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At least 811 records · Page 45Linked to original sources
PubMed2004

A conserved leucine that constricts the pore through the capsid fivefold cylinder plays a central role in parvoviral infection.

The atomic structure of the DNA-containing T = 1 particle of the parvovirus minute virus of mice (MVM) reveals cylindrical projections at each fivefold symmetry axis, each containing an 8 Angstrom pore through which runs 10 amino acids of a single VP2 N-terminus. The tightest constriction of this pore is formed at its inner end by the juxtaposition of leucine side chains from position 172 of five independent VP2 molecules. To test whether L172 modulates the extrusion of VP N-termini, we constructed and analyzed a complete set of amino acid substitution mutants at this highly conserved residue. All but one mutant produced DNA-containing virions, but only two, L172V and L172I, were infectious, the others being blocked for viral entry. Several mutants were significantly defective for assembly at 39 degrees C, but not at 32 degrees C. L172W significantly impaired genome encapsidation, indicating that the fivefold cylinder may also be the DNA packaging portal. Although tryptic cleavage of the VP2 N-terminus was not affected for the mutants, VP1 was degraded during proteolysis of mutant, but not wild-type, virions.

Amino Acid Sequence↗
PubMed2004

A fragile lattice: replacing bacteriophage lambda's head stability gene D with the shp gene of phage 21 generates the Mg2+-dependent virus, lambda shp.

Phage lambda DNA packaging is accompanied by prohead expansion, due to structural changes in gpE, the major capsid protein. Rearrangement of the gpE lattice creates binding sites for trimers of gpD, the head stabilization protein. lambda-Like phage 21's shp gene is homologous to lambda's D gene. gpD and gpShp share 49% amino acid identity. To ask whether gpShp could stabilize the lambda head shell, we replaced lambda's D gene with shp, creating lambda shp. Unlike lambda or 21, lambda shp was strictly dependent on the presence of 10(-2) M Mg2+, and lambda shp virions were very sensitive to chelating agents. Density gradient studies indicated that the lambda gpE lattice was underpopulated with gpShp. gpD's N-terminus has been proposed to contact gpE, and we found that lambda D/shp, which produces a chimeric protein with the N-terminus of gpD and the C-terminus of gpShp, was Mg2+-independent and more stable than lambda shp.

Bacteriophage lambda↗
PubMed2004

A single-LTR HIV-1 vector optimized for functional genomics applications.

The development of high-throughput methods of converting simple expression cassettes into lentiviral vectors and expediting the process of retrieving vector genomes that carry candidate genes from host DNA will facilitate the use of lentiviral vectors as an efficient means of screening novel gene function. To optimize lentiviral vectors for functional genomic applications we have developed a shuttle HIV-1 vector containing a single LTR. Incorporation of a LoxP site and the Sbfl restriction enzyme site into the vector LTR allowed for the rescue of integrated vector genomes into individual bacterial clones. Vector DNA isolated from bacteria was used for a second round of functional screening. Furthermore, we identified a continuous DNA sequence containing all the cis elements required for vector production. Incorporating the isolated sequence into expression cassettes resulted in the generation of HIV-1 vectors in a single cloning step, which imparts a simplified procedure for converting cDNA expression cassettes into single-LTR lentiviral vectors.

Base Sequence↗
PubMed2005

Thiocholesterol-based lipids for ordered assembly of bioresponsive gene carriers.

A series of thiocholesterol-based cationic lipids (TCL) has been designed and synthesized by the attachment of thiocholesterol to a cationic amine via a disulfide bond. TCL can be incorporated into liposomes and used to package DNA into a lipoplex, thereby protecting it from DNase digestion. DNA is rapidly released from the complex in the presence of low concentrations of reducing agents. The lipoplex mediated efficient transfection activity and had low cytotoxicity. To improve the biocompatibility of the cationic lipoplex, TCL were used as a component in the assembly of a nanolipoparticle (NLP). The particle surface was subsequently modified by disulfide exchange to replace the cationic group with a negatively charged (glutathione) or zwitterionic (cysteine) reducing agent. A cell-binding ligand (TAT peptide, sequence GRKKRRQRRRGYG) was then incorporated onto the particle surface to enhance the particle-cell recognition. The sequentially assembled cell-binding NLP with a zwitterionic surface gave a larger transfection yield than the cationic NLP at all concentrations tested. At low DNA concentrations, the enhancement was 80-fold. The disulfide cationic lipids and the sequential assembly strategy enable one to tailor the surface charge, hydrophilicity, and recognition elements of a nanosized gene carrier. This results in increased gene transfer activity in a biocompatible particle.

