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PubMed1998

Truncation of the nuclear localization signal of polyomavirus VP1 results in a loss of DNA packaging when expressed in the baculovirus system.

Using the pBlueBacIII baculovirus transfer vector, N11-VP1, a truncated form of the polyomavirus major capsid protein VP1, was cloned for expression in the baculovirus-insect cell expression system. The N11-VP1 protein is virtually identical to full-length, wild-type VP1, except that the first 11 amino acids have been deleted from the amino terminus of the protein. The N-terminal region of VP1 has previously been shown to contain the nuclear localization signal (NLS) of the protein and contains residues essential for both nuclear transport as well as DNA-binding functions. The 5-day infected Sf9 cellular lysate from the recombinant N11-VP1 preparation was purified by cesium chloride density gradient centrifugation. Capsid-like particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as Western blotting and was shown to have accurately expressed the N11-VP1 as cloned. Examination of the Coomassie-stained gels revealed that the capsid-like particles composed of the N11-VP1 protein did not contain any host-derived histones. The absence of the histones in the N11-VP1 capsid-like particles is indicative of the inability of these particles to package DNA, a feature which is observed when wild-type VP1 is treated in this manner. Electron microscopy of these particles substantiated this observation. To determine if the deletion of the NLS exhibited true in vivo characteristics, Sf9 insect cells were infected with the recombinant baculovirus carrying the N11-VP1 gene and examined early in infection (30 h post-infection) by indirect immunofluorescence. The N11-VP1 protein was not transported to the nucleus and remained in the cytoplasm. When the Sf9 cells were coinfected with N11-VP1 and polyomavirus VP2 and VP3 carrying baculoviruses, the N11-VP1 was transported to the nucleus by cooperation with the minor capsid proteins. These studies demonstrate that the N-terminal region of VP1, which contains the NLS and DNA-binding domains, is essential for VP1 nuclear transport and its ability to package Sf9 cellular DNA.

Baculoviridae↗
PubMed1992

Identification of a gene in Bacillus subtilis bacteriophage SPP1 determining the amount of packaged DNA.

The virulent Bacillus subtilis bacteriophage SPP1 encapsidates its DNA by a headful mechanism. Analyzing phage missense mutants, which package less DNA than SPP1 wild-type but show no other affected properties, we have identified a gene whose product is involved in the sizing of phage DNA during maturation. Characterization of this gene and its product provides an experimental access to the poorly understood mechanism of DNA sizing in packaging. The gene (gene 6 or siz) was cloned and sequenced. An open reading frame (ORF) coding for a 57.3 kDa polypeptide was identified. All the single nucleotide substitutions present in different siz mutants affect the net charge of that protein. The gene was further characterized by assignment of several nonsense mutations (sus) to the ORF. Phages carrying the latter type of mutations could be complemented in trans when gene 6 is provided by a plasmid.

Amino Acid Sequence↗
PubMed1987

Upper limit for DNA packaging by Bacillus subtilis bacteriophage phi 105: isolation of phage deletion mutants by induction of oversized prophages.

We have determined the upper size limit for DNA packaging in Bacillus subtilis bacteriophage phi 105 by examining the plaque-forming and transducing capabilities of lysates made from strains containing prophages of various sizes. The upper size limit for efficient packaging of the phage genome appears to be about 40.2 kb, which is about 1 kb larger than the wild-type genome. This places an upper limit of about 5 kb on the size of insertions that can be accommodated in phi 105 transfection cloning vectors, such as phi 105J27. Induction of prophages that exceed that upper limit, followed by selection for plaque formation or transduction, provides a powerful means of isolating phage deletion mutants. A comparison of the location of each deletion with the resultant phenotype has enabled us to identify non-essential regions of the phage genome, and regions that are required for tail biosynthesis and for host cell lysis.

Bacillus subtilis↗
PubMed1983

Stability and in vitro DNA packaging of bacteriophages: effects of dextrans, sugars, and polyols.

Attempts were made to increase the efficiency of infectious particle formation during the in vitro assembly of bacteriophage T7 from procapsids and DNA. It was found that dextrans and some smaller, related compounds (sucrose and sorbitol) increase this efficiency by a factor of 8 to 50. Dextrans also inhibited elevated temperature-induced emptying of DNA from bacteriophages T7, P22, and T4, suggesting that the stimulation of assembly is caused, at least in part, by the stabilization of packaged DNA in capsids. The data indicated that the sugars and polyols can slow DNA emptying from bacteriophages at elevated temperature whether they permeate the bacteriophage capsid or not. In contrast, the data suggested that permeation of some particle, probably a capsid, results in inhibition of in vitro T7 assembly.

