Encapsidation of heterologous RNAs by bacteriophage MS2 coat protein

Abstract

The RNA bacteriophages of E. coli specifically encapsidate a single copy of the viral genome in a protein shell composed mainly of 180 molecules of coat protein. Coat protein is also a translational repressor and shuts off viral replicase synthesis by interaction with a RNA stem-loop containing the replicase initiation codon. We wondered whether the translational operator also serves as the viral pac site, the signal which mediates the exclusive encapsidation of viral RNA by its interaction with coat protein. To test this idea we measured the ability of lacZ RNA fused to the translational operator to be incorporated into virus-like particles formed from coat protein expressed from a plasmid. The results indicate that the operator-lacZ RNA is indeed encapsidated and that nucleotide substitutions in the translational operator which reduce the tightness of the coat protein-operator interaction also reduce or abolish encapsidation of the hybrid RNA. When coat protein is expressed in excess compared to the operator-lacZ RNA, host RNAs are packaged as well. However, elevation of the level of operator-lacZ RNA relative to coat protein results in its selective encapsidation at the expense of cellular RNAs. Our results are consistent with the proposition that this single protein-RNA interaction accounts both for translational repression and viral genome encapsidation.

A novel mRNA affinity purification technique for the identification of interacting proteins and transcripts in ribonucleoprotein complexes

“…First, to avoid potential artifacts resulting from MS2-CP aggregation upon overexpression (Pickett and Peabody 1993), we placed MS2-CP-GFP-SBP under an inducible promoter in both yeast ( Fig. 2A) and mammalian cells (Figs.…”

Bio-distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo

“…Virus capsids are relatively rigid structures, making it feasible to display molecules or epitopes in precise spatial distributions at the nanoscale, a difficult task using inorganic or lipid materials [15,52]. Viral nanoparticles (VNPs) currently in development include cowpea mosaic virus (CPMV) [9,29,51,[58][59][60], cowpea chlorotic mottle virus (CCMV) [2], bacteriophages (MS2 [3,8,23,43], M13 [28,31,32] Qβ [4]) and polyoma virus [1]. A significant challenge in the design of new nanoparticle formulations is monitoring the plasma circulation, biodistribution and toxicity in vivo, which is required to establish the biocompatibility of new nanomaterials prior to extensive development of a formulation strategy.…”

In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

“…We have shown previously that the operator had rather small effect on the specificity of capsid contents for recombinant capsids, which are produced with and without GAoperator sequences in mRNAs (Strods et al, 2013), although many successful examples of use of MS2 operator have been elaborated (Pickett and Peabody, 1993;Pasloske et al, 1998;Legendre and Fastrez, 2005;Wei et al, 2008).…”