[In vitro packaging of phage DNA--a new method for detection of biologically active DNA (author's transl)].
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Coliphages↗
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BPV1 E2 protein enhances packaging of full-length plasmid DNA in BPV1 pseudovirions.
We studied determinants of efficient encapsidation of circular DNA, incorporating a PV early region DNA sequence (nt 584-1978) previously shown to enhance packaging of DNA within papillomavirus (PV)-like particles (VLPs). Insect coelomic cells (Sf-9) and cultured monkey kidney cells (Cos-1) were transfected with an 8-kb reporter plasmid incorporating the putative BPV packaging sequence and infected with BPV1 L1 and L2 recombinant baculovirus or vaccinia virus. Heavy (1.34 g/ml) and light (1.30 g/ml) VLPs were produced, and each packaged some of the input plasmid. In light VLPs, truncated plasmids, which nevertheless incorporated the PV-derived DNA packaging sequence, were more common than full-length plasmids. Packaging efficiency of the plasmid was estimated at 1 plasmid per 10(4) VLPs in both Cos-1 and Sf-9 cells. In each cell type, expression of the BPV1 early region protein E2 in trans doubled the quantity of heavy but not light VLPs and also increased the packaging efficiency of full-length circular plasmids by threefold in heavy VLPs. The resultant pseudovirions incorporated significant amounts of E2 protein. Pseudovirions, comprising plasmids packaged within heavy VLPs, mediated the delivery of packaged plasmid into Cos-1 cells, whereby "infectivity" was blocked by antisera to BPV1 L1, but not antisera to BPV1 E4. We conclude that (a) packaging of DNA within PV L1+L2 pseudovirions is enhanced by BPV1 E2 acting in trans, (b) E2 may be packaged with the pseudovirion, and (c) E2-mediated enhancement of packaging favors 8-kb plasmid incorporation over incorporation of shorter DNA sequences.
Animals↗
Regulation of adenovirus packaging.
The application of fundamental concepts about the packaging of the adenovirus genome has contributed significantly to the development of therapeutic viral vectors for gene therapy. The packaging of adenovirus DNA into virus particles requires a cis-acting domain at the left end of the genome. This region contains a series of repeated sequences, termed A repeats due to their AT-rich character, that direct the packaging process. A repeats are believed to represent the binding sites for viral and cellular factors that mediate viral DNA packaging. This review will focus on fundamental aspects of adenovirus DNA packaging as well as how this information has been used and may be used to augment the selectivity of viral DNA packaging in applications pertaining to gene therapy vectors.
Adenoviridae↗
In vitro packaging of mature phage DNA by Salmonella phage P22.
Mature, headful-sized DNA extracted from the Salmonella phages P22 and L, and P22/L-hybrid phages can be encapsulated in vitro by means of a packaging system for exogenous DNA. The probability of packaging reaches about 10(-3) per headful-sized molecule. The absence of in vitro recombination was demonstrated, to eliminate the possibility that such a process had created concatemers. The endonucleolytic cut at the pac site, which initiates sequential packaging in vivo, does not occur with the mature DNA substrate in vitro. The position of pac on the molecule is not important but the pac-recognizing phage protein gp3 is indispensable for in vitro encapsulation.
DNA, Viral↗
In vitro maturation and encapsidation of the DNA of transposable Mu-like phage D108.
Mu and D108 are related, temperate, transposable coliphages with unusual modes of DNA replication (transposition) and virion DNA maturation. These double-stranded DNA genomes replicate intrachromosomally and are matured and encapsidated linked to DNA sequences flanking the dispersed, integrated phage genomes. We have developed an in vitro system that employs crude lysates prepared from cells late in the Mu lytic cycle and that is proficient for both maturation and encapsidation of D108 DNA. Different forms of phage DNA were packaged at different efficiencies, with a circular pSC101::D108cts10 plasmid being most efficient, linearized plasmid less so, and mature virion DNA a poor substrate. The addition of purified D108 Ner protein to the reaction had no effect, whereas D108 repressor (c protein) inhibited the reaction. Escherichia coli integration host factor and D108 transposase proteins exerted an inhibitory effect on circular DNA substrates but had little effect on linear DNA packaging. This in vitro system, coupled with that developed for transposition, can now be used to biochemically dissect the protein and substrate requirements of these phages' DNA maturation pathway and the nature of the molecular switch between DNA transposition and encapsidation.
Bacteriophage mu↗
Nucleosomes positioned by ORC facilitate the initiation of DNA replication.
