Structure and assembly of bacteriophage PRD1, and Escherichia coli virus with a membrane

This article describes the structure and assembly of bacteriophage PRD1, a lipid-containing virus able to infect Escherichia coli. This phage, with an approximate diameter of 65 nm, is composed of an outer protein shell surrounding a lipid-protein membrane which, in turn, encloses the nucleic acid. The phage genome consists of a single linear dsDNA molecule of about 15 kb that has a protein covalently linked to each of its 5' ends. This protein is used as a primer in DNA replication. During assembly membrane proteins are inserted into the host cytoplasmic membrane while major capsid protein multimers are found in the cytoplasm. Capsid multimers, assisted by two nonstructural assembly factors, are capable of translocating the virus-specific membrane resulting in the formation of cytoplasmic empty particles. Subsequent DNA packaging leads to the formation of infections virus.     

Cysteine residues in the transmembrane regions of M13 procoat protein suggest that oligomeric coat proteins assemble onto phage progeny

The M13 phage assembles in the inner membrane of Escherichia coli. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the -6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process.     

Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages

The filamentous phage protein pIV is required for assembly and secretion of the virus and possesses regions homologous to those found in a number of Gram-negative bacterial proteins that are essential components of a widely distributed extracellular protein-export system. These proteins form multimers that may constitute an outer membrane channel that allows phage/protein egress. Three sets of f1 gene IV mutants were isolated at positions that are absolutely (G355 and P375) or largely (F381) conserved amongst the 16 currently known family members. The G355 mutants were non-functional, interfered with assembly of plV⁺ phage, and made Escherichia coli highly sensitive to deoxycholate. The P375 mutants were non-functional and defective in multimerization. Many of the F381 mutants retained substantial function, and even those in which charged residues had been introduced supported some phage assembly. Some inferences about the roles of these conserved amino acids are made from the mutant phenotypes.     

A trans-envelope protein complex needed for filamentous phage assembly and export

Assembly and export of filamentous phage requires four non-capsid proteins: the outer membrane protein, pIV; the inner membrane proteins, pI and pXI; and a cytoplasmic host factor, thioredoxin. Chemical cross-linking of intact cells demonstrates a trans-membrane complex containing pI and pIV. Formation of the complex protects pI from proteolytic cleavage by an endogenous protease. This protection also requires pXI, which is identical to the C-terminal portion of pI. This indicates that pXI, which is required for phage assembly in its own right, is also part of the complex. This complex forms in the absence of any other phage proteins or the DNA substrate; hence, it represents the first preinitiation step of phage morphogenesis. On the basis of protease protection data, we propose that the preinitiation complex is converted to an initiation complex by binding phage DNA, thioredoxin and the initiating minor coat protein(s).     

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.     

Membrane-associated assembly of a phage T4 DNA entrance vertex structure studied with expression vectors

The DNA entrance vertex of the phage head is critical for prohead assembly and DNA packaging. A single structural protein comprises this dodecameric ring substructure of the prohead. Assembly of the phage T4 prohead occurs on the cytoplasmic membrane through a specific attachment at or near the gp20 DNA entrance vertex. An auxiliary head assembly gene product, gp40, was hypothesized to be involved in assembling the gp20 substructure. T4 genes 20, 40 and 20 + 40 were cloned into expression vectors under lambda pL promoter control. The corresponding T4 gene products were synthesized in high yield and were active as judged by their ability to complement the corresponding infecting T4 mutants in vivo. The cloned T4 gene 20 and gene 40 products were inserted into the cytoplasmic membrane as integral membrane proteins; however, gp20 was inserted into the membrane only when gp40 was also synthesized, whereas gp40 was inserted in the presence or absence of gp20. The gp20 insertion required a membrane potential, was not dependent upon the Escherichia coli groE gene, and assumed a defined membrane-spanning conformation, as judged by specific protease fragments protected by the membrane. The inserted gp20 structure could be probed by antibody binding and protein A-gold immunoelectron microscopy. The data suggest that a specific gp20-gp40-membrane insertion structure constitutes the T4 prohead assembly initiation complex.     

Structure and assembly of filamentous bacteriophages

Filamentous bacteriophages are interesting paradigms in structural molecular biology, in part because of the unusual mechanism of filamentous phage assembly. During assembly, several thousand copies of an intracellular DNA-binding protein bind to each copy of the replicating phage DNA, and are then displaced by membrane-spanning phage coat proteins as the nascent phage is extruded through the bacterial plasma membrane. This complicated process takes place without killing the host bacterium.     

