Identification of an interacting coat-external scaffolding protein domain required for both the initiation of phiX174 procapsid morphogenesis and the completion of DNA packaging

The phiX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven alpha-helices. The protein's core, alpha-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are phiX174, G4, and alpha3) external scaffolding proteins. On the other hand, the primary sequences of the first alpha-helices have diverged. The results of previous studies with alpha3/phiX174 chimeric external scaffolding proteins suggest that alpha-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and phiX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. phiX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.     

Scaffolding proteins altered in the ability to perform a conformational switch confer dominant lethal assembly defects

In the phiX174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D(1)D(2) and D(3)D(4), in a non-quasi-equivalent structure. To achieve this arrangement, alpha-helix 3 assumes two different conformations: (i) kinked 30 degrees at glycine residue 61 in subunits D(1) and D(3) and (ii) straight in subunits D(2) and D(4). Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in alpha-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein.     

Foreign and chimeric external scaffolding proteins as inhibitors of Microviridae morphogenesis

Viral assembly is an ideal system in which to investigate the transient recognition and interplay between proteins. During morphogenesis, scaffolding proteins temporarily associate with structural proteins, stimulating conformational changes that promote assembly and inhibit off-pathway reactions. Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and an external species. The external scaffolding protein is the most conserved protein within the Microviridae, whose canonical members are phiX174, G4, and alpha3. However, despite 70% homology on the amino acid level, overexpression of a foreign Microviridae external scaffolding protein is a potent cross-species inhibitor of morphogenesis. Mutants that are resistant to the expression of a foreign scaffolding protein cannot be obtained via one mutational step. To define the requirements for and constraints on scaffolding protein interactions, chimeric external scaffolding proteins have been constructed and analyzed for effects on in vivo assembly. The results of these experiments suggest that at least two cross-species inhibitory domains exist within these proteins; one domain most likely blocks procapsid formation, and the other allows procapsid assembly but blocks DNA packaging. A mutation conferring resistance to the expression of a chimeric protein (chiD(r)) that inhibits DNA packaging was isolated. The mutation maps to gene A, which encodes a protein essential for packaging. The chiD(r) mutation confers resistance only to a chimeric D protein; the mutant is still inhibited by the expression of foreign D proteins. The results presented here demonstrate how closely related proteins could be developed into antiviral agents that specifically target virion morphogenesis.     

Conformational switching by the scaffolding protein D directs the assembly of bacteriophage phiX174

The three-dimensional structure of bacteriophage phiX174 external scaffolding protein D, prior to its interaction with other structural proteins, has been determined to 3.3 angstroms by X-ray crystallography. The crystals belong to space group P4(1)2(1)2 with a dimer in the asymmetric unit that closely resembles asymmetric dimers observed in the phiX174 procapsid structure. Furthermore, application of the crystallographic 4(1) symmetry operation to one of these dimers generates a tetramer similar to the tetramer in the icosahedral asymmetric unit of the procapsid. These data suggest that both dimers and tetramers of the D protein are true morphogenetic intermediates and can form independently of other proteins involved in procapsid morphogenesis. The crystal structure of the D scaffolding protein thus represents the state of the polypeptide prior to procapsid assembly. Hence, comparison with the procapsid structure provides a rare opportunity to follow the conformational switching events necessary for the construction of complex macromolecular assemblies.     

The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus phiX174.

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage phiX174. Assembly of the phiX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a "closed" procapsid particle has been determined to 3.5 A resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A resolution electron microscopic reconstruction of the "open" procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly alpha-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.     

Structural studies of bacteriophage alpha3 assembly

Bacteriophage alpha3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include phiX174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108S empty procapsid that requires the external D and internal B scaffolding proteins for its formation.The alpha3 "open" procapsid structural intermediate was determined to 15A resolution by cryo-electron microscopy (cryo-EM). Unlike the phiX174 "closed" procapsid and the infectious virion, the alpha3 open procapsid has 30A wide pores at the 3-fold vertices and 20A wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid. Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30A wide pores.The structure of the alpha3 mature virion was solved to 3.5A resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the alpha3 and phiX174 virion structures are in the spike and the DNA-binding proteins. The alpha3 pentameric spikes have a rotation of 3.5 degrees compared to those of phiX174. The alpha3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the phiX174 J protein, retains its carboxy-terminal-binding site on the internal surface of the capsid protein. The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in alpha3 compared to phiX174, allowing the building of about 10% of the ribose-phosphate backbone.     

Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells.

The polymerization of protein subunits into precursor shells empty of DNA is a critical process in the assembly of double-stranded DNA viruses. For the well-characterized icosahedral procapsid of phage P22, coat and scaffolding protein subunits do not assemble separately but, upon mixing, copolymerize into double-shelled procapsids in vitro. The polymerization reaction displays the characteristics of a nucleation limited reaction: a paucity of intermediate assembly states, a critical concentration, and kinetics displaying a lag phase. Partially formed shell intermediates were directly visualized during the growth phase by electron microscopy of the reaction mixture. The morphology of these intermediates suggests that assembly is a highly directed process. The initial rate of this reaction depends on the fifth power of the coat subunit concentration and the second or third power of the scaffolding concentration, suggesting that pentamer of coat protein and dimers or trimers of scaffolding protein, respectively, participate in the rate-limiting step.     

Eliminating the requirement of an essential gene product in an already very small virus: scaffolding protein B-free øX174, B-free

Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage ?X174 morphogenesis. The external scaffolding protein D is highly ordered in the atomic structure and proper function is very sensitive to mutation. In contrast, the internal scaffolding protein B is relatively unordered and extensive alterations do not eliminate function. Despite this genetic laxity, protein B is absolutely required for virus assembly. Thus, this system, with its complex arrangements of overlapping reading frames, can be regarded as an example of "irreducible complexity." To address the biochemical functions of a dual scaffolding protein system and the evolution of complexity, progressive and targeted genetic selections were employed to lessen and finally eliminate B protein-dependence. The biochemical and genetic bases of adaptation were characterized throughout the analysis that led to the sextuple mutant with a B-independent phenotype, as evaluated by plaque formation in wild-type cells. The primary adaptation appears to be the over-expression of a mutant external scaffolding protein. Progeny production was followed in lysis-resistant cells. The ability to produce infectious virions does not require all six mutations. However, the lag phase before progeny production is shortened as mutations accumulate. The results suggest that the primary function of the internal scaffolding protein may be to lower the critical concentration of the external scaffolding protein needed to nucleate procapsid formation. Moreover, they demonstrate a novel mechanism by which a stringently required gene product can be bypassed, even in a system encoding only eight strictly essential proteins.     

Chlamydiaphage Chp2, a skeleton in the phiX174 closet: scaffolding protein and procapsid identification

Chlamydiaphage Chp2 is a member of the family Microviridae, of which bacteriophage phiX174 is the type species. Although grouped in the same family, the relationship between the Microviridae coliphages and the Chp2-like viruses, which infect obligate intracellular parasitic bacteria, is quite distant, with major differences in structural protein content and scaffolding protein dependence. To investigate the morphogenesis of Chp2, large particles were isolated from infected Chlamydophila abortus by equilibrium and rate zonal sedimentation. A monoclonal antibody that recognizes only assembled viral coat proteins was used in these detection assays. Thus, the detected particles represent virions and/or postcapsid formation assembly intermediates. Two distinct particle types were detected, differing in both protein and DNA content. Filled particles lacked VP3, the putative internal scaffolding protein, whereas empty particles contained this protein. These results indicate that VP3 is a scaffolding protein and that the isolated VP3-containing particles most likely represent Chp2 procapsids.     

Effects of an Early Conformational Switch Defect during ?X174 Morphogenesis Are Belatedly Manifested Late in the Assembly Pathway

C-terminal, aromatic amino acids in the ϕX174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B⁻ mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B⁻ assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.     

Detection of intermediates and kinetic control during assembly of bacteriophage P22 procapsid

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter ≈ 50 nm). In this work, we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibrium (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering, which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild-type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly.     

