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At least 757 records · Page 42Linked to original sources
PubMed1984

Bacteriophage P22 capsids with a subgenome length of packaged DNA.

Bacteriophage P22 assembles a DNA-free procapsid that subsequently packages P22 DNA. To study the packaging of bacteriophage P22 DNA, attempts were made to isolate P22 capsids with a subgenome length of packaged DNA. With the use of cesium chloride buoyant density sedimentation and agarose gel electrophoresis, the following capsids with a subgenome length of packaged DNA were isolated and characterized: (i) a capsid with the solid-support-free electrophoretic mobility and radius of the DNA-free P22 procapsid; (ii) a capsid with the solid-support-free electrophoretic mobility and radius of the mature P22 bacteriophage; and (iii) a capsid with a solid-support-free electrophoretic mobility and possibly a radius intermediate to those of the procapsid and bacteriophage.

Bacteriophages↗
PubMed1985

A noninverting genome of a viable herpes simplex virus 1: presence of head-to-tail linkages in packaged genomes and requirements for circularization after infection.

The wild-type herpes simplex virus 1 genome consists of two components, L and S, which invert relative to each other, giving rise to four isomers. Previously we reported the construction of a herpes simplex virus 1 genome, HSV-1(F)I358, from which 15 kilobase pairs of DNA spanning the junction between L and S components were deleted and which no longer inverted (Poffenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2690-2694, 1983). Further studies on the structure of HSV-1(F)I358 revealed the presence of two submolar populations among packaged DNA. The first, comprising no more than 10% of total packaged DNA, consisted of defective genomes with a subunit size of 36 kilobase pairs. The results suggest that this population arose by recombination through a directly repeated sequence inserted in place of the deleted L-S junction. The second minor population consisted of HSV-1(F)I358 DNA linked head-to-tail. Analyses of the structure of HSV-1(F)I358 DNA after infection indicated that the fraction of total DNA linked head-to-tail increased to approximately 40 to 50% within 30 min after exposure of cells to virus. The formation of head-to-tail linkages did not require de novo protein synthesis. Our interpretation of the results is that the termini of full-length DNA molecules are held together during packaging, that a small fraction of the termini is covalently linked during or after packaging, and that the remainder is covalently joined after the release of viral DNA from the infecting virus by either host or viral factors introduced into the cell during infection.

Cell Line↗
PubMed1985

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: prohead restoration for DNA-gp3 packaging and assembly.

The DNA-protein complex DNA-gp3 of phi 29 is efficiently packaged into purified proheads with the aid of plasmid-derived gp16. The filled heads can be assembled to phage by addition of an extract providing the products for neck-tail assembly (Bjornsti et al., J. Virol. 50:766-772, 1984). However, purified proheads lost their competence to package DNA-gp3 upon storage for 2 months at 4 degrees C. Competence was restored by complementation with extracts of certain mutant-infected cells, and these experiments demonstrated that late proteins were not involved; restoration obtained with 4-8-14--infected cells was indistinguishable from that obtained with 7-8-14--infected cells. 2-8-14- and 3-8-14- extracts restored about one-third of the capacity to package exogenous DNA-gp3. A 1-8-14- extracts restored activity to package 20.6% of the DNA-gp3 added, but phage were not produced.

Bacillus subtilis↗
PubMed1986

Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence.

Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence.

Animals↗
PubMed1986

Formation of deletions after initiation of simian virus 40 replication: influence of packaging limit of the capsid.

