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PubMed1994

Retroviral RNA packaging: a review.

In retroviruses, the "Gag" or core polyprotein is capable of assembling into virus particles and packaging the genomic RNA of the virus. How this protein recognizes viral RNA is not understood. Gag polyproteins contain a zinc-finger domain; mutants with changes in this domain assemble into virions, but a large fraction of these particles lack viral RNA. Thus, one crucial element in the RNA packaging mechanism is the zinc-finger domain. RNA sequences required for packaging ("packing signals") have been studied both by deletion analysis and by measuring encapsidation of nonviral mRNAs containing limited insertions of viral sequence. These experiments show that all or part of the packaging signal in viral RNA is located near the 5 end of the genome. These signals appear to be quite large, i.e., hundreds of nucleotides. Each virus particle actually contains a dimer of two identical, + strand genomic RNA molecules. The nature of the dimeric linkage is not understood. In some experimental situations (including zinc-finger mutants), only a small fraction of the particles in a virus preparation contain genomic RNA. It is striking that the genomic RNA packaged in these situations is dimeric. Because of this important observation, it is speculated that only dimers are packaged, and that the dimeric structure is an element of the packaging signal. It is also suggested that the dimers undergo a conformational change ("RNA maturation") after the virus is released from the cell, and that this change may depend upon the cleavage of the Gag polyprotein, a post-assembly event catalyzed by the virus-coded protease.

Base Sequence↗

PubMed1988

Packaging of transducing DNA by bacteriophage P1.

P1 transduces bacterial chromosomal markers with widely differing frequencies. We use quantitative Southern hybridisations here to show that, despite this, most markers are packaged at similar levels. Exceptions are a group of markers near 2 min and another at 90 min which seem to be packaged at levels two- to threefold higher. We thus conclude that certain marker frequency variations in transduction can be explained by differences in packaging level, but that most cannot. The limited range in packaging levels suggests that P1 can initiate the packaging of chromosomal DNA from many sites. This idea is supported by our failure to find any chromosomal sequences with homology to the phage pac site and by the occurrence of hybridising bands which seem to suggest sequential packaging from a large number of specific sites. We eliminate the possibility that chromosomal DNA packaging is the result of endonucleolytic cutting by the P1 res enzyme.

Autoradiography↗

PubMed1996

Environmental Assessment of Packaging: The Consumer Point of View

When marketing environmentally responsible packaged products, the producer is confronted with consumer beliefs concerning the environmental friendliness of packaging materials. When making environmentally conscious packaging decisions, these consumer beliefs should be taken into account alongside the technical guidelines. Dutch consumer perceptions of the environmental friendliness of packaged products are reported and compared with the results of a life-cycle analysis assessment. It is shown that consumers judge environmental friendliness mainly from material and returnability. Furthermore, the consumer perception of the environmental friendliness of packaging material is based on the postconsumption waste, whereas the environmental effects of production are ignored. From the consumer beliefs concerning environmental friendliness implications are deduced for packaging policy and for environmental policy.KEY WORDS: Consumer behavior; Environment; Food; Packaging; Perception; Waste

Journal Article↗

PubMed2004

Quantification of myocardial perfusion defects using three different software packages.

Software packages are widely used for quantification of myocardial perfusion defects. The quantification is used to assist the physician in his/her interpretation of the study. The purpose of this study was to compare the quantification of reversible perfusion defects by three different commercially available software packages. We included 50 consecutive patients who underwent myocardial perfusion single-photon emission tomography (SPET) with a 2-day technetium-99m tetrofosmin protocol. Two experienced technologists processed the studies using the following three software packages: Cedars Quantitative Perfusion SPECT, Emory Cardiac Toolbox and 4D-MSPECT. The same sets of short axis slices were used as input to all three software packages. Myocardial uptake was scored in 20 segments for both the rest and the stress studies. The summed difference score (SDS) was calculated for each patient and the SDS values were classified into: normal (< 4), mildly abnormal (4-8), moderately abnormal (9-13), and severely abnormal (> 13). All three software packages were in agreement that 21 patients had a normal SDS, four patients had a mildly abnormal SDS and one patient had a severely abnormal SDS. In the remaining 24 patients (48%) there was disagreement between the software packages regarding SDS classification. A difference in classification of more than one step between the highest and lowest scores, for example from normal to moderately abnormal or from mildly to severely abnormal, was found in six of these 24 patients. Widely used software packages commonly differ in their quantification of myocardial perfusion defects. The interpreting physician should be aware of these differences when using scoring systems.

