to the exocytic pathway, and, (ii) a translation product where 2A‐mediated translational recoding has occurred: the 2A‐like signal sequence is synthesized as a separate translation product and, therefore, the nascent (downstream) polypeptide lacks the 2A‐like signal sequence and is localized to the cytoplasm. This type of dual‐functional signal sequence results, therefore, in the partitioning of the translation products between the two sub‐cellular sites and represents a newly described form of dual protein targeting.

APE-Type Non-LTR Retrotransposons of Multicellular Organisms Encode Virus-Like 2A Oligopeptide Sequences, Which Mediate Translational Recoding during Protein Synthesis

2A oligopeptide sequences (“2As”) mediate a cotranslational recoding event termed “ribosome skipping.” Previously we demonstrated the activity of 2As (and “2A-like sequences”) within a wide range of animal RNA virus genomes and non-long terminal repeat retrotransposons (non-LTRs) in the genomes of the unicellular organisms Trypanosoma brucei (Ingi) and T. cruzi (L1Tc). Here, we report the presence of 2A-like sequences in the genomes of a wide range of multicellular organisms and, as in the trypanosome genomes, within non-LTR retrotransposons (non-LTRs)—clustering in the Rex1, Crack, L2, L2A, and CR1 clades, in addition to Ingi. These 2A-like sequences were tested for translational recoding activity, and highly active sequences were found within the Rex1, L2, CR1, and Ingi clades. The presence of 2A-like sequences within non-LTRs may not only represent a method of controlling protein biogenesis but also shows some correlation with such apurinic/apyrimidinic DNA endonuclease-type non-LTRs encoding one, rather than two, open reading frames (ORFs). Interestingly, such non-LTRs cluster with closely related elements lacking 2A-like recoding elements but retaining ORF1. Taken together, these observations suggest that acquisition of 2A-like translational recoding sequences may have played a role in the evolution of these elements.

Growing Uses of 2A in Plant Biotechnology

The combination, ‘pyramiding’ or ‘stacking’ of multiple genes in plants is a fundamental aspect of modern plant research and biotechnology. The most widely adopted stacked traits (herbi‐ cide tolerance and insect protection) provide growers with benefits of increased crop yield, simplified management of weed control and reduced insecticide use. The global acreage of stacked traits or more precisely genetically modified organisms bearing stacked traits is expected to increase rapidly in the near future, with the introduction of nutritional and/or industrial traits to satisfy the needs of consumers and producers [1]. Several approaches have been used to stack multiple genes into plant genomes and then to coordinate expression [2-4]. Stacking approaches include sexual crossing between plants carrying distinct transgenes [5,6], sequential re-transformation [7], and single-plasmid [8] or multiple-plasmid co-transforma‐ tion [9]. These strategies, however, suffer from the inherent weakness that co-expression of the heterologous proteins is unreliable.

Lost in translation: The biogenesis of non-LTR retrotransposon proteins.

“Young” APE-type non-LTR retrotransposons (non-LTRs) typically encode two open reading frames (ORFs 1 and 2). The shorter ORF1 translation product (ORF1p) comprises an RNA binding activity, thought to bind to non-LTR transcript RNA, protect against nuclease degradation and specify nuclear import of the ribonuclear protein complex (RNP). ORF2 encodes a multifunctional protein (ORF2p) comprising apurinic/apyrimidinic endonuclease (APE) and reverse-transcriptase (RT) activities, responsible for genome replication and re-integration into chromosomal DNA. However, some clades of APE-type non-LTRs only encode a single ORF—corresponding to the multifunctional ORF2p outlined above (and for simplicity referred-to as ORF2 below). The absence of an ORF1 correlates with the acquisition of a 2A oligopeptide translational recoding element (some 18–30 amino acids) into the N-terminal region of ORF2p. In the case of non-LTRs encoding two ORFs, the presence of ORF1 would necessarily downregulate the translation of ORF2. We argue that in the absence of an ORF1, 2A could provide the corresponding translational downregulation of ORF2. While multiple molecules of ORF1p are required to decorate the non-LTR transcript RNA in the cytoplasm, conceivably only a single molecule of ORF2p is required for target-primed reverse transcription/integration in the nucleus. Why would the translation of ORF2 need to be controlled by such mechanisms? An “excess” of ORF2p could result in disadvantageous levels of genome instability by, for example, enhancing short, interspersed, element (SINE) retrotransposition and the generation of processed pseudogenes. If so, the acquisition of mechanisms—such as 2A—to control ORF2p biogenesis would be advantageous.