Transfected DNA is frequently broken and rejoined in mammalian cells by recombination processes that depend on minimal nucleotide sequence homology. Although measurements of breakage and joining account reasonably well for the frequent formation of deletions during transfection, they are inadequate to explain the high frequency of deletion formation by simian virus 40 (SV40) genomes that are slightly larger than the packaging limit of the capsid. To investigate this anomaly, we constructed and transfected into CV-1 cells a series of modified SV40 genomes containing 136, 284, 460, and 656 extra base pairs in the intron of the gene encoding T antigen. These experiments indicate that the effective packaging limit of an SV40 capsid lies between 284 and 460 extra base pairs. Further analysis of these transfections suggests that molecules just above the effective packaging limit may be encapsidated and transmitted between cells at low efficiency, thereby allowing multiple rounds of replication and multiple opportunities to generate and package genomes that contain deletions. The junctional sequences in several such deletions were determined; they were similar to the junctions in deletions that were formed before replication began, suggesting that the enzymatic machinery responsible for both types of deletion may be similar.

Animals↗
PubMed1986

Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA.

Newly replicated herpes simplex virus (HSV) DNA consists of head-to-tail concatemers which are cleaved to generate unit-length genomes bounded by the terminally reiterated a sequence. Constructed defective HSV vectors (amplicons) containing a viral DNA replication origin and the a sequence are similarly replicated into large concatemers which are cleaved at a sequences punctuating the junctions between adjacent repeat units, concurrent with the packaging of viral DNA into nucleocapsids. In the present study we tested the ability of seed amplicons containing specific deletions in the a sequence to become cleaved and packaged and hence be propagated in virus stocks. These studies revealed that two separate signals, located within the Ub and Uc elements of the a sequence, were essential for amplicon propagation. No derivative defective genomes were recovered from seed constructs which lacked the Uc signal. In contrast, propagation of seed constructs lacking the Ub signal resulted in the selection of defective genomes with novel junctions, containing specific insertions of a sequences derived from the helper virus DNA. Comparison of published sequences of concatemeric junctions of several herpesviruses supported a uniform mechanism for the cleavage-packaging process, involving the measurement from two highly conserved blocks of sequences (pac-1 and pac-2) which were homologous to the required Uc and Ub sequences. These results form the basis for general models for the mechanism of cleavage-packaging of herpesvirus DNA.

Animals↗
PubMed1988

Development of avian sarcoma and leukosis virus-based vector-packaging cell lines.

We have constructed an avian leukosis virus derivative with a 5' deletion extending from within the tRNA primer binding site to a SacI site in the leader region. Our aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1 psi-, to develop vector-packaging cell lines. We show that RAV-1 psi- can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, we have demonstrated that QT6-derived lines expressing RAV-1 psi- can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-1 psi--expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. We show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.

Animals↗
PubMed1990

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes.

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.

Animals↗
PubMed1990

Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus.

To identify RNA and protein sequences involved in packaging of human immunodeficiency virus type 1 (HIV-1), various mutations were introduced into the viral genome. Portions of the human immunodeficiency virus type 1 genome between the first splice donor site and the gag initiation codon were deleted to investigate the RNA packaging site (psi). Point mutations that alter cysteine residues in one or both zinc finger motifs of p7, a cleavage product of the gag precursor, were created to study the role of the gag zinc fingers in packaging. The psi site mutants and the gag mutants exhibited similar phenotypes. Cells transfected with the mutant genomes, while expressing normal levels of human immunodeficiency virus type 1 RNA and proteins, produced viral particles that were normal in protein content but lacked detectable viral RNA. These mutant virions were unable to productively infect cells. The combination of human immunodeficiency virus type 1 packaging mutations should minimize fortuitous assembly of infectious virus and may provide a means to produce noninfectious particles for candidate vaccines.

Amino Acid Sequence↗
PubMed1992

The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure.

Interaction of cis-acting RNA sequences with nucleocapsid proteins is one of the critical events leading to retroviral genomic RNA packaging. We have derived a potentially stable secondary structure for the packaging signal region of human immunodeficiency virus strain IIIB, using a combination of biochemical analysis and computer modelling. This region encompasses the major splice donor (SD), which is found in a highly structured conserved stem-loop. Comparison with other published human immunodeficiency virus type 1 sequences shows almost absolute nucleotide conservation in base-paired regions required to maintain this structure. Presently and previously described packaging-defective mutants would disrupt the structure. The structure depends on base pairing between nucleotide sequences 5' of the major SD which are common to both genomic and subgenomic RNAs and sequences 3' of SD which are unique to the unspliced RNA. This may explain how in retroviruses such as Rous sarcoma virus, mutations in regions common to genomic and subgenomic RNA might prevent packaging of the unspliced mRNA by disrupting a signal structure which can exist only in the genomic RNA species.