Capsid↗
PubMed1990

Bacteriophage T7 DNA packaging. III. A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction.

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.

Base Sequence↗
PubMed1985

Alterations of the bacteriophage T7 and T3 DNA packaging pathway in Escherichia coli mutant TSN B.

Data previously obtained indicate that, during assembly of the related bacteriophages T7 and T3, a DNA-free procapsid (capsid I) is produced and that subsequently capsid I: (1) binds to a longer than mature (concatemeric) DNA and then becomes structurally altered to a particle isolated as a capsid (capsid II) physically resembling the mature bacteriophage capsid more than the procapsid (initiation phase of packaging), (2) draws DNA to its interior (entry phase of packaging), (3) participates in cutting the concatemeric DNA to mature size. It was found that, after infection of Escherichia coli mutant tsnB (selected for a deficiency in plating T7; M. Chamberlin [1974], J. Virol. 14, 509-516), T7 and T3 capsid I is assembled at a rate not significantly different from its rate of assembly in the wild-type host. However, the conversion of capsid I to capsid II was slowed in E. coli tsnB, suggesting that the tsnB mutation interferes with the initiation of DNA packaging. Although some T3 and T7 DNA enters capsids and is cut to mature size in the tsnB mutant, the data further suggest that the entry rate of DNA into capsid II is lower in the tsnB mutant than it is in an unaltered host. T7 capsid II-concatemeric DNA complexes accumulate during infection of the tsnB mutant. These observations suggest that use of the tsnB mutant as a host will simplify studies of bacteriophage T7 and T3 DNA packaging.

Capsid↗
PubMed2005

Obligate heterodimerization of the archaeal Alba2 protein with Alba1 provides a mechanism for control of DNA packaging.

Organisms growing at elevated temperatures face a particular challenge to maintain the integrity of their genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of the Alba (Sac10b) gene. Alba is an abundant, dimeric, highly basic protein that binds cooperatively and at high density to DNA. Sulfolobus solfataricus encodes a second copy of the Alba gene, and the Alba2 protein is expressed at approximately 5% of the level of Alba1. We demonstrate by NMR, ITC, and crystallography that Alba2 exists exclusively as a heterodimer with Alba1 at physiological concentrations and that heterodimerization exerts a clear effect upon the DNA packaging, as observed by EM, potentially by changing the interface between adjacent Alba dimers in DNA complexes. A functional role for Alba2 in modulation of higher order chromatin structure and DNA condensation is suggested.

Algorithms↗
PubMedDate not supplied

Role of the major capsid protein of phage T4 in DNA packaging from structure-function and site-directed mutagenesis studies.

Heat cleavage of asp-pro peptide bonds was used to probe the primary structures of the Phage T4 major capsid protein precursor, gp23, its mature capsid form gp23*, and a DNA-dependent ATPase, called capsizyme. This analysis suggests that capsizyme is a gp23** resulting from the N-terminal processing found in gp23* as well as shortening at the C-terminus. Photoaffinity labeling with Azido-ATP and BrU-DNA, followed by heat cleavage, suggests binding sites for these compounds toward the C-terminus of gp23**, suggesting localization of functions within the gp23 primary sequence. Site-directed mutagenesis experiments were targeted therefore to the C-terminal end of g23 as well as to its processing sites. N-terminal processing site modification supports the consensus gp21 proteinase cleavage rule, whereas mutagenesis at the C-terminus suggests that the C-terminal alteration is unlikely to result from a gp21-morphogenesis proteinase cleavage. Amino acid replacements in gp23 at newly introduced amber sites reveal a new g23 mutant phenotype, defective partially DNA-filled heads, in support of the hypothesis that gp23 and its products function directly in the DNA packaging mechanism.

Adenosine Triphosphatases↗
PubMed1997

A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging.

Adenoviruses (Ads) are intermediate-sized mammalian DNA viruses with a double-stranded linear genome of 36 kb. The icosohedral virion has been shown to accommodate up to 105% of the wild-type genome length, and genomes larger than this size are either unpackageable or extremely unstable, frequently undergoing DNA rearrangement. Here we show that the Ad virion also has a lower packaging limit of approximately 75% of the wild-type genome length. We have constructed a series of vectors with sizes ranging from 15.1 to 33.6 kb and used these to show that in our Cre/loxP helper-dependent system (R. J. Parks, L. Chen, M. Anton, U. Sankar, M. A. Rudnicki, and F. L. Graham, Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996), vectors with genomes greater than or equal to 27.7 kb are packaged with equal efficiencies, whereas vectors with smaller genomes are inefficiently packaged. A 15.1-kb vector, approximately half the size of the wild-type adenovirus genome, was packaged with an efficiency intermediate between that of the small (21.3- to 25.7-kb) and large (27.7- to 33.5-kb) vectors. Analysis of vector DNA after amplification in helper virus-infected cells showed that vectors below 75% of the Ad genome had undergone DNA rearrangements, whereas larger vectors were unaltered. The 15.1-kb vector was recovered primarily as a mix of head-to-tail and tail-to-tail covalent dimers, with a size of 30 kb. We conclude that the Ad virion can efficiently accommodate viral DNA of greater than 75% of the viral genome but that smaller viral genomes tend to undergo rearrangement, resulting in a final size of greater than approximately 27 kb before they can be efficiently packaged. Knowledge of the lower limit to Ad DNA packaging should allow for the design of better and more stable vectors.

