Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coli.

Abstract

The bidirectional self-assembly of tobacco mosaic virus (TMV, common or Ul strain) has been studied extensively in vitro. Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS,432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical pseudovirus particles. To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants. The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications. Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA. U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties. However, in vivo coexpression of CP and singlestranded RNAs (up to =2 kb) containing the TMV OAS gave high yields of helical pseudovirus particles of the predicted length (up to 7.4 ± 1.4 jpg/mg of total bacterial protein). If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of pseudovirus assembly was blocked. In vivo, E. coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants.Tobacco mosaic virus (TMV) particles are extremely stable and retain infectivity for decades. Over 2100 copies of a 17.6-kDa coat protein (CP) fully protect the 6.4-kb singlestranded RNA (ssRNA) genome against degradation by RNases. TMV was the model system of choice for early studies on the spontaneous "self-assembly" of multimeric, biological structures in vitro (1-3). TMV assembly is initiated by a specific interaction between a prefabricated (20 S) protein aggregate (the "disk" or protohelix) and an RNA sequence centered either 0.4 kb or 0.9 kb from the 3' end of the genomic RNA (4). An origin-of-assembly sequence (OAS) was characterized (5) and proposed to exist as three stem-loop structures, before the complete 6395-nt genome of the common (or Ul) strain of TMV had been sequenced (6). The structures ofthe prefabricated, oligomeric forms ofTMV CP used for efficient assembly initiation and/or bidirectional elongation, and the precise assembly pathway (i.e., which CP form is used in which RNA direction, and whether simultaneously or sequentially) remain controversial (7)(8)(9), and the data continue to evade a consensus model (10). Two simple protocols exist to isolate CP from virions for subsequent encapsidation of ssRNA in vitro (11,12).In addition to native TMV RNA, or 3' coterminal nested genome fragments, TMV CP was shown to package chimeric ssRNAs efficiently (13, 14) in a length-and sequenceindependent manner, provided that a contiguous viral s...

Functional Analysis of a DNA-Shuffled Movement Protein Reveals That Microtubules Are Dispensable for the Cell-to-Cell Movement of Tobacco mosaic virus

“…van Regenmortel). After incubation with protoplast supernatant samples, bound coat protein was detected using rabbit polyclonal antiserum (Hwang et al, 1994) followed by alkaline phosphatase-conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO). All antibodies were used at a dilution of 1:2000.…”

Plant virus directed fabrication of nanoscale materials and devices

“…Viral nucleic acids encode origin of assembly (OAS) sequences that function in association with the CP to initiate virion assembly (Basnayake et al, 2009;Butler, 1999). In addition, OAS sequences are modular and when engineered into a heterologous nucleic acid sequence can direct the encapsidation of that sequence (Hwang et al, 1994). Interestingly, Loo et al attached an oligonucleotide mimic of the RCNMV OAS to Au, CdSe and CoFe 2 O 4 nanoparticles, 3-15 nm in diameter (Loo et al, 2007).…”

Block copolymer nanotemplating of tobacco mosaic and tobacco necrosis viruses

“…The tendency was for the virus to be weakly stained, accompanied by a loss in contrast from the metal-containing microstructure of the copolymer. A successful alternative was to stain the block copolymer/virus system with a 1% solution of uranyl acetate (UA) [20]. When applied to the block copolymer/virus surface, UA was able to clearly show virus particles while not interfering with the visualization of the block copolymer microstructure.…”