Cholesterol↗
PubMed1980

Regulation of coat protein polymerization by the scaffolding protein of bacteriophage P22.

In the morphogenesis of double stranded DNA phages, a precursor protein shell empty of DNA is first assembled and then filled with DNA. The assembly of the correctly dimensioned precursor shell (procapsid) of Salmonella bacteriophage P22 requires the interaction of some 420 coat protein subunits with approximately 200 scaffolding protein subunits to form a double shelled particle with the scaffolding protein on the inside. In the course of DNA packaging, all of the scaffolding protein subunits exit from the procapsid and participate in further rounds of procapsid assembly (King and Casjens. 1974. Nature (Lond.). 251:112-119). To study the mechanism of shell assembly we have purified the coat and scaffolding protein subunits by selective dissociation of isolated procapsids. Both proteins can be obtained as soluble subunits in Tris buffer at near neutral pH. The coat protein sedimented in sucrose gradients as a roughly spherical monomer, while the scaffolding protein sedimented as if it were an elongated monomer. When the two proteins were mixed together in 1.5 M guanidine hydrochloride and dialyzed back to buffer at room temperature, procapsids formed which were very similar in morphology, sedimentation behavior, and protein composition to procapsids formed in vivo. Incubation of either protein alone under the same conditions did not yield any large structures. We interpret these results to mean that the assembly of the shell involves a switching of both proteins from their nonaggregating to their aggregating forms through their mutual interaction. The results are discussed in terms of the general problem of self-regulated assembly and the control of protein polymerization in morphogenesis.

Bacterial Proteins↗
PubMed1999

Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.

Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid.

Bacteriophage P22↗
PubMed1998

Purification, crystallization and preliminary X-ray diffraction studies of the bacteriophage phi29 connector particle.

The connector or portal particle from double-stranded DNA bacteriophage phi29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu-C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino- and carboxy-terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a = 416.86 A, b = 227.62 A, c = 236.68 A and beta = 96.3 degrees and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P4(2)2(1)2 with cell dimensions a = b = 170.2 A, c = 156.9 A and contain half a particle per asymmetric unit. X-ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 A respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two-dimensional crystals analyzed previously by electron microscopy.

Bacillus Phages↗
PubMed2003

Transcriptional and post-transcriptional control of cold-shock genes.

A mesophile like Escherichia coli responds to abrupt temperature downshifts (e.g. from 37 degrees C to 10 degrees C) with an adaptive response that allows cell survival and eventually resumption of growth under the new unfavorable environmental conditions. During this response, bulk transcription and translation slow or come to an almost complete stop, while a set of about 26 cold-shock genes is preferentially and transiently expressed. At least some of the proteins encoded by these genes are essential for survival in the cold, but none plays an exclusive role in cold adaptation, not even the "major cold-shock protein" CspA and none is induced de novo. The majority of these proteins binds nucleic acids and are involved in fundamental functions (DNA packaging, transcription, RNA degradation, translation, ribosome assembly, etc.). Although cold-induced activation of specific promoters has been implicated in upregulating some cold-shock genes, post-transcriptional mechanisms play a major role in cold adaptation; cold stress-induced changes of the RNA degradosome determine a drastic stabilization of the cold-shock transcripts and cold shock-induced modifications of the translational apparatus determine their preferential translation in the cold. This preferential translation at low temperature is due to cis elements present in the 5' untranslated region of at least some cold-shock mRNAs and to trans-acting factors whose levels are increased substantially by cold stress. Protein CspA and the three translation initiation factors (IF3 in particular), whose stoichiometry relative to the ribosomes is more than doubled during the acclimation period, are among the trans elements found to selectively stimulate cold-shock mRNA translation in the cold.

Bacterial Proteins↗
PubMed1983

Gene 20 product of bacteriophage T4. II. Its structural organization in prehead and bacteriophage.

The location of gene 20 product of bacteriophage T4 in phage and phage percursors has been determined by immunochemical analysis of polyacrylamide gels, immunoturbidimetry and immunoelectron microscopy. The protein is present at the membrane attachment site of the prehead, a head precursor, and is accessible to the antibodies in the solution. It is present at the tail attachment site of the capsid, partially buried in the structure. In complete phage particles it is totally buried in the structure. It is in contact with the major shell proteins, gp23 and gp23*, respectively, in preheads and capsids, as revealed by partial crosslinking experiments. It forms the upper collar of the neck in necked tails. The lower collar is constructed from other gene products. On the basis of these data a structural model of the neck region of the phage has been derived. This model is consistent with a number of events in phage assembly, such as the role of gp20 in head assembly and DNA packaging, prehead detachment from the bacterial membrane and head-tail attachment. The symmetry mismatch known to occur between head and tail has been localized at the gp20-gp23* contact area.