Amino Acid Sequence↗
PubMed1995

Aura alphavirus subgenomic RNA is packaged into virions of two sizes.

The alphavirus genome is 11.8 kb in size. During infection, a 4.2-kb subgenomic RNA is also produced. Most alphaviruses package only the genomic RNA into virions, which are enveloped particles with icosahedral symmetry, having a triangulation number (T) = 4. Aura virus, however, packages both the genomic RNA and the subgenomic RNA into virions. The genomic RNA is primarily packaged into a virion that has a diameter of 72 nm and which appears to be identical to the virions produced by other alphaviruses. The subgenomic RNA is packaged into two major, regular particles with diameters of 72 and 62 nm. The 72-nm-diameter particle appears to be identical in construction to virions containing genomic RNA. The 62-nm-diameter particle probably has T = 3. The large and small Aura virions can be partially separated in sucrose gradients. In addition to these two major classes of particles, there are other particles produced that appear to arise from abortive assembly. From these results and from previous studies of alphavirus assembly, we suggest that during assembly of alphavirus nucleocapsids in the infected cell there is a specific initiation event followed by recruitment of additional capsid subunits into the complex, that the triangulation number of the complex is not predetermined but depends upon the size of the RNA and interactions that occur during assembly, and that budding of assembled nucleocapsids results in the acquisition of an envelope containing glycoproteins arranged in a manner determined by the nucleocapsid.

Alphavirus↗
PubMed1996

Inducible human immunodeficiency virus type 1 packaging cell lines.

Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well.

Cell Line↗
PubMed1997

Magnesium-induced conformational change of packaging RNA for procapsid recognition and binding during phage phi29 DNA encapsidation.

Bacteriophage phi29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during maturation. An intriguing feature of phi29 assembly is that a virus-encoded RNA (pRNA) is required for the packaging of its genomic DNA. Psoralen cross-linking, primer extension, and T1 RNase partial digestion revealed that pRNA had at least two conformations; one was able to bind procapsids, and the other was not. In the presence of Mg2+, one stretch of pRNA, consisting of bases 31 to 35, was confirmed to be proximal to base 69, as revealed by its efficient cross-linking by psoralen. Two cross-linking sites in the helical region were identified. Mg2+ induced a conformational change of pRNA that exposes the portal protein binding site by promoting the refolding of two strands of the procapsid binding region, resulting in the formation of pRNA-procapsid complexes. The procapsid binding region in this binding-competent conformation could not be cross-linked with psoralen. When the two strands of the procapsid binding region were fastened by cross-linking, pRNA could neither bind procapsids nor package phi29 DNA. A pRNA conformational change was also discernible by comparison of migration rates in native EDTA and Mg2+ polyacrylamide gel electrophoresis and was revealed by T1 RNase probing. The Mg2+ concentration required for the detection of a change in pRNA cross-linking patterns was 1 mM, which was the same as that required for pRNA-procapsid complex formation and DNA packaging and was also close to that in normal host cells.

Bacillus Phages↗
PubMed1997

Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system.