Adult↗

PubMed1987

Recognition and cleavage of the bacteriophage P1 packaging site (pac). I. Differential processing of the cleaved ends in vivo.

The packaging of bacteriophage P1 DNA into viral capsids is initiated at a specific DNA site called pac. During packaging, that site is cleaved and at least one of the resulting ends is encapsidated into a P1 virion. We show here that pac is located on a 620 base-pair fragment of P1 DNA (EcoRI-20). When that fragment is inserted into the chromosome of cells that are then infected with P1, packaging of host DNA into phage particles is initiated at pac and proceeds down the chromosome, unidirectionally, for about five to ten P1 "headfuls" (about 5 X 10(5) to 10 X 10(5) bases of DNA). Using an assay for pac cleavage that does not depend on DNA packaging, we have identified a set of five amber mutations that are mapped adjacent to pac, and that define a gene (gene 9) essential for pac cleavage. Amber mutations that are located in genes necessary for viral capsid formation (genes 4, 8 and 23), or in a gene necessary for "late" protein synthesis (gene 10), do not affect pac cleavage. The latter result suggests that the synthesis of the pac cleavage protein is not regulated co-ordinately with other phage morphogenesis proteins. The products of pac cleavage were analyzed using two different DNA substrates. In one case, a single copy of pac was placed in the chromosome of P1-sensitive cells. When those cells were infected with P1, we could detect the cleavage of as much as 70% of the pac-containing DNA. The pac end destined to be packaged in the virion was detected five to 20 times more efficiently than was the other end. Since this result is obtained whether or not the infecting P1 phage can encapsidate the cut pac site, the differential detection of pac ends is not simply a consequence of one end being packaged and the other not. In a second case, pac was located in cells on a small (5 X 10(3) bases) multicopy plasmid. When those cells were infected with P1, neither pac end was detected efficiently after P1 infection, unless the cells carried a recBCD- mutation. In recBCD- cells, the results with plasmid-pac substrates were similar to those obtained with chromosomally integrated pac substrates. We interpret these results to mean that, following pac cleavage, the end destined to be packaged is protected from cellular nucleases while the other end is degraded by the action of at least two nucleases, one of which is the product of the host recBCD gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophages↗

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↗

PubMed1985

Isolation and characterization of bacteriophage T3/T7 hybrids and their use in studies on molecular basis of DNA-packaging specificity.

In vitro DNA-packaging systems of bacteriophages T3 and T7 packaged homologous DNA more efficiently than heterologous DNA. Packaging of phage DNA proceeds by way of concatemeric intermediates (H. Fujisawa, J. Miyazaki, and T. Minagawa (1978), Virology 87, 394-400). The conversion of mature homologous and heterologous DNAs to concatemers was efficient in both the T3- and T7-packaging systems. In vitro complementation experiments indicate that the gene 19 product (gp19) specifies which DNA enters the capsid. To identify DNA regions recognized by the packaging systems, T3/T7 hybrids were constructed and physical maps of the hybrid DNAs were determined by restriction enzyme analysis. By comparing restriction maps and in vitro packaging of hybrid DNAs, it is concluded that the sequence responsible for specificity of DNA packaging is confined within 5% of the ends of the T3 and T7 genomes.

Base Sequence↗

PubMed1987

On the molecular mechanism of DNA translocation during in vitro packaging of bacteriophage T3 DNA.

The process of packaging of bacteriophage T3 DNA in a defined in vitro system can be separated into two stages: formation of a precursor complex (50 S complex) in the presence of adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S) and subsequent translocation of DNA into the head by the addition of ATP. Packaged DNA exits when DNA translocation is interrupted by the addition of ATP-gamma-S (M. Shibata, H. Fujisawa, and T. Minagawa, 1987, Virology, in press; M. Shibata, H. Fujisawa, and T. Minagawa, 1987, J. Mol. Biol., in press). The in vitro system packaged nicked and cross-linked DNAs but did not package single-stranded DNA. DNA packaging was inhibited by intercalating reagents such as ethidium bromide, acridine orange, and 4',6-diamino-2-phenylindole dihydrochloride. The inhibitory effect was proportional to the ability of intercalating agents to unwind DNA. Ethidium bromide did not inhibit the formation of 50 S complex but blocked translocation of DNA into and out of the capsid. DNA packaging was inhibited by actinomycin D and distamycin A which bind to the minor groove of the DNA helix. From these results, we conclude that DNA packaging mechanism utilizes the exterior structure of duplex DNA for translocating the DNA into the capsid.