Adenoviruses, Human↗
PubMed1993

A novel terminase activity associated with the DNA packaging protein gp17 of bacteriophage T4.

The mechanism of generation of circularly permuted ends in bacteriophage T4 by a strictly headful packaging process has remained unresolved since its proposal by Streisinger et al. (1967, Proc. Natl. Acad. Sci. USA 57, 292-295). In this paper, we show that the phage T4 DNA packaging proteins gp16 and gp17 act as T4 terminase. Expression of gp16 and gp17 in Escherichia coli resulted in extensive cleavage of both plasmid as well as E. coli genomic DNAs. Analysis of a number of recombinant terminase constructs, and mutants in gene 17, demonstrated that the active site for cleavage of DNA is located in gp17, but not in gp16. Consistent with the circularly permuted nature of phage T4 ends, cleavage by gp17 occurred in a sequence independent manner generating random ends. The terminase cutting occurred preferentially on a DNA substrate that is transcriptionally active. We propose that a structural feature in the transcriptionally active region provides a site for attachment of terminase to DNA following which the terminase moves along the DNA, and cleaves at a random sequence.

Bacteriophage T4↗
PubMed2004

Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses.

In previous studies, novel putative viral pathogens designated that asinine herpesvirus 4 (AsHV4) and asinine herpesvirus 5 (AsHV5) were associated with fatal interstitial pneumonia in donkeys (Equus asinus). Nucleotide sequence analysis of a portion of the DNA polymerase gene identified these putative pathogens as herpesviruses and possibly as members of the Gammaherpesvirinae subfamily. Although similar to equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5), sequence diversity was observed among the detected viruses. In this study, novel sequence is reported for a DNA-packaging protein gene of EHV5 plus AsHV4, AsHV5, and a newly described putative pathogen herein designated asinine herpesvirus 6 (AsHV6). Phylogenetic analysis of these sequences suggested that the equine gammaherpesviruses may form a separate clade within the Gammaherpesvirinae subfamily. Based on the sequence of EHV2 and the novel sequences reported in this study, a PCR assay was developed to detect equine gammaherpesviruses. Products of the predicted size were produced after amplification of DNA from EHV2, EHV5, AsHV4, AsHV5, and AsHV6. This nonnested assay was shown to consistently amplify approximately 10 genomic copies of EHV2. Amplification products were not produced from DNA template of other alpha- and gammaherpesviruses. Because the role of gammaherpesviruses has not been well defined in equine disease, it is envisioned that a single, sensitive PCR assay to detect these potential pathogens will facilitate further assessment of their role in disease.

Amino Acid Sequence↗
PubMed1999

Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein.

Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.

Bacteriophage lambda↗
PubMed2000

Topology of the components of the DNA packaging machinery in the phage phi29 prohead.

Chromosome condensation inside dsDNA viral particles is a complex process requiring the coordinated action of several viral components. The similarity of the process in different viral systems has led to the suggestion that there is a common underlying mechanism for DNA packaging, in which the portal vertex or connector plays a key role. We have studied the topology of the packaging machinery using a number of antibodies directed against different domains of the connector. The charged amino-terminal, the carboxyl-terminal, and the RNA binding domain are accessible areas in the connector assembled into the prohead, while the domains corresponding to the 12 large appendages of the connector are buried inside the prohead. Furthermore, while the antibodies against the carboxyl and amino-terminal do not affect the packaging reaction, incubation of proheads with antibodies against the RNA binding domain abolishes the packaging activity. The comparison of the three-dimensional reconstructions of bacteriophage phi29 proheads with proheads devoid of their specific pRNA by RNase treatment shows that this treatment removes structural elements of the distal vertex of the portal structure, suggesting that the pRNA required for packaging is located at the open gate of the channel in the narrow side of the connector.

Antibodies, Viral↗
PubMedDate not supplied

Role of RNA in bacteriophage phi 29 DNA packaging.

