Analysis of phage MS2 coat protein mutants expressed from a reconstituted phagemid reveals that proline 78 is essential for viral infectivity.
A full-length cDNA copy of the RNA genome of bacteriophage MS2 was assembled by the in-frame ligation of the central portion of the genome into a plasmid containing the 5' and 3' ends. Upon transformation of the ligation reaction into Escherichia coli, infectious phage particles were released into the medium. The plaquing ability of the phage produced from the cDNA construct was assessed against various bacterial strains confirming that the bacteriophage produced were male-specific. Sensitivity to RNase in agar overlay was used to confirm that the phage contained RNA. In addition, the phage were unable to infect piliated cells overexpressing MS2 coat protein, a resistance conferred by the binding of recombinant coat protein to the infecting strand of RNA at the replicase initiation region, thus preventing translation of the replicase gene. The phage capsids were visualised after negative staining by transmission electron microscopy, and appeared as spherical particles of approximately 25 nm diameter. The capsid proteins were examined by Western blotting, confirming the presence of a single protein of approximately 14 kDa, which bound anti-MS2 coat protein antibodies. The genomic RNA from single plaques was analysed by reverse transcription-PCR and the presence of the MS2 coat protein gene confirmed by DNA sequencing. The production of replicative MS2 phage from cDNA fragments was used to assess the viability of MS2 coat protein mutants, which had previously been shown to assemble into T = 3 capsid-like particles when expressed in vivo from a bacterial vector. The E76D mutation did not appear to affect phage viability, whilst replacement of the completely conserved P78 residue with asparagine abolished the production of infectious particles, suggesting that P78 may be involved in interactions with the phage maturation protein.
Hill
H R
HR
Department of Biology, University of Leeds, UK.
Stonehouse
N J
NJ
Fonseca
S A
SA
Stockley
P G
PG
D001483:Base Sequence; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism; D036022:Capsid Proteins; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D005814:Genes, Viral; D016679:Genome, Viral; D017909:Levivirus / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011392:Proline; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D010948:Viral Plaque Assay; D014779:Virus Replication
D036022:Capsid Proteins; D017931:DNA Primers; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D011392:Proline