The packaging of eukaryotic DNA into nucleosomes is a critical regulator of nuclear events. To address the interplay between chromatin and replication initiation, we have assessed the determinants and function of the nucleosomal configuration of S. cerevisiae replication origins. Using in vitro and in vivo assays, we demonstrate that the yeast initiator, the origin recognition complex (ORC), is required to maintain the nucleosomal configuration adjacent to origins. Disruption of the ORC-directed nucleosomal arrangement at an origin interferes with initiation of replication, but does not alter the association of ORC with the origin. Instead, the nucleosomes positioned by ORC are important for prereplicative complex formation. These findings suggest that origin-proximal nucleosomes facilitate replication initiation, and that local chromatin structure affects origin function.
Chromatin↗
A defined in vitro system for packaging of bacteriophage T3 DNA.
Using purified components, we have constructed an in vitro system for packaging of mature phage T3 DNA. In addition to mature T3 DNA, the system contained T3 proheads and the products of gene 18 (gp18) and gene 19 (gp19). The reaction required Mg2+, ATP, and polyvinyl alcohol. Spermidine was stimulatory but not absolutely required for the packaging reaction. Polyvinyl alcohol could be replaced by polyethylene glycol. The packaging efficiency decreased with decreasing molecular weight of the polymer, and low molecular weight polyols such as sucrose, sorbitol, and glycerol were inactive. The packaging reaction exhibited a sigmoidal relationship with respect to the concentration of ATP with the concentration for half maximal activity about 15 microM. A nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate), inhibited the packaging reaction.
Adenosine Triphosphate↗
Lambda phage DNA sequences affecting the packaging process.
Our previous work identified a minimal region of bacteriophage lambda DNA that is necessary for packaging into phage particles. It consists of 40 bp of the right arm and 45 bp of the left arm [Miwa and Matsubara, Gene 20 (1982)267-279]. A part of this region, 22 bp of the right arm and 38 bp of the left arm, is sufficient for cutting at cos lambda (the minimal sequence for cos lambda cutting). An 84-bp region to its right contains a binding site for lambda terminase, a complex of Nu1 and A gene products. This second region, which we called the enhancing region for packaging, stimulates cutting at cos lambda as well as packaging. This region is not active if it is physically separated from the minimal packaging region. The enhancing region has 15-bp inverted repeats. These sequences are conserved in the corresponding region of bacteriophage phi 80, which has the same packaging specificity as lambda.
Bacteriophage lambda↗
Linker insertion mutations in the herpes simplex virus type 1 UL28 gene: effects on UL28 interaction with UL15 and UL33 and identification of a second-site mutation in the UL15 gene that suppresses a lethal UL28 mutation.
The UL28 protein of herpes simplex virus type 1 (HSV-1) is one of seven viral proteins required for the cleavage and packaging of viral DNA. Previous results indicated that UL28 interacts with UL15 and UL33 to form a protein complex (terminase) that is presumed to cleave concatemeric DNA into genome lengths. In order to define the functional domains of UL28 that are important for DNA cleavage/packaging, we constructed a series of HSV-1 mutants with linker insertion and nonsense mutations in UL28. Insertions that blocked DNA cleavage and packaging were found to be located in two regions of UL28: the first between amino acids 200 to 400 and the second between amino acids 600 to 740. Insertions located in the N terminus or in a region located between amino acids 400 and 600 did not affect virus replication. Insertions in the carboxyl terminus of the UL28 protein were found to interfere with the interaction of UL28 with UL33. In contrast, all of the UL28 insertion mutants were found to interact with UL15 but the interaction was reduced with mutants that failed to react with UL33. Together, these observations were consistent with previous conclusions that UL15 and UL33 interact directly with UL28 but interact only indirectly with each other. Revertant viruses that formed plaques on Vero cells were detected for one of the lethal UL28 insertion mutants. DNA sequence analysis, in combination with genetic complementation assays, demonstrated that a second-site mutation in the UL15 gene restored the ability of the revertant to cleave and package viral DNA. The isolation of an intergenic suppressor mutant provides direct genetic evidence of an association between the UL28 and UL15 proteins and demonstrates that this association is essential for DNA cleavage and packaging.
Amino Acid Sequence↗
A coupled in vitro system for the formation and packaging of concatemeric phage T1 DNA.
Extracts derived from E. coli cells infected non-permissively with phage T1 amber mutants were used in an in vitro system to investigate the packaging of T1 DNA into phage heads. The standard extract used infections with amber mutants in genes 1 and 2 (g1- g2-) which are defective in T1 DNA synthesis but can synthesis the proteins required for particle morphogenesis. g1- g2- extracts packaged T1+ virion DNA molecules with an efficiency of 3 X 10(5) pfu/micrograms DNA. Extracts from cells infected with phage also defective in DNA synthesis but carrying additional mutations in genes 3.5 or 4 which are required for concatemer formation in vivo (g1- g3.5- and g1- g4- extracts) package T1 virion DNA at substantially lower efficiencies. Analysis of the DNA products from these in vitro reaction showed that concatemeric DNA is formed very efficiently by g1- g2- extracts but not by g1- g3.5- or g1- g4- extracts. These results are interpreted as evidence that the T1 in vitro DNA packaging system primarily operates in a similar manner to the in vivo headful mechanism. This is achieved in vitro by the highly efficient conversion of T1 virion DNA into concatemers which are then packaged with a much lower efficiency into heads to form infectious particles. A secondary pathway for packaging T1 DNA into heads and unrelated to the headful mechanism may also exist.
