Assembly in vitro of bacteriophage P22 procapsids from purified coat and scaffolding subunits

The coat and scaffolding proteins of bacteriophage P22 procapsids have been purified in soluble form. By incubating both purified proteins with a mutant-infected cell extract lacking procapsids, but competent for DNA packaging in vitro (Poteete et al., 1979), we were able to obtain assembly of biologically active procapsids in vitro. The active species for complementation in vitro in both protein preparations copurified with the soluble subunits, indicating that these subunits represent precursors in procapsid polymerization.When the purified coat and scaffolding subunits were mixed directly, they polymerized into double-shelled procapsid-like structures during dialysis from 1.5 m-guanidine hydrochloride to buffer. When dialyzed separately under the same conditions, the scaffolding subunits did not polymerize but remained as soluble subunits, as did most of the coat subunits. No evidence was found for self-assembly of the scaffolding protein in the absence of the coat protein.The unassembled coat subunits sedimented at 3.9 S and the unassembled scaffolding subunits sedimented at 2.4 S in sucrose gradients. The Stokes' radius, determined by gel filtration, was 25 Å for the coat subunits and 34 Å for the scaffolding subunits. These results indicate that the scaffolding subunits are relatively slender elongated molecules, whereas the coat subunits are more globular.The experiments suggest that the procapsid is built by copolymerization of the two protein species. Their interaction on the growing surface of the shell structure, and not in solution, appears to regulate successive binding interactions.     

2-Aminopurine induced mutations in T4 bacteriophage

Summary Treatment of E. coli B bacteria by the base analogue 2-aminopurine, before infection with T4 phages, induces mutations of the transition type into the virus. Treatment of the phage-bacterium complex, only during the first four minutes of the latent period i.e. at a moment where no phage DNA is synthetized, is also mutagenic. The kinetics of acquisition and loss by treated bacteria of mutagenicity and the action of various metabolic inhibitors show that the base analogue is stored into the bacterial or the phage messenger RNA from which it is reused to be incorporated into the phage DNA.This investigation was supported by the Centre National de la Recherche Scientifique (L. A. No 86 et 136), l'Institut Pasteur, la Délégation Générale à la Recherche Scientifique and la Fondation pour la Recherche Medicale.     

Identification of subunit-subunit interactions in bacteriophage P22 procapsids by chemical cross-linking and mass spectrometry

Viral capsids are dynamic structures which self-assemble and undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Chemical cross-linking analyzed by mass spectrometry was used to identify residue specific inter- and intra-subunit interactions in the P22 procapsids. All the intersubunit cross-links occurred between residues clustered in a loop region (residues 157-207) which was previously identified by mass spectrometry based on hydrogen/deuterium exchange and biochemical experiments. DSP and BS3 which have similar distance constraints (12 angstroms and 11.4 angstroms, respectively) cross-linked the same residues between two subunits in the procapsids (K183-K183), whereas DST, a shorter cross-linker, cross-linked lysine 175 in one subunit to lysine 183 in another subunit. The replacement of threonine with a cysteine at residue 182 immediately adjacent to the K183 cross-linking site resulted in slow spontaneous disulfide bond formation in the procapsids without perturbing capsid integrity, thus suggesting flexibility within the loop region and close proximity between neighboring loop regions. To build a detailed structure model, we have predicted the secondary structure elements of the P22 coat protein, and attempted to thread the prediction onto identified helical elements of cryoEM 3D reconstruction. In this model, the loop regions where chemical cross-linkings occurred correspond to the extra density (ED) regions which protrude upward from the outside of the capsids and face one another around the symmetry axes.     

Isolation and characterization of the smallest bacteriophage P4 derivatives packaged into P4-size head in bacteriophage P2-P4 system

Bacteriophage P4, a satellite phage of coliphage P2, is a very useful experimental tool for the study of viral capsid assembly and cos-cleavage. For an in vitro cos-cleavage reaction study of the P2-P4 system, new shortened and selectable markers containing P4 derivative plasmids were designed as a substrate molecules. They were constructed by swapping the non-essential segment of P4 DNA for either the kanamycin resistance (kmr) gene or the ampicillin resistance (apr) gene. The size of the genomes of the resulting markers were 82% (P4 ash8 delRI:: kmr) and 79% (P4 ash8 delRI:: apr) of the wild type P4 genome. To determine the lower limit of genome size that could be packaged into the small P4-size head, these shortened P4 plasmids were converted to phage particles with infection of the helper phage P2. The conversion of plasmid P4 derivatives to bacteriophage particles was verified by the heat stability test and the burst size determination experiment. CsCl buoyant equilibrium density gradient experiments confirmed not only the genome size of the viable phage form of shortened P4 derivatives, but also their packaging into the small P4-size head. P4 ash8 delRI:: apr turned out to be the smallest P4 genome that can be packaged into P4-sized head.     

Clone size distributions of mutations induced by ethyl methanesulfonate in bacteriophage T4

Size distributions of mutant clones can reveal important aspects of the mutation process. Previously published data on mutant clones induced by ethyl methanesulfonate (EMS) in bacteriophage T4 generated a distribution that was essentially flat, implying a mutagenic mechanism involving only rare mispairing by reacted bases. Here, methods for estimating the spontaneous component of such a distribution are used to generate a corrected distribution. The corrected distribution is strongly peaked, implying frequent (but not obligatory) mispairing. Frequent mispairing is in accord with current views of the fates of DNA lesions believed to mediate EMS-induced mutagenesis.     