Capsid↗

PubMed1988

Concatemerization and packaging of bacteriophage T7 DNA in vitro: determination of the concatemers' length and appearance kinetics by use of rotating gel electrophoresis.

During its morphogenesis both in intact infected cells (in vivo) and in lysates of infected cells (in vitro), bacteriophage T7 forms end-to-end concatemers of its mature DNA, a linear, nonpermuted, terminally repetitious DNA. During morphogenesis, in vivo T7 concatemers are packaged in preformed capsids and cut to mature size. In the present study the lengths and appearance kinetics of concatemers formed in vitro from mature T7 DNA have been determined. The following procedures are used here for the first time: (a) 20-35% efficient in vitro concatemerization and packaging of T7 DNA; the mixture used for packaging contained two lysates that together had all T7 gene products, and (b) fractionation of concatemers by rotating gel electrophorsis (RGE), which improves the resolution by length of concatemer-length DNA. Concatemerization at 30 degrees was so fast that some other process must be rate limiting for packaging. The concatemers formed were linear and joined left-end to right-end by complementary base pairing, not by blunt-end ligation. Concatemers formed at 30 degrees were reconverted to mature DNA by packaging in vitro. Reducing the temperature to 0 degrees both slowed concatemerization to the time scale (minutes) needed for control of the extent of concatemerization and reduced packaging to insignificant levels, thereby also uncoupling packaging from concatemerization. At both 30 degrees and 0 degrees bands of discrete-length concatemers were observed by RGE. The lengths were n times the length of mature T7 DNA; n was found to be any integer from 2 to 15. The bands were stronger at 0 degrees than they were at 30 degrees in comparison to a background of heterogeneous DNA. No evidence for the favoring of any value of n was found. In addition, it was found by two-dimensional agarose gel electrophoresis that a comparatively small amount of circular DNA was produced in vitro.

Cold Temperature↗

PubMed1991

Dissection of functional domains of the packaging protein of bacteriophage T3 by site-directed mutagenesis.

Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are joined together in head-to-tail fashion through terminally redundant sequences. During packaging of DNA, mature monomers are cut from the concatemer. The cutting is obligatorily coupled to DNA packaging. The packaging of phage DNA is under the control of a pair of noncapsid proteins, called packaging proteins, gp 18 and gp19. gp19 is an ATP-binding protein that plays multiple roles in DNA packaging. gp19 is predicted, from the sequence of its gene, to contain 586 amino acids, and has consensus sequences for an ATP binding site. To dissect structure-function relationships of gp19, mutations were introduced into the ATP binding domain and the mutant proteins were overproduced, purified and characterized. Mutant gp19 with a Gly-to-Asp mutation at amino acid 61 (gp19 G61D) was defective in DNA packaging due to an altered interaction with ATP. Gp19 G424E, with a change in another putative ATP binding domain, was active in DNA packaging but was defective in DNA cutting. A second mutation in the latter domain, gp19 K430T, and a mutation at 553 (to give gp19 H553L), within a putative Mg2+ binding domain, had only minor effects on gp19 activities.

Amino Acid Sequence↗

PubMed1991

Characterization of a versatile in vitro DNA-packaging system based on hybrid lambda/phi 29 proheads.

We have studied the assembly of bacteriophage lambda head proteins on the phage phi 29 connector to produce in vitro chimeric proheads, whose ability to package different types of DNA depends on the physical integrity of the phi 29 connector. Terminal protein-free phi 29 as well as nonviral DNAs have been shown to be efficiently packaged by this hybrid system. An RNA, that can be provided by any of the extracts used in the complementation mixture, was required for DNA packaging, both by the hybrid system as well as by the homologous lambda system. The DNA-packaging activity of RNase-treated proheads can be restored by adding a mixture of ribosomal RNAs. There is also a requirement for a minimal length of DNA to be stably packaged. The packaging protein p16 of phi 29 can replace the lambda terminase complex in the in vitro packaging system, both with the chimeric as well as genuine lambda proheads.

Bacteriophage lambda↗

PubMed1995

Examination of conditions affecting the efficiency of HVS-1 amplicon packaging.