A novel bacteriophage phi 29 RNA of 174 nucleotides is essential for the in vitro packaging of the DNA-terminal protein complex into proheads. The RNA, bound to the prohead portal vertex (connector), participates in assembly and function of the DNA translocating ATPase and in recognition of the DNA left-end during the course of the packaging reaction. The RNA is present in related phages and varies widely in primary sequence, but its secondary structure, as deduced by phylogenetic analysis, is both highly conserved and unique among small RNAs.

Adenosine Triphosphatases↗
PubMed1999

Comparative sequence analysis of the DNA packaging, head, and tail morphogenesis modules in the temperate cos-site Streptococcus thermophilus bacteriophage Sfi21.

The temperate Streptococcus thermophilus bacteriophage Sfi21 possesses 15-nucleotide-long cohesive ends with a 3' overhang that reconstitutes a cos-site with twofold hyphenated rotational symmetry. Over the DNA packaging, head and tail morphogenesis modules, the Sfi21 sequence predicts a gene map that is strikingly similar to that of lambdoid coliphages in the absence of any sequence similarity. A nearly one to one gene correlation was found with the phage lambda genes Nu1 to H, except for gene B-to-E complex, where the Sfi21 map resembled that of coliphage HK97. The similarity between Sfi21 and HK97 was striking: both major head proteins showed an N-terminal coiled-coil structure, the mature major head proteins started at amino acid positions 105 and 104, respectively, and both major head genes were preceded by genes encoding a possible protease and portal protein. The purported Sfi21 protease is the first viral member of the ClpP protease family. The prediction of Sfi21 gene functions by reference to the gene map of intensively investigated coliphages was experimentally confirmed for the major head and tail gene. Phage Sfi21 shows nucleotide sequence similarity with Lactococcus phage BK5-T and a lactococcal prophage and amino acid sequence similarity with the Lactobacillus phage A2 and the Staphylococcus phage PVL. PVL is a missing link that connects the portal proteins from Sfi21 and HK97 with respect to sequence similarity. These observations and database searches, which demonstrate sequence similarity between proteins of phage from gram-positive bacteria, proteobacteria, and Archaea, constrain models of phage evolution.

Amino Acid Sequence↗
PubMed2004

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.

Bacteriophages↗
PubMed2000

Structure of the bacteriophage phi29 DNA packaging motor.

Motors generating mechanical force, powered by the hydrolysis of ATP, translocate double-stranded DNA into preformed capsids (proheads) of bacterial viruses and certain animal viruses. Here we describe the motor that packages the double-stranded DNA of the Bacillus subtilis bacteriophage phi29 into a precursor capsid. We determined the structure of the head-tail connector--the central component of the phi29 DNA packaging motor--to 3.2 A resolution by means of X-ray crystallography. We then fitted the connector into the electron densities of the prohead and of the partially packaged prohead as determined using cryo-electron microscopy and image reconstruction analysis. Our results suggest that the prohead plus dodecameric connector, prohead RNA, viral ATPase and DNA comprise a rotary motor with the head-prohead RNA-ATPase complex acting as a stator, the DNA acting as a spindle, and the connector as a ball-race. The helical nature of the DNA converts the rotary action of the connector into translation of the DNA.

Adenosine Triphosphatases↗
PubMed2000

Shape and DNA packaging activity of bacteriophage SPP1 procapsid: protein components and interactions during assembly.

The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7. The protein stoichiometry suggests a T=7 symmetry for the SPP1 procapsid. Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures. Co-production of gp11, gp13 and gp6 is essential for assembly of procapsids competent for DNA packaging in vitro. Presence of gp7 in the procapsid increases the yield of viable phages assembled during the reaction in vitro five- to tenfold. Formation of closed procapsid-like structures requires uniquely the presence of the major head protein and the scaffolding protein. The two proteins interact only when co-produced but not when mixed in vitro after separate synthesis. Gp11 controls the polymerization of gp13 into normal (T=7) and small sized (T=4?) procapsids. Predominant formation of T=7 procapsids requires presence of the portal protein. This implies that the portal protein has to be integrated at an initial stage of the capsid assembly process. Its presence, however, does not have a detectable effect on the rate of procapsid assembly during SPP1 infection. A stable interaction between gp6 and the two major procapsid proteins was only detected when the three proteins are co-produced. Efficient incorporation of a single portal protein in the procapsid appears to require a structural context created by gp11 and gp13 early during assembly, rather than strong interactions with any of those proteins. Gp7, which binds directly to gp6 both in vivo and in vitro, is not necessary for incorporation of the portal protein in the procapsid structure.

Bacillus↗