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PubMed1982

Cosmid DNA packaging in vivo.

The packaging of cosmid DNA into phage particles during phage lambda growth is described. Evidence is presented supporting the work of others that cosmid transducing phages contain linear multimers of cosmid DNA in which the number of cosmid copies is that required to make a packagable DNA length (greater than 0.77 of the lambda DNA length). The yield of cosmid transducing phages declines sharply as the number of cosmid copies required to make a packagable DNA length increases. The cosmid DNA replication that produces the packaging substrate shares with lambda rolling-circle replication a dependence on the lambda gam gene product.

Bacteriophage lambda↗
PubMed1990

RNA dependence of the bacteriophage phi 29 DNA packaging ATPase.

The activity of the DNA packaging adenosine triphosphatase (ATPase) of the Bacillus subtilis bacteriophage phi 29 is dependent upon prohead RNA. The 174 nucleotide viral-encoded RNA is positioned on the head-tail connector at the portal vertex of the phi 29 precursor shell (prohead). Here, the RNA interacts with the ATP-binding gene 16 product (gp16) to constitute the DNA-packaging ATPase and initiate DNA packaging in vitro. Both the prohead connector (gene 10 product, gp10) and gp16 may utilize an RNA recognition motif characteristic of a number of RNA-associated proteins, and the binding of gp16 by proheads shields the prohead RNA from RNase A. The ATPase activity of gp16 is stimulated fourfold by RNA and tenfold by proheads with RNA. RNA is needed continuously for the gp16/RNA ATPase activity and is essential for the gp16/prohead ATPase activity. The prohead, with its connector, RNA and associated gp16 in an assembly-regulated configuration, hydrolyzes ATP and drives phi 29 DNA translocation.

Adenosine Triphosphatases↗
PubMed2004

The portal protein plays essential roles at different steps of the SPP1 DNA packaging process.

A large number of viruses use a specialized portal for entry of DNA to the viral capsid and for its polarized exit at the beginning of infection. These families of viruses assemble an icosahedral procapsid containing a portal protein oligomer in one of its 12 vertices. The viral ATPase (terminase) interacts with the portal vertex to form a powerful molecular motor that translocates DNA to the procapsid interior against a steep concentration gradient. The portal protein is an essential component of this DNA packaging machine. Characterization of single amino acid substitutions in the portal protein gp6 of bacteriophage SPP1 that block DNA packaging identified sequential steps in the packaging mechanism that require its action. Gp6 is essential at early steps of DNA packaging and for DNA translocation to the capsid interior, it affects the efficiency of DNA packaging, it is a central component of the headful sensor that determines the size of the packaged DNA molecule, and is essential for closure of the portal pore by the head completion proteins to prevent exit of the DNA encapsidated. Functional regions of gp6 necessary at each step are identified within its primary structure. The similarity between the architecture of portal oligomers and between the DNA packaging strategies of viruses using portals strongly suggests that the portal protein plays the same roles in a large number of viruses.

Amino Acid Substitution↗
PubMed1987

Early events in DNA packaging in a defined in vitro system of bacteriophage T3.

We have developed a defined in vitro system for packaging phage T3 DNA which is composed of purified proheads and the noncapsid proteins gp18 and gp19, products of genes 18 and 19. The reaction requires Mg2+, ATP, and polyethylene glycol and is inhibited by a nonhydrolyzable ATP analog, adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S) (K. Hamada, H. Fujisawa, and T. Minagawa, 1986, Virology 151, 119-123). About 30% of added mature T3 DNA was packaged into heads in the defined system. A complex with a sedimentation coefficient of about 50 S (50 S complex) accumulated in the reaction mixture containing ATP-gamma-S. The 50 S complex was DNase sensitive and was converted to filled heads by a second reaction in the presence of ATP without addition of DNA, proheads, gp18, and gp19. These results indicate that during early stages of DNA packaging, formation of precursor complexes proceeds by an allosteric mechanism with ATP acting as effector. The movement of DNA into the head is driven by the energy released by hydrolysis of ATP. gp18 formed a complex with DNA without addition of ATP-gamma-S and gp19. gp18-DNA complex was DNase sensitive and did not bind gp19; it was converted to filled heads by way of a second reaction after addition of ATP, gp19, and proheads. gp19 formed a functional complex with prohead in the presence of ATP-gamma-S or ATP. The complex did not bind gp18 but was converted to filled heads by incubation with ATP, gp18, and DNA. In the absence of ATP-gamma-S, gp19 formed complexes with prohead that were abortive in DNA packaging. Formation of the 50 S complex occurred in a reaction mixture containing gp18-DNA and gp19-prohead complexes in the presence of ATP-gamma-S. From these results, we propose details of the molecular mechanism of DNA packaging in the defined in vitro system.

