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 ass embled by the in-frame ligation of the central portion of the genome i nto a plasmid containing the 5' and 3' ends. Upon transformation of th e ligation reaction into Escherichia coli, infectious phage particles were released into the medium. The plaquing ability of the phage produ ced from the cDNA construct was assessed against various bacterial str ains confirming that the bacteriophage produced were male-specific. Se nsitivity to RNase in agar overlay was used to confirm that the phage contained RNA. In addition, the phage were unable to infect piliated c ells overexpressing MS2 coat protein, a resistance conferred by the bi nding of recombinant coat protein to the infecting strand of RNA at th e replicase initiation region, thus preventing translation of the repl icase gene. The phage capsids were visualised after negative staining by transmission electron microscopy, and appeared as spherical particl es of approximately 25 nm diameter. The capsid proteins were examined by Western blotting, confirming the presence of a single protein of si milar to 14 kDa, which bound anti-MS2 coat protein antibodies. The gen omic RNA from single plaques was analysed by reverse transcription-PCR and the presence of the MS2 coat protein gene confirmed by DNA sequen cing. The production of replicative MS2 phage from cDNA fragments was used to assess the viability of MS2 coat protein mutants, which had pr eviously been shown to assemble into T = 3 capsid-like particles when expressed in vivo from a bacterial vector. The E76D mutation did not a ppear to affect phage viability, whilst replacement of the completely conserved P78 residue with asparagine abolished the production of infe ctious particles, suggesting that P78 may be involved in interactions with the phage maturation protein. (C) 1997 Academic Press Limited.