Defective herpes simplex virus type 1 vectors (HSV amplicons) have been used as vehicles for efficiently delivering foreign genes into non-dividing cells such as neutrons in vitro and in vivo. This system is useful for studying neuronal physiology and may have potential for human gene therapy of neuronal disorders. The preparation of infectious amplicon particles is normally achieved by transfecting amplicon plasmid DNA, which contains the HSV replication origin and packaging signal, into mammalian cell lines followed by infection of the cells with HSV helper virus. This allows for replication and packaging of both viral and amplicon plasmid DNA. To improve the packaging efficiency of amplicons, several parameters involved in the packaging process were investigated. By introducing the SV40 DNA replication origin into an amplicon plasmid and prereplicating it before HSV infection, it was demonstrated that the existing amount of amplicon DNA prior to infection in the cells is not a rate-limiting step during HSV packaging. In addition, it was shown that the yield of the packaged amplicon particles can be improved by: (1) using a relatively small amount of HSV helper virus up to multiplicity of infection (m.o.i.) equal to 0.1 at infection; (2) infecting with HSV helper virus at 2 or 3 days post-transfection; and (3) passaging the initial packaged amplicon stocks 1-2 times on fresh host cells.

Animals↗

PubMed2003

Defining the bacteriophage T4 DNA packaging machine: evidence for a C-terminal DNA cleavage domain in the large terminase/packaging protein gp17.

Double-stranded DNA packaging in bacteriophage T4 and other viruses occurs by translocation of DNA into an empty prohead by a packaging machine assembled at the portal vertex. Coordinated with this complex process is the cutting of concatemeric DNA to initiate and terminate DNA packaging and encapsidate one genome-length viral DNA. The catalytic site responsible for cutting, and the mechanisms by which cutting is precisely coordinated with DNA translocation remained as interesting open questions. Phage T4, unlike the phages with defined ends (e.g. lambda, T3, T7), packages DNA in a strictly headful manner, and exhibits no strict sequence specificity to initiate or terminate DNA packaging. Previous evidence suggests that the large terminase protein gp17, a key component of the T4 packaging machine, possesses a non-specific DNA cutting activity. A histidine-rich metal-binding motif, H382-X(2)-H385-X(16)-C402-X(8)-H411-X(2)-H414-X(15)-H430-X(5)-H436, in the C-terminal half of gp17 is thought to be involved in the terminase cleavage. Here, exhaustive site-directed mutagenesis revealed that none of the cysteine and histidine residues other than the H436 residue is critical for function. On the other hand, a cluster of conserved residues within this region, D401, E404, G405, and D409, are found to be critical for function. Biochemical analyses showed that the D401 mutants exhibited a novel phenotype, showing a loss of in vivo DNA cutting activity but not the DNA packaging activity. The functional nature of the critical residues and their disposition in the conserved loop region between two predicted beta-strands suggest that these residues are part of a metal-coordinated catalytic site that cleaves the phosphodiester bond of DNA substrate. The data suggest that the T4 terminase consists of at least two functional domains, an N-terminal DNA-translocating ATPase domain and a C-terminal DNA-cutting domain. Although the DNA recognition mechanisms may be distinct, it appears that T4 and other phage terminases employ a common catalytic paradigm for phosphodiester bond cleavage that is used by numerous nucleases.

Adenosine Triphosphatases↗

PubMed2005

Dispensability of 3' tRNA-like sequence for packaging cowpea chlorotic mottle virus genomic RNAs.

The 3' ends of three genomic RNAs (gRNAs) of cowpea chlorotic mottle virus (CCMV) terminate in a highly conserved tRNA-like structure (3'TLS). To examine the intrinsic role played the 3'TLS in packaging, the competence of each gRNA lacking the 3' TLS (DeltaTLS-gRNA) to interact with dissociated coat protein (CP) subunits and form virions was assayed in vitro. In contrast to the well established requirement for the participation of either viral 3'TLS or host-tRNAs in the assembly of RNA-containing virions in brome mosaic virus (BMV; Choi, Y, G., Dreher, T. W., Rao, A. L. N. 2002. tRNA elements mediate the assembly of an icosahedral RNA virus. Proc. Natl. Acad. Sci. 99, 655-660), CCMV CP does not require the presence of viral TLS in cis or in trans. Similar in vitro assembly assays showed that CCMV CP subunits also packaged BMV RNAs lacking 3' TLS as well as two other non-bromoviral RNAs although with lesser efficiency. To characterize sequences of CCMV RNA3 (C3) required for packaging, a series deletions was engineered into C3 and their effect on virus assembly was examined. It was observed that, unlike BMV RNA3 whose packaging requires a bipartite signal (Choi, Y. G., Rao, A. L. N. 2003. Packaging of brome mosaic virus RNA3 is mediated through a bipartite signal. J. Virol. 77, 9750-9757), packaging of C3 is independent of either movement protein (MP) ORF or CP ORF or 3' non-coding regions. Based on the differential prerequisites identified in this study for the assembly of BMV and CCMV, we hypothesize that the adaptive condition for movement in monocotyledonous host has made packaging a necessary co-requirement for BMV.

