Impact of in vitro assembly defects on in vivo function of the phage P22 portal.
The podovirus P22, which infects O-antigen strains of Salmonella, incorporates a dsDNA translocating channel (portal dodecamer) at a unique vertex of the icosahedral capsid. The portal subunit (gp1, 82.7 kDa) exhibits multiple S-Hcdots, three dots, centeredX hydrogen bonding states for cysteines 153, 173, 283 and 516 and these interactions are strongly perturbed by portal ring formation. Here, we analyze in vivo activities of wild type (wt) and Cys-->Ser mutant portals, demonstrate that in vivo activity is correlated with in vitro assembly kinetics, and suggest mechanistic bases for the observed assembly defects. The C283S portal protein, which assembles into rings at about half the rate of wt, exhibits significantly diminished infectivity ( approximately 50% of wt) and manifests its defect prior to DNA packaging, most likely at the stage of procapsid assembly. Conversely, the C516S mutant, which assembles at twice the rate of wt, is more severely deficient in vivo ( approximately 20% of wt) and manifests its defect subsequent to capsid maturation and DNA packaging. Both C153S and C173S portals function at levels close to wt. The results suggest that C283S and C516S mutations may be exploited for improved characterization of the folding and assembly pathway of P22 portal protein.
Sun
Ying
Y
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, MO 64110, USA.
Overman
Stacy A
SA
Thomas
George J
GJ
D019943:Amino Acid Substitution; D017100:Bacteriophage P22 / Q000235:genetics* / Q000254:growth & development*; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D012475:Salmonella / Q000737:chemistry / Q000821:virology*; D013059:Spectrum Analysis, Raman; D014764:Viral Proteins / Q000032:analysis; D014771:Virion / Q000648:ultrastructure; D019065:Virus Assembly / Q000235:genetics / Q000502:physiology*
D036022:Capsid Proteins; D014764:Viral Proteins; C079841:portal protein, bacteriophage P22