A highly conserved basic motif in the wing domain of portal protein is necessary for oligomerization and incorporation in phage P22.

The dodecameric portal ring complex plays a critical role in the assembly of most double-stranded DNA (dsDNA) viruses and bacteriophages. The fidelity of ring oligomerization and incorporation of a single 12-mer portal ring is driven by interactions with a viral scaffolding protein. Structural evidence indicates that the wing domain of the portal is a crucial site for scaffolding protein interactions. In this study, the functional significance of the highly conserved 272KRRR275 motif in the portal wing domain of bacteriophage P22 is investigated. Alteration of the entire motif to alanines results in a reduction in infectious phage production owing to a decrease in portal incorporation into procapsids in vivo. Further interrogation of the variant portal protein in vitro showed that it assembles into oligomeric assemblies larger than dodecamers, as determined by charge detection mass spectrometry. Though variant monomers have an altered secondary structure, they are still capable of assembling into rings in the presence of scaffolding protein, but it is likely that these rings adopt a modified conformation that is unfit for incorporation. These findings indicate that the 272KRRR275 motif is necessary for critical intra-subunit interactions that regulate the portal monomer's folding into the correct conformation for oligomerization into stable 12-mer rings.IMPORTANCETo be infectious, most double-stranded DNA (dsDNA) viruses and bacteriophages package their DNA into the capsid via the dodecameric portal protein vertex. This first requires that portal monomers assemble into a portal ring that becomes incorporated into the capsid, but this oligomerization process is not well defined. Here, we show that mutating a highly conserved basic motif, 272KRRR275, in the bacteriophage P22 portal protein wing domain impairs proper ring assembly and incorporation, despite not being involved in inter-subunit contacts. This defect arises from altered secondary structure of the portal monomer, emphasizing the importance of specific intra-subunit interactions necessary for portal oligomerization. These results further our understanding of essential protein-protein interactions involved in viral assembly and provide insight into key interactions that can aid the development of antiviral drugs targeting portal oligomerization and incorporation.