title: 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.
authors: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors: Aleksander A
authors: AA
authors: Demidenko
authors: Joseph N
authors: JN
authors: Blattman
authors: Negin N
authors: NN
authors: Blattman
authors: Philip D
authors: PD
authors: Greenberg
authors: Max L
authors: ML
authors: Nibert
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000818:Animals; D005260:Female; D015683:Gene Products, gag / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D016679:Genome, Viral; D051379:Mice; D008810:Mice, Inbred C57BL; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D012330:RNA, Double-Stranded / Q000378:metabolism; D012087:Reoviridae / Q000378:metabolism*; D015302:Simian Immunodeficiency Virus; D020080:Tandem Repeat Sequences / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; C066594:Gag protein p27, Simian immunodeficiency virus; D015683:Gene Products, gag; D010455:Peptides; D012330:RNA, Double-Stranded
keywords: Orthoreovirus; Reoviridae; dsRNA virus; viral vector
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