DNA Replication↗
Defective infectious particles and rare packaged genomes produced by cells carrying terminal-repeat-negative epstein-barr virus.
The Epstein-Barr virus (EBV) lytic program includes lytic viral DNA replication and the production of a viral particle into which the replicated viral DNA is packaged. The terminal repeats (TRs) located at the end of the linear viral DNA have been identified as the packaging signals. A TR-negative (TR(-)) mutant therefore provides an appropriate tool to analyze the relationships between EBV DNA packaging and virus production. Here, we show that supernatants from lytically induced 293 cells carrying TR mutant EBV genomes (293/TR(-)) contain large amounts of viral particles devoid of viral DNA which are nevertheless able to bind to EBV target cells. This shows that viral DNA packaging is not a prerequisite for virion formation and egress. Rather surprisingly, supernatants from lytically induced 293/TR(-) cells also contained rare infectious viruses carrying the viral mutant DNA. This observation indicates that the TRs are important but not absolutely essential for virus encapsidation.
Cell Line↗
Analysis of functional domains of the packaging proteins of bacteriophage T3 by site-directed mutagenesis.
Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed together in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of non-capsid proteins, gp18 and gp19. We have developed a defined system, composed of purified gp18, gp19 and proheads, and a crude system, composed of lysates of T3 infected cells, for in vitro packaging of T3 DNA. The defined system displays an ATPase activity which is composed of DNA packaging-dependent and -independent ATPases (pac- and nonpac-ATPases, respectively). In the crude system, DNA is packaged by a way of concatemer as an intermediate. gp19 has ATP binding activity and three ATP binding and two Mg2+ binding consensus motifs in its amino acid sequence. We have expanded the previous studies on the roles of these domains in the DNA packaging reaction by more extensive analysis by site-directed mutagenesis. gp19 mutants, including the previously isolated four mutants, were divided into four groups according to the DNA packaging activity in the defined and crude systems: group 1 mutants were defective in both systems (gp19-G61D, which is a gp19 mutant with Gly to Asp at amino acid 61 and so on, and gp19-H344D); the group 2 mutant had decreased activity in both systems (gp19-G429R); group 3 mutants were active in the defined system but defective in the crude system (gp19-G63D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-G424E); group 4 mutants had almost the same activity as gp19-wt (gp19-K64T, gp19-K370I, gp19-G429L, gp19-K430T and gp19-H553L). Group 1 mutants had an altered conformation, resulting in defective interaction with ATP and in abortive binding to the prohead, and lost specifically the pac-ATPase activity. The group 2 mutant had an increased pac-ATPase activity in spite of the decreased DNA packaging activity, indicating that this mutant is inefficient in coupling of ATP hydrolysis to DNA translocation. The inability of the group 3 mutants except gp19-H347R to package DNA in the crude system would be due to a defect in processing of concatemer DNA. gp19-H347R would be a mutant defective in the initiation event(s) of DNA packaging.
Adenosine Triphosphatases↗
Capsid transformation during packaging of bacteriophage lambdaDNA.
Assembly pathways of complex viruses might not be simple additions of one protein after another with rigid tertiary structure. It might in fact involve shifts in subunit structure, movement of subunits relative to each other to form new arrangements, transient action of proteins and protein segments, involvement of structure forming 'microenvironments' of the host. Thus morphogenesis of the bacteriophage lambda head starts with the formation of a core-containing DNA-free petit lambda particle. In a first transition, and dependent on a host function, the core is released, minor protein components of the capsid are processed and the particle's structure is altered, as shown by a change of its hydrodynamic properties. The resulting 'prehead' undergoes a second transition triggered by a complex of DNA and recognition protein (A-protein). This transition is more drastic than the first one. The particle doubles its volume without increasing in protein mass, the shell becomes thinner, and the surface structure is changed. Concomitantly with this process, the DNA becomes packaged and the particle becomes able to bind the small 'D-protein' in amounts equimolar to the capsid protein, which it could not do before. The D-protein addition probably causes another shift of the capsid structure. DNA packaging is completed, and the DNA is cut from concatemeric precursors to unit length molecules. Binding sites are created for the tail connector molecules which in turn allow the independently assembled tail to attach. Research on these processes proceeds along several lines: comparison of physical and chemical properties of particles accumulating in mutants; pulse-chase experiments on assembly precursors; morphogenesis in vitro; and model transitions of aberrant lambda polyheads.
Capsid↗
Morphology of a human-derived YAC in yeast meiosis.