Using the p2Bac dual multiple cloning site transfer vector, the polyomavirus major capsid protein gene VP1 was cloned for expression in the baculovirus-insect cell expression system. The 5-day-infected cellular lysate from this recombinant 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 and was shown to have accurately expressed the polyomavirus VP1 protein as cloned. It was found that the preparation revealed the presence of host histones in the stained gels, which is indicative of DNA packaging. To determine if cellular DNA was being packaged in the particles, Sf9 insect cells were prelabeled with [3H] thymidine. The label was removed, and the cells were subsequently infected with a recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) carrying the polyomavirus VP1 gene. Upon purification through three cesium chloride gradients and DNase I treatment, capsid-like particles, containing [3H]thymidine-labeled DNA, were isolated which were found to coincide with hemagglutination activity. Studies have indicated that the AcMNPV appears to have the ability to fragment Sf9 cellular DNA. When infected with the recombinant AcMNPV carrying the VP1 gene of polyomavirus, these host DNA fragments are being packaged by the VPI major capsid protein; further, these DNA fragments have been shown to be approximately 5 kb in size, which corresponds to the size of the native polyomavirus genome. These studies demonstrate that the recombinant polyomavirus VP1 protein has the ability to package DNA in the absence of the minor structural proteins VP2 and VP3 and independently of the polyomavirus T antigens.

Animals↗
PubMed1998

Sequences within the herpesvirus-conserved pac1 and pac2 motifs are required for cleavage and packaging of the murine cytomegalovirus genome.

The DNA sequence motifs pac1 [an A-rich region flanked by poly(C) runs] and pac2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of pac1 and pac2 were essential for cleavage whereas others were not. Within pac1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within pac2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.

3T3 Cells↗
PubMed1998

Retrovirus packaging cells expressing the Mus dunni endogenous virus envelope facilitate transduction of CHO and primary hematopoietic cells.

Mus dunni endogenous virus (MDEV) infects a wide variety of cell types from many different species. To take advantage of this broad host range, we have constructed packaging cells (PD223) that produce virions bearing the MDEV envelope. PD223 cells are able to package Moloney murine leukemia virus-based vectors at a titer of 4 x 10(5) infectious units per ml in the absence of contaminating replication-competent retrovirus. Vectors packaged by PD223 cells are able to transduce CHO cells, which are resistant to transduction by many retroviruses, at >/=25-fold higher efficiency than vectors having other pseudotypes. A vector packaged by PD223 was found to be among the most efficient for transducing primary baboon and human CD34(+) cells.

3T3 Cells↗
PubMed1998

Herpes simplex virus DNA cleavage and packaging: association of multiple forms of U(L)15-encoded proteins with B capsids requires at least the U(L)6, U(L)17, and U(L)28 genes.

The U(L)15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against U(L)15-encoded protein recognized three proteins with apparent Mrs of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-Mr protein was detected in type C capsids and comigrated with the product of a U(L)15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-Mr proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(deltaU(L)15), a virus lacking an intact U(L)15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3' end of U(L)15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent Mrs of 83,000, 80,000, and 79,000 are products of U(L)15 with identical C termini. The 79,000-, 80,000-, and 83,000-Mr proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that U(L)15-encoded proteins are integral components of B capsids. Only the 83,000-Mr protein was detected in B capsids purified from cells infected with viruses lacking the U(L)6, U(L)17, or U(L)28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-Mr proteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-Mr U(L)15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.

Animals↗
PubMed1999

ATP depletion blocks herpes simplex virus DNA packaging and capsid maturation.

During herpes simplex virus (HSV) assembly, immature procapsids must expel their internal scaffold proteins, transform their outer shell to form mature polyhedrons, and become packaged with the viral double-stranded (ds) DNA genome. A large number of virally encoded proteins are required for successful completion of these events, but their molecular roles are poorly understood. By analogy with the dsDNA bacteriophage we reasoned that HSV DNA packaging might be an ATP-requiring process and tested this hypothesis by adding an ATP depletion cocktail to cells accumulating unpackaged procapsids due to the presence of a temperature-sensitive lesion in the HSV maturational protease UL26. Following return to permissive temperature, HSV capsids were found to be unable to package DNA, suggesting that this process is indeed ATP dependent. Surprisingly, however, the display of epitopes indicative of capsid maturation was also inhibited. We conclude that either formation of these epitopes directly requires ATP or capsid maturation is normally arrested by a proofreading mechanism until DNA packaging has been successfully completed.

Adenosine Triphosphate↗