Open membranes are the precursors for assembly of large DNA viruses

Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double‐stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convexside of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an ‘open‐eight’ conformation with a characteristic diameter of 90 nm for Mimi‐ and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis.     

In vivo assembly of phage phi 29 replication protein p1 into membrane-associated multimeric structures

The mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown. In the case of the Bacillus subtilis phage 29, the viral protein p1 enhances the rate of in vivo viral DNA replication. Previous work showed that p1 generates highly ordered structures in vitro. We now show that protein p1, like integral membrane proteins, has an amphiphilic nature. Furthermore, immunoelectron microscopy studies reveal that p1 has a peripheral subcellular location. By combining in vivo chemical cross-linking and cell fractionation techniques, we also demonstrate that p1 assembles in infected cells into multimeric structures that are associated with the bacterial membrane. These structures exist both during viral DNA replication and when 29 DNA synthesis is blocked due to the lack of viral replisome components. In addition, protein p1 encoded by plasmid generates membrane-associated multimers and supports DNA replication of a p1-lacking mutant phage, suggesting that the pre-assembled structures are functional. We propose that a phage structure assembled on the cell membrane provides a specific site for 29 DNA replication.     

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full‐length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.     

The TolQRA Proteins Are Required for Membrane Insertion of the Major Capsid Protein of the Filamentous Phage f1 during Infection

Infection of Escherichia coli by the filamentous bacteriophage f1 is initiated by interaction of the end of the phage particle containing the gene III protein with the tip of the F conjugative pilus. This is followed by the translocation of the phage DNA into the cytoplasm and the insertion of the major phage capsid protein, pVIII, into the cytoplasmic membrane. DNA transfer requires the chromosomally encoded TolA, TolQ, and TolR cytoplasmic membrane proteins. By using radiolabeled phages, it can be shown that no pVIII is inserted into the cytoplasmic membrane when the bacteria contain null mutations in tolQ, -R and -A. The rate of infection can be varied by using bacteria expressing various mutant TolA proteins. Analysis of the infection process in these strains demonstrates a direct correlation between the rate of infection and the incorporation of infecting bacteriophage pVIII into the cytoplasmic membrane.     

Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.     

Multimers of the precursor of a type IV pilin-like component of the general secretory pathway are unrelated to pili

Both the mature and precursor forms of PulG, a type IV pilin-like component of the general secretory pathway of Klebsiella oxytoca, can be chemically cross-linked into multimers similar to those obtained by cross-linking the components of type IV pili. To explore the possibility that the PulG precursor could form a pilus-like structure, the PulG sequence was altered in a variety of ways, including (i) replacement of the characteristic hydrophobic region, which is required for the assembly of type IV pilins by the MalE signal peptide, or (ii) fusion of β-lactamase (βlaM) to the C-terminus. Neither of these changes affected multimerization. PulG precursor could be post-translationally processed by pre-pilin peptidase (PulO), indicating that the N-terminus of pre PulG remains on the cytoplasmic side of the cytoplasmic membrane where it is accessible to the catalytic site of this enzyme. Finally, precursor and mature forms of PulG could be efficiently cross-linked in a mixed dimer, indicating that at least a subpopu-lation of the two forms of the protein are probably located in clusters in the cytoplasmic membrane. These results provide further evidence that the cross-linked multimers of the precursor form of PulG are unrelated to type IV pilus-like structures. It is still unclear whether a subpopulation of processed PulG can be assembled into a rudimentary pilus-like structure.     

Functional analysis of the adsorption protein of two filamentous phages with different host specificities

The gene 3 coding for one minor coat protein (adsorption protein) of phage IKe was cloned into an expression plasmid and overproduced. The presence of a promoter for this gene could be demonstrated as well as the incorporation of the IKe gene 3 protein (g3p) into the cytoplasmic membrane of host cells. When 110 carboxy-terminal amino acids were deleted, the truncated protein was translocated across the cytoplasmic membrane into the periplasm. Thus the deleted amino acids bear a membrane anchor domain. In contrast to the partly homologous g3p of the Ff phages, IKe g3p did not alter the membrane properties of its host. IKe g3p was not incorporated into Ff phage particles in amounts detectable by our assays although the presence of IKe g3p may affect the efficiency of Ff phage production. The existence of a structural feature necessary for the specific recognition of the respective g3p during phage assembly is deduced.     