Mode of Host Cell Penetration by Bacteriophage φX174

Bacteriophage phiX174 is an icosahedral phage which attaches to host cells without the aid of a complex tail assembly. When phiX174 was mixed with cell walls isolated from the bacterial host, the virions attached to the wall fragments and the phage deoxyribonucleic acid (DNA) was released. Attachment was prevented if the cell walls were treated with chloroform. Release of phage DNA, but not viral attachment, was prevented if the cell walls were incubated with lysozyme or if the virions were inactivated with formaldehyde. Treatment of the cell walls with lysozyme released structures which were of uniform size (6.5 by 25 nm). These structures attached phiX174 at the tip of one of its 12 vertices, but the viral DNA was not released. The virions attached to these structures were oriented with their fivefold axis of symmetry normal to the long axis of the structure. No virions were attached to these structures by more than one vertex. Freeze-etch preparations of phiX174 adsorbed to intact bacteria showed that the virions were submerged to one half their diameter into the host cell wall, and the fivefold axis of symmetry was normal to the cell surface. A second cell could not be attached to the outwardly facing vertex of the adsorbed phage and thus the phage could not cross-link two cells. When the virions were labeled with (3)H-leucine, purified, and adsorbed to Escherichia coli cells, about 15% of the radioactivity was recovered as low-molecular-weight material from spheroplasts formed by lysozyme-ethylenediaminetetraacetic acid. Other experiments revealed that about 7% of the total parental virus protein label could be recovered in newly formed progeny virus.     

Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro

Coat and scaffolding subunits derived from P22 procapsids have been purified in forms that co-assemble rapidly and efficiently into icosahedral shells in vitro under native conditions. The half-time for this reaction is approximately five minutes at 21 degrees C. The in vitro reaction exhibits the regulated features observed in vivo. Neither coat nor scaffolding subunits alone self-assemble into large structures. Upon mixing the subunits together they polymerize into procapsid-like shells with the in vivo coat and scaffolding protein composition. The subunits in the purified coat protein preparations are monomeric. The scaffolding subunits appear to be monomeric or dimeric. These results confirm that P22 procapsid formation does not proceed through the assembly of a core of scaffolding, which then organizes the coat, but requires copolymerization of coat and scaffolding. To explore the mechanisms of the control of polymerization, shell assembly was examined as a function of the input ratio of scaffolding to coat subunits. The results indicated that scaffolding protein was required for both initiation of shell assembly and continued polymerization. Though procapsids produced in vivo contain about 300 molecules of scaffolding, shells with fewer subunits could be assembled down to a lower limit of about 140 scaffolding subunits per shell. The overall results of these experiments indicate that coat and scaffolding subunits must interact in both the initiation and the growth phases of shell assembly. However, it remains unclear whether during growth the coat and scaffolding subunits form a mixed oligomer prior to adding to the shell or whether this occurs at the growing edge.     

Quantitative analysis of multi-component spherical virus assembly: scaffolding protein contributes to the global stability of phage P22 procapsids

Assembly of the hundreds of subunits required to form an icosahedral virus must proceed with exquisite fidelity, and is a paradigm for the self-organization of complex macromolecular structures. However, the mechanism for capsid assembly is not completely understood for any virus. Here we have investigated the in vitro assembly of phage P22 procapsids using a quantitative model specifically developed to analyze assembly of spherical viruses. Phage P22 procapsids are the product of the co-assembly of 420 molecules of coat protein and approximately 100-300 molecules of scaffolding protein. Scaffolding protein serves as an assembly chaperone and is not part of the final mature capsid, but is essential for proper procapsid assembly. Here we show that scaffolding protein also affects the thermodynamics of assembly, and for the first time this quantitative analysis has been performed on a virus composed of more than one type of protein subunit. Purified coat and scaffolding proteins were mixed in varying ratios in vitro to form procapsids. The reactions were allowed to reach equilibrium and the proportion of the input protein assembled into procapsids or remaining as free subunits was determined by size exclusion chromatography and SDS-PAGE. The results were used to calculate the free energy contributions for individual coat and scaffolding proteins. Each coat protein subunit was found to contribute -7.2(+/-0.1)kcal/mol and each scaffolding protein -6.1(+/-0.2)kcal/mol to the stability of the procapsid. Because each protein interacts with two or more neighbors, the pair-wise energies are even less. The weak protein interactions observed in the assembly of procapsids are likely important in the control of nucleation, since an increase in affinity between coat and scaffolding proteins can lead to kinetic traps caused by the formation of too many nuclei. In addition, we find that adjusting the molar ratio of scaffolding to coat protein can alter the assembly product. When the scaffolding protein concentration is low relative to coat protein, there is a correspondingly low yield of proper procapsids. When the relative concentration is very high, too many nuclei form, leading to kinetically trapped assembly intermediates.     

