title: Assembly of tobacco mosaic virus and TMV-like pseudovirus particles in Escherichia coli.
abstract: High-level expression of plant viral proteins, including coat protein (CP), is possible in Escherichia coli. Native tobacco mosaic virus (TMV) CP expressed in E. coli remains soluble but has a non-acetylated N-terminal Ser residue and following extraction, is unable to package TMV RNA in vitro under standard assembly conditions. Changing the Ser to Ala or Pro by PCR-mutagenesis did not confer assembly competence in vitro, despite these being non-acetylated N-termini present in two natural strains of TMV. All TMV CPs made in E. coli formed stacked cylindrical aggregates in vitro at pH 5.0 and failed to be immunogold-labelled using a mouse monoclonal antibody specific for helically assembled TMV CP. TMV self-assembly has been studied extensively in vitro, and an origin of assembly sequence (OAS) mapped internally on the 6.4 kb ssRNA genome. Pseudovirus particles can be assembled mono- or bi-directionally in vitro using virus-derived CP and chimeric ssRNAs containing the cognate TMV OAS, but otherwise of unlimited length and sequence. Studies on plant virus assembly in vivo would be facilitated by a model system amenable to site-directed mutagenesis and rapid recovery of progeny particles. When chimeric transcripts containing the TMV OAS were co-expressed with TMV CP in vivo for 2-18 h, helical TMV-like ribonucleoprotein particles of the predicted length were formed in high yield (up to 7.4 micrograms/mg total bacterial protein). In addition to providing a rapid, inexpensive and convenient system to produce, protect and recover chimeric gene transcripts of any length or sequence, this E. coli system also offers a rapid approach for studying the molecular requirements for plant virus "self-assembly" in vivo. Transcription of a full-length cDNA clone of TMV RNA also resulted in high levels of CP expression and assembly of sufficient intact genomic RNA to initiate virus infection of susceptible tobacco plants.
authors.affiliation: AgBiotech Center, Cook College, Rutgers University, New Brunswick, New Jersey.
authors.forename: D J
authors.initials: DJ
authors.lastname: Hwang
authors.forename: I M
authors.initials: IM
authors.lastname: Roberts
authors.forename: T M
authors.initials: TM
authors.lastname: Wilson
mesh_terms: D002213:Capsid / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D016253:Microscopy, Immunoelectron; D008961:Models, Structural; D009024:Morphogenesis; D016297:Mutagenesis, Site-Directed; D012313:RNA / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014027:Tobacco Mosaic Virus / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure*
chemical_list: D012367:RNA, Viral; C039919:RNA, recombinant; D011994:Recombinant Proteins; D012313:RNA