3' Untranslated Regions↗

PubMed2004

Mutational analysis of the predicted secondary RNA structure of the Mason-Pfizer monkey virus packaging signal.

The 5' end of the Mason-Pfizer monkey virus (MPMV) genomic RNA has been predicted to fold into a complex stem/loop structure that is thought to play a role in specific RNA encapsidation. In this study, we used a set of mutations that either abrogated or recreated the first four stem loops predicted within the 5' untranslated region (5' UTR) for effects on RNA packaging. Test of these mutations in our biological assay revealed that only stem loop 1 (SL1) was important for the packaging potential of MPMV, while mutations in none of the other stem loops affected packaging significantly. Interestingly, it was the primary sequence of SL1 RNA and not its secondary structure that affected packaging since compensatory mutations that reformed SL1 were unable to restore the packaging efficiency of the retroviral vector. Additionally, our mutational analysis reveals that stem loop 4, predicted to be the major packaging determinant of MPMV, does not seem to have a significant role in packaging. Finally, results of the biological effects of the structural mutations are discussed in relation to their effects on the folding potential of the various stem loops.

5' Untranslated Regions↗

PubMed2004

Packaging, replication and recombination of the segmented genome of bacteriophage Phi6 and its relatives.

The genomes of bacteriophage Phi6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented dsRNA genomes into preformed polyhedral structures called procapsids or inner cores. The packaging requires hydrolysis of NTPs and takes place in the order S:M:L. Minus strand synthesis begins after the completion of the plus strand packaging. The packaging and replication reactions can be studied in vitro with purified components. A model has been presented that proposes that the program of serially dependent packaging is determined by the conformational changes at the surface of the procapsid due to the amount of RNA packaged at each step. The in vitro packaging and replication system has facilitated the application of reverse genetics and the study of recombination in the family of Cystoviridae.

Bacteriophage phi 6↗

PubMed1999

Stable expression of the ecotropic retrovirus receptor in amphotropic packaging cells facilitates the transfer of recombinant vectors and enhances the yield of retroviral particles.

Retroviral vectors are used widely as gene transfer vehicles. Vector particles are generated by packaging cell lines, which supply the structural proteins gag, pol and env needed to package the retroviral vector RNA. The most efficient way to introduce the vector genome into the packaging cell line is cross-infection with a retroviral vector. Since the infection of a packaging cell line by the produced virus is blocked due to the down regulation of the retrovirus receptor by the envelope glycoprotein, the vector genome should be introduced by a virus with a host tropism different from the one of the packaging cell line. The murine ecotropic retrovirus receptor was expressed in the human amphotropic packaging cell line FLYA13 to generate a cell line which can be infected by murine ecotropic retroviruses. Vector transfer can now be facilitated by cross-infection with the appropriate ecotropic retroviral vectors and provides a simple and efficient method for the generation of amphotropic packaging lines.

3T3 Cells↗

PubMed2001

Enhanced reporter gene expression in the rat brain from helper virus-free HSV-1 vectors packaged in the presence of specific mutated HSV-1 proteins that affect the virion.

Herpes simplex virus (HSV-1) gene expression is hypothesized to shut off promoters in HSV-1 vectors, but in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus, recombinant gene expression remains short-term in the absence of approximately 99% of the HSV-1 genome. To resolve this paradox, we hypothesized that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. This study evaluated expression from HSV-1 vectors, containing neuronal-specific promoters, that were packaged in the presence of specific mutated HSV-1 proteins that affect the virion. The mutated HSV-1 proteins that were examined included two protein kinases (U(L)13 and U(S)3), the virion host shut-off factor (vhs), the transactivator of immediate early promoters (VP16), and a virion protein that affects RNA metabolism (U(S)11). Helper virus-free packaging could occur in the presence of each mutated protein alone or specific combinations of two or three mutated proteins. In BHK and PC12 cells, vectors packaged in the presence of each mutated protein increased ( approximately 2-fold) the level of expression per cell, and vectors packaged in the presence of specific combinations of mutated proteins supported larger (4-7-fold) increases. In the rat striatum, vectors packaged in the presence of a mutated U(S)3 displayed enhanced gene transfer (13-18-fold increases in the number of cells at 4 days), and vectors packaged in the presence of mutated U(L)13 or VP16 enhanced long-term expression (2 months). Vectors packaged in the presence of mutated vhs or U(S)11 displayed minimal changes in expression.

Animals↗