The functions of the N terminus of the phiX174 internal scaffolding protein, a protein encoded in an overlapping reading frame in a two scaffolding protein system

phiX174 utilizes two scaffolding proteins during morphogenesis, an internal protein (B) and an external protein (D). The B protein induces a conformational change in coat protein pentamers, enabling them to interact with both spike and external scaffolding proteins. While functions of the carboxyl terminus of protein B have been defined, the functions of the amino terminus remain obscure. To investigate the morphogenetic functions of the amino terminus, several 5' deleted genes were constructed and the proteins expressed in vivo. The DeltaNH(2) B proteins were assayed for the ability to complement an ochre B mutant and defects in the morphogenetic pathway were characterized. The results of the biochemical, genetic and second-site genetic analyses indicate that the amino terminus induces conformational changes in the viral coat protein and facilitates minor spike protein incorporation. Defects in conformational switching can be suppressed by substitutions in the external scaffolding protein, suggesting some redundancy of function between the two proteins.     

Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.

Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid.     

The phiX174 protein J mediates DNA packaging and viral attachment to host cells

Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages phiX174, G4, and alpha3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The phiX174 DNA-binding protein, J, is 13 amino acid residues longer than the alpha3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus. Chimeric phiX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric alpha3 and G4 phages, containing the phiX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of alpha3 packaged with phiX174 J protein was determined to 3.5A resolution and compared with the previously determined structures of phiX174 and alpha3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type alpha3 virion proteins. The amino-terminal region of the phiX174 J protein, which is missing from wild-type alpha3 virions, is mostly disordered in the alpha3 chimera. The differences observed between solution properties of wild-type phiX174, wild-type alpha3, and alpha3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the phiX174 J protein in the chimera and the results of mutational analyses suggest that an evolutionary correlation may exist between the size of the J protein and the stoichiometry of the DNA pilot protein H, required in the initial stages of infection. Hence, the function of the J protein is to facilitate DNA packaging, as well as to mediate surface properties such as cell attachment and infection.     

Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function

Escherichia coli strains lacking PriA are severely compromised in their ability to repair UV-damaged DNA and to perform homologous recombination. These phenotypes arise because of a lack of PriA-directed replication fork assembly at recombination intermediates such as D-loops. Naturally arising suppressor mutations in dnaC restore strains carrying the priA2::kan null allele to wild-type function. We have cloned one such gene, dnaC810, and overexpressed, purified, and characterized the DnaC810 protein. DnaC810 can support a PriA-independent synthesis of phiX174 complementary strand DNA. This can be attributed to its ability, unlike wild-type DnaC, to catalyze a SSB-insensitive general priming reaction with DnaB and DnaG on any SSB-coated single-stranded DNA. Gel mobility shift analysis revealed that DnaC810 could load DnaB directly to SSB-coated single-stranded DNA as well as to D loop DNA. This explains the ability of DnaC810 to bypass the requirement for PriA, PriB, PriC, and DnaT during replication fork assembly at recombination intermediates.     

Molecular genetics of bacteriophage P22 scaffolding protein's functional domains

The assembly intermediates of the Salmonella bacteriophage P22 are well defined but the molecular interactions between the subunits that participate in its assembly are not. The first stable intermediate in the assembly of the P22 virion is the procapsid, a preformed protein shell into which the viral genome is packaged. The procapsid consists of an icosahedrally symmetric shell of 415 molecules of coat protein, a dodecameric ring of portal protein at one of the icosahedral vertices through which the DNA enters, and approximately 250 molecules of scaffolding protein in the interior. Scaffolding protein is required for assembly of the procapsid but is not present in the mature virion. In order to define regions of scaffolding protein that contribute to the different aspects of its function, truncation mutants of the scaffolding protein were expressed during infection with scaffolding deficient phage P22, and the products of assembly were analyzed. Scaffolding protein amino acids 1-20 are not essential, since a mutant missing them is able to fully complement scaffolding deficient phage. Mutants lacking 57 N-terminal amino acids support the assembly of DNA containing virion-like particles; however, these particles have at least three differences from wild-type virions: (i) a less than normal complement of the gene 16 protein, which is required for DNA injection from the virion, (ii) a fraction of the truncated scaffolding protein was retained within the virions, and (iii) the encapsidated DNA molecule is shorter than the wild-type genome. Procapsids assembled in the presence of a scaffolding protein mutant consisting of only the C-terminal 75 amino acids contained the portal protein, but procapsids assembled with the C-terminal 66 did not, suggesting portal recruitment function for the region about 75 amino acids from the C terminus. Finally, scaffolding protein amino acids 280 through 294 constitute its minimal coat protein binding site.