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Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression.

ABSTRACT: We tested a strategy for engineering recombinant mammalian reoviruses (rMRVs) to express exogenous polypeptides. One important feature is that these rMRVs are designed to propagate autonomously and can therefore be tested in animals as potential vaccine vectors. The strategy has been applied so far to three of the 10 MRV genome segments: S3, M1, and L1. To engineer the modified segments, a 5' or 3' region of the essential, long ORF in each was duplicated, and then exogenous sequences were inserted between the repeats. The inner repeat and exogenous insert were positioned in frame with the native protein-encoding sequences but were separated from them by an in-frame "2A-like" sequence element that specifies a cotranslational "stop/continue" event releasing the exogenous polypeptide from the essential MRV protein. This design preserves a terminal region of the MRV genome segment with essential activities in RNA packaging, assortment, replication, transcription, and/or translation and alters the encoded MRV protein to a limited degree. Recovery of rMRVs with longer inserts was made more efficient by wobble-mutagenizing both the inner repeat and the exogenous insert, which possibly helped via respective reductions in homologous recombination and RNA structure. Immunogenicity of a 300-aa portion of the simian immunodeficiency virus Gag protein expressed in mice by an L1-modified rMRV was confirmed by detection of Gag-specific T-cell responses. The engineering strategy was further used for mapping the minimal 5'-terminal region essential to MRV genome segment S3.

SUBMITTER: Demidenko AA

PROVIDER: S-EPMC3657830 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression.

Demidenko Aleksander A AA Blattman Joseph N JN Blattman Negin N NN Greenberg Philip D PD Nibert Max L ML

Proceedings of the National Academy of Sciences of the United States of America 20130429 20

We tested a strategy for engineering recombinant mammalian reoviruses (rMRVs) to express exogenous polypeptides. One important feature is that these rMRVs are designed to propagate autonomously and can therefore be tested in animals as potential vaccine vectors. The strategy has been applied so far to three of the 10 MRV genome segments: S3, M1, and L1. To engineer the modified segments, a 5' or 3' region of the essential, long ORF in each was duplicated, and then exogenous sequences were insert ...[more]

PMID: 23630248

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Project description:BackgroundTandem repeats are ubiquitous and abundant in higher eukaryotic genomes and constitute, along with transposable elements, much of DNA underlying centromeres and other heterochromatic domains. In maize, centromeric satellite repeat (CentC) and centromeric retrotransposons (CR), a class of Ty3/gypsy retrotransposons, are enriched at centromeres. Some satellite repeats have homology to retrotransposons and several mechanisms have been proposed to explain the expansion, contraction as well as homogenization of tandem repeats. However, the origin and evolution of tandem repeat loci remain largely unknown.ResultsCRM1TR and CRM4TR are novel tandem repeats that we show to be entirely derived from CR elements belonging to two different subfamilies, CRM1 and CRM4. Although these tandem repeats clearly originated in at least two separate events, they are derived from similar regions of their respective parent element, namely the long terminal repeat (LTR) and untranslated region (UTR). The 5' ends of the monomer repeat units of CRM1TR and CRM4TR map to different locations within their respective LTRs, while their 3' ends map to the same relative position within a conserved region of their UTRs. Based on the insertion times of heterologous retrotransposons that have inserted into these tandem repeats, amplification of the repeats is estimated to have begun at least ~4 (CRM1TR) and ~1 (CRM4TR) million years ago. Distinct CRM1TR sequence variants occupy the two CRM1TR loci, indicating that there is little or no movement of repeats between loci, even though they are separated by only ~1.4 Mb.ConclusionsThe discovery of two novel retrotransposon derived tandem repeats supports the conclusions from earlier studies that retrotransposons can give rise to tandem repeats in eukaryotic genomes. Analysis of monomers from two different CRM1TR loci shows that gene conversion is the major cause of sequence variation. We propose that successive intrastrand deletions generated the initial repeat structure, and gene conversions increased the size of each tandem repeat locus.

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Dataset Information

Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression.

Publications

Engineering recombinant reoviruses with tandem repeats and a tetravirus 2A-like element for exogenous polypeptide expression.

Similar Datasets

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