In meiosis of human males DNA is packaged along pachytene chromosomes about 20 times more compactly than in meiosis of yeast. Nevertheless, a human-derived yeast artificial chromosome (YAC) shows the same degree of compaction of DNA as endogenous chromosomes in meiotic prophase nuclei of yeast. This suggests that in yeast meiosis, human and yeast DNA adopt a similar organization of chromatin along the pachytene chromosome cores. Therefore meiotic chromatin organization does not seem to be an inherent chromosomal property but is governed by the host-specific cellular environment. We suggest that there is a correlation between the less dense DNA packaging and the increased rate of recombination that has been reported for human-derived YACs as compared with human DNA in its natural environment.
Chromosomes, Artificial, Yeast↗
Subunit conformational changes accompanying bacteriophage P22 capsid maturation.
In double-stranded DNA bacteriophages, packaging of dsDNA requires the transformation of a precursor procapsid into a mature viral capsid. Lattice expansion and release of scaffolding subunits accompanying DNA packaging. Three-dimensional structures of procapsid and mature phage lattices demonstrate that the capsid transformation involves substantial changes in subunit environment. Since this transformation occurs without subunit dissociation, it represents a transition between at least two stable subunit conformations. Using Raman spectroscopy, we have identified changes in coat protein secondary structure and side-chain environments which accompany the capsid transformation. The subunits of procapsid shells contain only 2.0 +/- 0.4% more alpha-helix and less beta-sheet than those of mature capsids; however, numerous side chains are substantially altered by the transformation, including tyrosines, tryptophans, phenylalanines, and aliphatics, which are widely distributed through the subunit sequence. We propose, therefore, that procapsid expansion is accomplished through the relative motion of coat subunit domains with little change in secondary structure. Such hinge-bending conformational transitions may couple ATP-dependent dsDNA condensation with shell expansion.
Bacteriophage P22↗
Assembly pathway of avian infectious laryngotracheitis virus.
Infectious laryngotracheitis virus (ILTV) is the causative agent of a highly contagious upper respiratory tract infection in chickens. At present, ILTV vaccines are not satisfactory because of development of a latent carrier status in vaccinated birds. Development of recombinant virus vaccines has been hampered by the limited information available on the molecular level and organization of this virus. We isolated 3 assembly intermediates, designated A, B, and C from ILTV-infected cells. Analysis of [3H]thymidine-and [35S]methionine-labeled particles, and electron microscopic studies indicated that particle A was the empty capsid, particle B was the procapsid containing scaffolding protein, and particle C was the DNA-filled capsid. The ILTV procapsids could only be found in the nucleus, which indicated that procapsids could not translocate through the nuclear membrane until they packaged the DNA. The DNA-filled capsids migrated through the nuclear membrane and obtained an envelope from the inner membrane of the nucleus. The enveloped particles then migrated through the lumen of the endoplasmic reticulum into vacuoles in the cytoplasm. Infective virions were isolated from within the infected cells, indicating that budding through the cytoplasmic membrane is not a necessary step in ILTV maturation. Abundant arrays composed of tubules about 45 to 50 nm wide were found in the cytoplasm of chicken embryonic liver cells about 30 to 38 hours after infection. Comparison of the assembly intermediates and the DNA packaging pathway of ILTV with that of bacteriophage pi 29 indicates that similarity exists.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Identification of the elemental packing unit of DNA in mammalian sperm cells by atomic force microscopy.
DNA is packaged within the sperm cell nuclei of many vertebrates and all mammals in a highly condensed state by small basic proteins (protamines). Despite continuous investigation for nearly half a century, the actual packing arrangement of the DNA has remained unresolved. Atomic force and electron microscopy studies described in this report provide evidence that the fundamental packing unit for sperm DNA is a toroidal structure, 900A in outside diameter with a 150A diameter hole, which contains up to 60kb of DNA. Although the results presented here are based primarily upon investigations of mammalian sperm cells, they are expected to be valid for all sperm cells which utilize protamines to package DNA.
Animals↗
Heterogeneity of the procapsid of bacteriophage T3.
Like other double-stranded DNA bacteriophages, bacteriophage T3 assembles a DNA-free procapsid that subsequently packages the bacteriophage DNA. By agarose gel electrophoresis, it has been found that the T3 procapsid has a negative electrophoretic mobility (mu) that progressively increases in magnitude by as much as 3% after assembly of the procapsid. This increase is (i) caused by an increase in the solid support-free mu (muo) of the procapsid, not a decrease in its radius, and (ii) not prevented by either genetically or chemically (use of proflavine) blocking DNA packaging. However, inhibition of the formation of high-energy compounds with a mixture of cyanide and fluoride ions does block the time-dependent increase in the magnitude of muo. This increase appears to be accompanied by addition of an unidentified T3 protein to the procapsid.
Capsid↗