Adenosine Triphosphate↗
PubMed1985

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144.

Bacillus subtilis↗
PubMed1996

Complete inhibition of virion assembly in vivo with mutant procapsid RNA essential for phage phi 29 DNA packaging.

A highly efficient method for the inhibition of bacteriophage phi 29 assembly was developed with the use of mutant forms of the viral procapsid (or packaging) RNA (pRNA) indispensable for phi 29 DNA packaging. Phage phi 29 assembly was severely reduced in vitro in the presence of mutant pRNA and completely blocked in vivo when the host cell expressed mutant pRNA. Addition of 45% mutant pRNA resulted in a reduction of infectious virion production by 4 orders of magnitude, indicating that factors involved in viral assembly can be targets for efficient and specific antiviral treatment. The mechanism leading to the high efficiency of inhibition was attributed to two pivotal features. First, the pRNA contains two separate, essential functional domains, one for procapsid binding and the other for a DNA-packaging role other than procapsid binding. Mutation of the DNA-packaging domain resulted in a pRNA with no DNA-packaging activity but intact procapsid binding competence. Second, multiple copies of the pRNA were involved in the packaging of one genome. This higher-order dependence of pRNA in viral replication concomitantly resulted in its higher-order inhibitory effect. This finding suggested that the collective DNA-packaging activity of multiple copies of pRNA could be disrupted by the incorporation of perhaps an individual mutant pRNA into the group. Although this mutant pRNA could not be used for the inhibition of the replication of other viruses directly, the principle of using molecules with two functional domains and multiple-copy involvement as targets for antiviral agents could be applied to certain viral structural proteins, enzymes, and other factors or RNAs involved in the viral life cycle. This principle also implies a strategy for gene therapy, intracellular immunization, or construction of transgenic plants resistant to viral infection.

Bacillus Phages↗
PubMed1992

Conformation of DNA packaged in bacteriophage T7. Analysis by use of ultraviolet light-induced DNA-capsid cross-linking.

The conformation of the linear, double-stranded, 39,936 kilobase-pair DNA packaged in the protein capsid of bacteriophage T7 is investigated here by use of short wavelength ultraviolet light-induced DNA-capsid cross-linking. To detect both DNA-capsid and DNA-DNA cross-links, DNA is expelled from the T7 capsid and the products of expulsion are analyzed by use of Nycodenz buoyant density centrifugation, followed by either pulsed field gel electrophoresis or invariant field gel electrophoresis. Short wavelength ultraviolet light is found to progressively induce both DNA-DNA and DNA-protein cross-links in intact bacteriophage T7, but not in T7 from which DNA had been expelled before exposure to ultraviolet light. Protein-protein cross-links are not induced. When DNA expelled from previously cross-linked T7 is cleaved with restriction endonuclease (1 to 3 sites cleaved), analysis of the resulting fragments reveals no regions on T7 DNA that are excluded from cross-linking to the capsid. However, the efficiency of cross-linking decreases as the distance from the left end (last end packaged) of the packaged DNA increases. Electron microscopy of negatively stained capsid-DNA complexes reveals no DNA-retaining structure other than the outer shell of the capsid. Together with previously reported data that indicate lack of protein-based specificity for ultraviolet light-induced cross-linking, these observations are interpreted by the assumptions that, within the limits of resolution of these experiments: (1) no region of packaged T7 DNA is excluded from contact with the outer shell of the T7 capsid; (2) the probability of contacting the outer shell decreases as the distance from the left end of packaged T7 DNA increases. Thus, T7 DNA packaging concentrates the last end packaged near the inner surface of the outer shell of the T7 capsid.

Capsid↗
PubMed2001

The N-terminal ATPase site in the large terminase protein gp17 is critically required for DNA packaging in bacteriophage T4.

