24is packaged into a precursor capsid through a 12-mer ring-shaped protein complex 25 called the portal protein, located at a unique 5-fold vertex. In several phages and 26 viruses, including T4, Φ29, and HSV-1, the dodecameric portal protein forms a 27 nucleation complex with scaffolding proteins to initiate procapsid assembly, thereby 28 ensuring incorporation of only one portal complex per capsid. However, for 29 bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is 30 unclear. We recently developed an in vitro P22 assembly assay where portal protein is 31 co-assembled into procapsid-like particles. We also showed that scaffolding protein 32 catalyzes oligomerization of monomeric portal protein into 12-mer rings, and possibly 33 forming a scaffolding-protein nucleation complex that results in one portal complex per 34 P22 procapsid. Here, we present evidence substantiating that P22 portal protein, similar 35 to the other dsDNA viruses, can act as an assembly nucleator. We find that the 36 presence of P22 portal protein is able to increase the rate of particle assembly. 37 Additionally, we show that P22 portal protein proper contributes to proper morphology of 38 the assembled particles. Our results highlight a key function of portal protein as an 39 assembly initiator, a feature likely conserved among these classes of dsDNA viruses. 40 41 Importance 42The existence of a single portal complex is crucial to the formation of mature infectious 43 virions in the tailed dsDNA phages, herpesviruses and adenoviruses, and as such is a 44 3 viable therapeutic target. How only one portal complex is selectively incorporated at a 45 unique vertex is unclear. In many well-characterized dsDNA virus and phages, the 46 portal acts as an assembly nucleator but early work by Bazinet and King (1) indicated 47 that portal protein did not function this way in P22 procapsid assembly, leading to the 48 suggestion that P22 might use a unique mechanism for portal incorporation. Here we 49 show that portal protein does nucleate assembly of P22 procapsid-like particles.
50Addition of portal protein rings to an assembly reaction increases the rate of formation, 51 the yield of particles, and corrects improper morphology. Our data suggest that 52 procapsid assembly may universally initiate with a portal:scaffolding protein complex. 53 54 55 Double stranded DNA (dsDNA) tailed bacteriophages, herpesviruses, some dsDNA 56 archaeal viruses and adenoviruses have a pseudo-icosahedral capsid with a portal 57 protein complex incorporated at a unique five-fold symmetry axis (2-4). The portal 58 complex is a ring of 12 identical subunits constituting an axial channel for the active 59 packaging and ejection of the dsDNA viral genome (1, 5-8). The existence of a single 60 portal complex is crucial to the formation of infectious virions. Evidence from dsDNA 61 bacteriophages such as T4, SPP1, Φ29, and HSV-1 suggests that portal protein 62 complexes play a key role in the nucleation and assembly of proper pro...