Herpes simplex virus type 1 cytoplasmic envelopment requires functional Vps4

The assembly and egress of herpesviruses are complex processes that require the budding of viral nucleocapsids into the lumen of cytoplasmic compartments to form mature infectious virus. This envelopment stage shares many characteristics with the formation of luminal vesicles in multivesicular endosomes. Through expression of dominant-negative Vps4, an enzyme that is essential for the formation of luminal vesicles in multivesicular endosomes, we now show that Vps4 function is required for the cytoplasmic envelopment of herpes simplex virus type 1. This is the first example of a large enveloped DNA virus engaging the multivesicular endosome sorting machinery to enable infectious virus production.     

Secretion and membrane integration of a filamentous phage-encoded morphogenetic protein

The filamentous phage-encoded gene IV protein is required at high levels for virus assembly, although it is not a constituent of the virion. It is an integral membrane protein that does not contain an extended hydrophobic region of the kind often required for stable integration in the inner membrane. Rather, like a number of Escherichia coli outer membrane proteins, pIV is rich in charged amino acid residues and is predicted to consist of extensive beta-sheet structures. In phage-producing cells, pIV is primarily detected in the outer membrane, while in cells that produce it from the cloned gene, pIV is found in both the inner and outer membranes. The protein is synthesized as a precursor. Following cleavage of the signal sequence and translocation into the periplasm, the mature form is initially found as a soluble species. Soluble pIV then integrates into the membrane with a half-time of one to two minutes. Neither phage assembly nor other phage proteins are needed for this membrane integration, and phage assembly does not require the presence of the soluble form. The gene IV protein may be part of the structure through which the assembling phage is extruded.     

The minor capsid protein gp7 of bacteriophage SPP1 is required for efficient infection of Bacillus subtilis

Gp7 is a minor capsid protein of the Bacillus subtilis bacteriophage SPP1. Homologous proteins are found in numerous phages but their function remained unknown. Deletion of gene 7 from the SPP1 genome yielded a mutant phage (SPP1del7) with reduced burst-size. SPP1del7 infections led to normal assembly of virus particles whose morphology, DNA and protein composition was undistinguishable from wild-type virions. However, only approximately 25% of the viral particles that lack gp7 were infectious. SPP1del7 particles caused a reduced depolarization of the B. subtilis membrane in infection assays suggesting a defect in virus genome traffic to the host cell. A higher number of SPP1del7 DNA ejection events led to abortive release of DNA to the culture medium when compared with wild-type infections. DNA ejection in vitro showed that no detectable gp7 is co-ejected with the SPP1 genome and that its presence in the virion correlated with anchoring of released DNA to the phage particle. The release of DNA from wild-type phages was slower than that from SPP1del7 suggesting that gp7 controls DNA exit from the virion. This feature is proposed to play a central role in supporting correct routing of the phage genome from the virion to the cell cytoplasm.     

Structure and assembly of filamentous bacterial viruses.

Filamentous bacterial viruses are flexible nucleoprotein rods, about 6 nm in diameter by 1000-2000 nm in length (depending on the virus strain). A protein shell encloses a central core of single-stranded circular DNA. The coat protein subunits forming the shell are largely alpha-helix, elongated in an axial direction, and also sloping radially, so as to overlap each other and give an arrangement of subunits reminiscent of scales on a fish. This arrangement of alpha-helices is rather like some models of myosin filaments. An early step in assembly of the virion is the formation of a complex between the viral DNA and an intracellular packaging protein that is not found in completed virions. Newly synthesized coat protein becomes associated with the plasma membrane of the cell. During the final steps of assembly, the packaging protein is displaced from the DNA and replaced by coat protein as the virion passes out through the plasma membrane of the host cell.     

Phage shock proteins B and C prevent lethal cytoplasmic membrane permeability in Yersinia enterocolitica

The bacterial phage shock protein (Psp) stress response system is activated by events affecting the cytoplasmic membrane. In response, Psp protein levels increase, including PspA, which has been implicated as the master effector of stress tolerance. Yersinia enterocolitica and related bacteria with a defective Psp system are highly sensitive to the mislocalization of pore-forming secretin proteins. However, why secretins are toxic to psp null strains, whereas some other Psp inducers are not, has not been explained. Furthermore, previous work has led to the confounding and disputable suggestion that PspA is not involved in mitigating secretin toxicity. Here we have established a correlation between the amount of secretin toxicity in a psp null strain and the extent of cytoplasmic membrane permeability to large molecules. This leads to a morphological change resembling cells undergoing plasmolysis. Furthermore, using novel strains with dis-regulated Psp proteins has allowed us to obtain unequivocal evidence that PspA is not required for secretin-stress tolerance. Together, our data suggest that the mechanism by which secretin multimers kill psp null cells is by causing a profound defect in the cytoplasmic membrane permeability barrier. This allows lethal molecular exchange with the environment, which the PspB and PspC proteins can prevent.