Double-stranded DNA packaging in bacteriophages is apparently driven by the most powerful molecular motor ever measured. Although it is widely accepted that a translocating ATPase powers the DNA packaging machine, the identity of the ATPase that generates this driving force is unknown. Evidence suggests that the large terminase protein gp17, which possesses two consensus ATP binding motifs and an ATPase activity, is a strong candidate for the translocating ATPase in bacteriophage T4. This hypothesis was tested by a PCR-directed combinatorial mutagenesis approach in which mutant libraries consisting of all possible codon combinations were constructed at the signature residues of the ATP binding motifs. The impact on gp17 function of each randomly selected mutant was evaluated by phenotypic analysis following recombinational transfer into the viral genome. The precise mutation giving rise to a particular phenotype was determined by DNA sequencing. The data showed that the N-terminal ATP binding site I (SRQLGKT(161-167)), but not the ATP binding site II (TAAVEGKS(299-306)), is critical for gp17 function. Even conservative substitutions such as G165A, K166R, and T167A were not tolerated at the GKT signature residues, which are predicted to interact with the ATP substrate. Biochemical analyses of the mutants showed a complete loss of in vitro DNA packaging activity but not the terminase (DNA-cutting) activity. The purified K166G mutant showed a loss of gp17-ATPase activity. The data, for the first time, implicated a specific ATPase center in the viral dsDNA packaging.

Adenosine Triphosphatases↗
PubMed1995

Virus DNA packaging: the strategy used by phage lambda.

Phage lambda, like a number of other large DNA bacteriophages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is co-ordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the lambda DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric lambda DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN to complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endonuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNu1, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in lambda has aspects that differ from other lambda DNA transactions. Unlike the site-specific recombination system of lambda, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complex, and unlike the DNA replication system of lambda, the same protein machinery is used for both initiation and translocation during lambda DNA packaging.

Amino Acid Sequence↗
PubMed1993

RNA-mediated specificity of DNA packaging into hybrid lambda/phi 29 proheads.

A small RNA (pRNA, 174 nt) is known to be essential for DNA packaging in bacteriophage phi 29. However, in an in vitro DNA packaging system based on hybrid lambda/phi 29 proheads (made up of head proteins from phage lambda and connectors from phage phi 29), the specificity of DNA packaging is lost, and different RNA molecules fulfil the requirements for DNA packaging, albeit with less efficiency than phi 29 pRNA. Competition assays with RNAs from different sources have shown that phi 29 connectors bind preferentially pRNA. An increase in the efficiency of phi 29 DNA packaging into hybrid proheads induced by phi 29 pRNA is observed because, when phi 29 pRNA is incubated with hybrid proheads, phi 29 DNA is packaged more efficiently than other DNAs of similar length. Furthermore, when hybrid proheads carrying phi 29 pRNA are incubated with a mixture of DNAs from different sources, phi 29 DNA is selectively packaged, thus indicating that phi 29 pRNA determines the specificity of DNA packaging.

Amino Acid Sequence↗
PubMed1985

Ion etching bacteriophage T4: support for a spiral-fold model of packaged DNA.

Ion etching of bacteriophage T4 erodes virus components progressively from the outside to the inside while preserving the overall structure. The terminal portion of the T4 DNA molecule packaged can be specifically radiolabeled and was found to be eroded more rapidly than the remainder of the DNA. This strongly suggests that the first DNA to enter the prohead is condensed in the center of the capsid and is therefore shielded from the ion beam by the surrounding last packaged DNA. The results support a "spiral-fold" model for the arrangement of DNA within the icosahedral bacteriophage head. According to this model, phage T4 DNA strands run parallel to the long axis of the phage, with sharp (180 degrees) bends at the top and bottom of the capsid. The folds themselves are arranged radially about the long axis of the head in spirally organized shells.

DNA, Viral↗
PubMed2003

Use of acetone to attain highly active and soluble DNA packaging protein Gp16 of Phi29 for ATPase assay.