The structure of the ATPase that powers DNA packaging into bacteriophage T4 procapsids

Packaging the viral genome into empty procapsids, an essential event in the life cycle of tailed bacteriophages and some eukaryotic viruses, is a process that shares features with chromosome assembly. Most viral procapsids possess a special vertex containing a dodecameric portal protein that is used for entry and exit of the viral genome. The portal and an ATPase are parts of the genome-packaging machine. The ATPase is required to provide energy for translocation and compaction of the negative charges on the genomic DNA. Here we report the atomic structure of the ATPase component in a phage DNA-packaging machine. The bacteriophage T4 ATPase has the greatest similarity to monomeric helicases, suggesting that the genome is translocated by an inchworm mechanism. The similarity of the packaging machines in the double-stranded DNA (dsDNA) bacteriophage T4 and dsRNA bacteriophage varphi12 is consistent with the evolution of many virions from a common ancestor.     

Physical mapping of bacteriophage P2 mutations and their relation to the genetic map

Three new deletion mutants and an insertion mutant of E. coli bacteriophage P2, del2, vir79, del4 and sig5, were mapped by the electron microscope heteroduplex method. The deletions were found to cover 45.5-51.6%, 75.6-76.7% and 92.3-99.3% respectively of P2 DNA while sig5 represented a 3.7% insertion at 78.6% from the left end. The region covering 75.9-76.7% of P2 DNA is also deleted in the two previously characterized immunity insensitive variants of P2, vir22 and Hy dis. This region may identify the portion of the genome responsible for immunity. The physical and genetic maps of P2 were previously found to be colinear with respect to the two mutations vir22 and vir37. This relationship is confirmed by the position of del2.     

Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.     

Head length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail.Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1¹, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII¹ is present in reduced amounts as compared to wild type. The precursor to B1¹ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved.The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).     

Location of DNA ends in P2, 186, P4 and lambda bacteriophage heads

When mature phage particles were suspended in a solution containing formaldehyde (0.07 m-Na⁺, pH 9.0, 10% HCHO for 10 min at 23 °C) and the mixture then spread for electron microscopy in the presence of 50% formamide and cytochrome c, the phage lysed and a high proportion of the DNA molecules were seen to be attached to phage tails. The phage tails were found to be attached at only one end of each DNA molecule and denaturation mapping showed that this end was unique for each of the phages P2, 186, P4 and λ. It is argued that in these mature phage particles one specific end of the DNA molecule is present at the head-tail attachment site.     

Thermodynamic stability and point mutations of bacteriophage T4 lysozyme

The thermodynamics of melting of bacteriophage T4 lysozyme and four of its mutants have been measured by van't Hoff methods. The effect of pH has been explored and utilized to obtain the dependence of the enthalpy on temperature as suggested by Privalov and co-workers. The enthalpy change is a steep linear function of temperature. ΔC_p is large and constant within experimental error. Changes in ΔH_u are as large as 30% for a single point mutation. Changes in enthalpy are largely compensated by changes in entropy. Changes in stability, as measured by the free energy of unfolding, are smaller than those of ΔH, but are very large in a relative sense, since ΔG is very much smaller than ΔH. Origins of the destabilization caused by mutations are discussed.     

Structure and function of the repressor of bacteriophage lambda

Summary The high-affinity mutant cI gene of cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of .     

Studies of opal mutations in bacteriophage T4B

Hydroxylamine-induced amber and opal mutants are localized on the map of gene 47 of bacteriophage T4B. The matched map of amber and opal mutations showed the presence of four paired sites which seemed to have arisen in the triplet coding for tryptophan.In growth studies o opal mutants in gene 47 in a series of Su⁺ strains the number of strains bearing a gene-suppressor for amber or ochre mutations also had a weak suppressor activity for some opal mutants. This suppressor acitivity is supposedly due to a second mutation in gene Su_uga.A comparative study of the phage yield with amber and opal mutations located in the same (paired) triplet in gene 47 has shown that the suppressor activity depends on the location of the mutant site along the gene.Experiments dealing with the induction of reversions by nitrous acid in amber and opal mutants with mutational sites located in the same triplet of gene 47 (mutant pairs) have shown the essential influence of the nucleotide sequence in the triplet on the frequency of induced reverse mutations at the given site.     

Structure and function of mutants in the P gene of bacteriophage lambda leading to the pi phenotype

The location of 14 independently isolated spontaneous pi A and pi B point mutants in the lambda P gene and their base exchanges were determined. It was found that the pi B mutation is one unique type mapping close to other pi A mutants. The number of possible pi A mutation sites could be estimated. The mutation sites are distributed asymmetrically in the gene. The N-terminal half of the protein is unchanged. It is assumed to be required for the interaction with the lambda O protein. The P protein can be changed by substitution of a limited number of amino acids at the C-terminus. All functional proteins of this type have pi character. pi proteins do not appear to have altered intracellular levels or stabilities as compared to wild-type P protein. The plating characteristics of our mutants on two groP- mutants located in the dnaJ and dnaK genes, respectively, are strikingly different.     

Genetic analysis of bacteriophage P4 using P4-plasmid ColE1 hybrids

Summary A set of plasmids that contain fragments of the bacteriophage P4 genome has been constructed by deleting portions of a P4-ColE1 hybrid. A P4 genetic map has been established and related to the physical map by examining the ability of these plasmids to rescue various P4 mutations. The P4 vir1 mutation and P4 genes involved in DNA replication (), activation of P2 helper genes ( and ), polarity suppression (psu) and head size determination (sid) have been mapped, as has the region responsible for synthesis of a nonessential P4 protein.One of the deleted plasmids contains only 5900 base pairs (52%) of P4 but will form plaques if additional DNA is added to increase its total size to near that of P4. This plasmid is also unique in that it will not form stable associations with P2 lysogens of E. coli which are recA ⁺. P4 mutants can be suppressed as a result of replication under control of the ColE1 part of the hybrid.