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Viable polioviruses that encode 2A proteins with fluorescent protein tags.

ABSTRACT: The 2A proteins of the Picornaviridae enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in cis and in several cellular proteins in trans. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease functions. We have generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insertion of Discosoma sp. red fluorescent protein (DsRed) after residue 144 of 2A, near the C terminus, produced plaques only slightly smaller than wild-type (wt) virus. The polyprotein harboring the 2A-DsRed fusion protein was efficiently and accurately cleaved; fluorescent 2A proteinase retained protease activity in trans and supported translation and replication of viral RNA, both in vitro and in infected cells. Intracellular membrane reorganization to support viral RNA synthesis was indistinguishable from that induced by wt virus. Infected cells exhibited strong red fluorescence from expression of the 2A-DsRed fusion protein, and the progeny virus was stable for three to four passages, after which deletions within the DsRed coding sequence began to accumulate. Confocal microscopic imaging and analysis revealed a portion of 2A-DsRed in punctate foci concentrated in the perinuclear region that colocalized with replication protein 2C. The majority of 2A, however, was associated with an extensive structural matrix throughout the cytoplasm and was not released from infected cells permeabilized with digitonin.

SUBMITTER: Teterina NL

PROVIDER: S-EPMC2812313 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Viable polioviruses that encode 2A proteins with fluorescent protein tags.

Teterina Natalya L NL Levenson Eric A EA Ehrenfeld Ellie E

Journal of virology 20091125 3

The 2A proteins of the Picornaviridae enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in cis and in several cellular proteins in trans. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease functions. We have generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insert ...[more]

PMID: 19939919

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Project description:BACKGROUND:Conventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs) are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP). RESULTS:As an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv) Ki-4(scFv) and the anti-MucI single-chain fragment variable M12(scFv). During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal was increased. Antibodies with W-tags generated stronger signals than the untagged construct. CONCLUSIONS:Our low-molecular-weight W-tags can be used to monitor the production of antibody fragments on-line. The binding specificity of the recombinant fusion protein is not affected, even though the binding activity decreases slightly with increasing number of tryptophan residues in the W-tags. Thus, the newly designed W-tags offer a versatile and generally applicable alternative to current fluorescent fusion tags.

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Viable polioviruses that encode 2A proteins with fluorescent protein tags.

Publications

Viable polioviruses that encode 2A proteins with fluorescent protein tags.

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