All the well-defined DNA-packaging motors of the dsDNA viruses contain one pair of nonstructural DNA-packaging enzymes. Studies on the mechanism of virus DNA packaging have been seriously hampered by their insolubility. Phi29's DNA-packaging enzyme, gp16, is also hydrophobic, insoluble, and self-aggregating. This article describes approaches to obtain affinity-purified, soluble, and highly active native gp16 with the aid of polyethylene glycol or acetone. The specific activity of this native gp16 was increased 3400-fold when compared with the traditional method. This unique approach made the ATP-gp16 interaction study feasible. Gp16 binds strongly to ATP, binds to ADP with a lower efficiency, and binds very weakly to AMP. The order of gp16-binding efficiency to the four ribonucleotides is, from high to low, ATP, GTP, CTP, and UTP. The ATP concentration level required to produce 50% of maximum virus yield exhibited during in vitro phi29 assembly is around 45 microM, which is close to the gp16 and ATP dissociation constant of 65 microM. Mutation studies revealed that changing only one conserved amino acid, whether R(17), G(24), G(27), G(29), K(30), or I(39), in the predicted Walker-A ATP motif of gp16 caused ATP hydrolysis and viral assembly to cease, while such mutation did not affect gp16's binding to ATP. However, mutation on amino acids G(248) and D(256) did not affect the function of gp16 in DNA packaging.

Acetone↗
PubMed1978

Visualization of the intracellular development of bacteriophage lambda, with special reference to DNA packaging.

To reveal intermediates in lambda DNA packaging, infected cells were osmotically ruptured and the cell lysates were deposited on electron microscope grids by sedimentation through a sucrose/formalin cushion. A fixation procedure that crosslinks head-related structures to DNA allowed us to study successive stages in the process of head filling. Three types of head-related structures can be distinguished: (i) empty heads (petit lambda), less angular in outline than complete lambda heads; (ii) heads partially filled with DNA (partially filled heads), having a roundish outline; and (iii) particles tightly packed with DNA (full heads), having a hexagonal outline. DNA-head complexes were bound either at the terminal end of a DNA thread or at a point intermediate along the thread. The terminal complexes were more abundant. No head-related structures could be found in an induced lambda mutant lysogen blocked in the synthesis of petit lambda (amber in lambda gene E). One type of mutant blocked in DNA packaging (amber in gene A) produces empty heads and free tails, whereas another (amber in gene D) produces partially filled heads in addition. Our data suggest that a DNA-petit lambda complex may be an early intermediate in packaging and that the lambda DNA substrate can be a cohesive-ended concatemer or a concatemer with double-stranded cohesive site sequences.

Coliphages↗
PubMed1996

DNA sequences responsible for specificity of DNA packaging and phage growth interference of bacteriophages T3 and T7.

T3 and T7 phages package recombinant plasmids carrying DNA necessary for DNA packaging (the pac sequences) of T3 and T7, respectively. Packaging is specific between T3 and T7. The pac sequence has a bipartite structure, consisting of target sequences for processing of concatemeric DNA (pac C) and its left side flanking sequence containing a promoter for phage RNA polymerase (pac B). To determine the sequences responsible for the specificity of plasmid DNA packaging, plasmids chimeric for the pac B and pac C sequences of T3 and T7 were constructed. Analysis of packaging of the chimeric plasmid DNAs showed that pac B is responsible for the packaging specificity of T3 and T7 DNAs. Plasmids carrying the genetic right end of T3 and T7 DNA interfered with the growth of T3 and T7 phages, respectively. Interference was specific between T3 and T7. pac B and sequences between pac B and pac C, but not pac C, were responsible for the interference. The specificity of interference was determined by pac B and sequences responsible for interference were partially defined.

Bacteriophage T3↗
PubMed1982

Bacteriophage lambda DNA packaging: scanning for the terminal cohesive end site during packaging.

Bacteriophage lambda packages the DNA of the related phage 21 poorly [Hohn, B. (1975) J. Mol. Biol. 98, 93--106]. To understand the nature of the packaging defect, the interaction of the cohesive end site (cos) specific for phage 21 (cos phi 21) with phage lambda terminase has been investigated. The ability of lambda terminase to cleave cos phi 21 was studied in vitro; lambda terminase cleaved cos phi 21 only 1% as well as it cleaved the phage lambda cohesive end site (cos lambda). In vitro packaging experiments showed that the lambda and 21 packaging specificities observed in vivo are also found in vitro. The cos cleavage reaction was modified so that competition experiments could be performed; these experiments showed that cos phi 21 was unable to bind lambda terminase, thus identifying the nature of the defect. Previous work [Feiss, M., Fisher, R. A., Siegele, D. A., Nichols, B. P. & Donelson, J. E. (1979) Virology 92, 56--67] has shown that the base pairs giving lambda or 21 packaging specificity are at the left end of the chromosome, outside the 22-base-pair symmetry region that includes the annealed cohesive ends. Therefore, terminase binding to cos requires interactions with base pairs to the Nu1 side of the cohesive end symmetry segment. The evidence supports the proposition that cos consists of adjacent sites for binding of terminase and for nicking by terminase. Because cos phi 21 can be cut by lambda terminase to terminate DNA packaging, it is proposed that the terminase that binds and nicks at the initial cos site is brought into contact with the terminal cos site by the packaging process. Terminase recognizes and nicks the cohesive end sequence of the terminal cos without requiring the binding site.

Bacteriophage lambda↗
PubMed1991

Nucleotide sequence of the bacteriophage P22 genes required for DNA packaging.

The mechanism of DNA packaging by dsDNA viruses is not well understood in any system. In bacteriophage P22 only five genes are required for successful condensation of DNA within the capsid. The products of three of these genes, the portal, scaffolding, and coat proteins, are structural components of the precursor particle, and two, the products of genes 2 and 3, are not. The scaffolding protein is lost from the structure during packaging, and only the portal and coat proteins are present in the mature virus particle. These five genes map in a contiguous cluster at the left end of the P22 genetic map. Three additional genes, 4, 10, and 26, are required for stabilizing of the condensed DNA within the capsid. In this report we present the nucleotide sequence of 7461 bp of P22 DNA that contains the five genes required for DNA condensation, as well as a nonessential open reading frame (ORF109), gene 4, and a portion of gene 10. N-terminal amino acid sequencing of the encoded proteins accurately located the translation starts of six genes in the sequence. Despite the fact that most of these proteins have striking analogs in the other dsDNA bacteriophage groups, which perform highly analogous functions, no amino acid sequence similarity between these analogous proteins has been found, indicating either that they diverged a very long time ago or that they are the products of spectacular convergent evolution.

Amino Acid Sequence↗
PubMed1995

Mutational analysis of the prohead binding domain of the large subunit of terminase, the bacteriophage lambda DNA packaging enzyme.

Terminase, the DNA packaging enzyme of bacteriophage lambda, is made up of two subunits, gpNul and gpA, the products of the Nu1 and A genes. The activities of terminase include DNA binding, cos cleavage and prohead binding. Specificity domains within the structure of terminase have previously been defined by genetic studies of lambda-21 hybrids. The prohead binding domain of terminase is localized to the last 32 amino acid residues of gpA. Mutations in the prohead binding domain of gpA were constructed by introducing the corresponding amino acids from gp2, the gpA analog of bacteriophage 21. The last five residues of gpA can be replaced with little effect on the burst size of lambda. A phage with a replacement of the last six residues of gpA with the corresponding residues of gp2 was unable to form plaques, indicating that the sixth-to-last residues of gpA is crucial for prohead binding. Site-specific mutagenesis of the sixth-to-last position of gpA indicated that the sixth-to-last residue of gpA must be hydrophobic, of the seven amino acids tested, only isoleucine and valine can substitute for leucine at this position. Although the last five residues of gp2 were functional when they replaced the last five residues of gpA, two results indicated that the last five residues of gpA functioned better than the corresponding residues of gp2. First, the presence of a valine residue at the sixth-to-last position of gpA allowed plaque formation, whereas replacement of the last six residues of gpA with those of gp2, which substitutes a valine residue at the sixth-to-last position, was lethal. The second set of results indicating that the last five residues of gpA function better than the gp2 residues were obtained by study of revertants of lethal substitution mutations. In constructing the replacement mutations, a short linker was inserted into the C terminus of the A gene; this insertion created a short duplication of the end of the A gene, so that the normal C-terminal codons were located downstream of the stop codon of the A gene in the substitution mutants. Revertants of the lethal substitution mutations were obtained in which a mutation in the stop codon resulted in addition of the last five residues of gpA to the end of the substitution terminase.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