Antibodies, Viral↗
PubMed2003

Cleavage leads to expansion of bacteriophage P4 procapsids in vitro.

Proteolytic cleavage of the structural proteins is an important part of the maturation process for most bacteriophages and other viruses. In the double-stranded DNA bacteriophages this cleavage is associated with DNA packaging, capsid expansion, and scaffold removal. To understand the role of protein cleavage in the expansion of bacteriophages P2 and P4, we have experimentally cleaved P4 procapsids produced by overexpression of the capsid and scaffolding proteins. The cleavage leads to particle expansion and scaffold removal in vitro. The resulting expanded capsid has a thin-shelled structure similar, but not identical, to that of mature virions.

Amino Acid Sequence↗
PubMed2004

Transcription in haploid male germ cells.

Major modifications in chromatin organization occur in spermatid nuclei, resulting in a high degree of DNA packaging within the spermatozoon head. However, before arrest of transcription during midspermiogenesis, high levels of mRNA are found in round spermatids. Some transcripts are the product of genes expressed ubiquitously, whereas some are generated from male germ cell-specific gene homologs of somatic cell genes. Others are transcript variants derived from genes with expression regulated in a testis-specific fashion. The haploid genome of spermatids also initiates the transcription of testis-specific genes. Various general transcription factors, distinct promoter elements, and specific transcription factors are involved in transcriptional regulation. After meiosis, spermatids are genetically but not phenotypically different, because of transcript and protein sharing through cytoplasmic bridges connecting spermatids of the same generation. Interestingly, different types of mRNAs accumulate in the sperm cell nucleus, raising the question of their origin and of a possible role after fertilization.

Animals↗
PubMed2003

Quantitative approaches to problems of eukaryotic gene expression.

During the past several years there has been intense interest in the mechanisms by which gene expression is regulated within the eukaryotic nucleus. We have made use of an avian beta-globin locus to study various aspects of this problem, some of which are amenable to quantitative analysis. In the course of this work we have identified the transcription factor GATA-1, which is an essential regulatory factor for virtually all erythroid-specific genes, and studied the structure of its complex with its specific DNA binding site. The way in which GATA-1 forms tight interactions with DNA led to an understanding of how other zinc finger proteins of this class bind to DNA. We have extended such studies to examine interactions with DNA packaged as chromatin, and to studies of chromatin structure and function at higher levels of organization.

Animals↗
PubMed1997

Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22.

A microdialysis flow cell has been developed for time-resolved Raman spectroscopy of biological macromolecules and their assemblies. The flow cell permits collection of Raman spectra concurrent with the efflux of small solute molecules into a solution of macromolecules and facilitates real-time spectroscopic detection of structural transitions induced by the effluent. Additionally, the flow cell is well suited to the investigation of hydrogen-isotope exchange phenomena that can be exploited as dynamic probes of viral protein folding and solvent accessibility along the assembly pathway. Here, we describe the application of the Raman dynamic probe to the maturation of the icosahedral capsid of bacteriophage P22, a double-stranded DNA virus. The P22 virion is constructed from a capsid precursor (procapsid) consisting of 420 coat subunits (gp5) in an outer shell and a few hundred scaffolding subunits (gp8) within. Capsid maturation involves expulsion of scaffolding subunits coupled with shell expansion at the time of DNA packaging. Raman static and dynamic probes reveal that the scaffolding subunit is highly alpha-helical and highly thermolabile, and lacks a typical hydrophobic core. When bound within the procapsid, the alpha-helical fold of gp8 is thermostabilized; however, this stabilization confers no apparent protection against peptide NH-->ND exchange. A molten globule model is proposed for the native scaffolding subunit that functions in procapsid assembly. Accompanying capsid expansion, a small conformational change (alpha-helix-->beta-strand) is also observed in the coat subunit. Domain movement mediated by hinge bending is proposed as the mechanism of capsid expansion. On the basis of these results, a molecular model is proposed for assembly of the P22 procapsid.

Bacteriophage P22↗
PubMed1997

A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome.

Adeno-associated virus (AAV) vectors have a limited capacity for packaging DNA. To insert both a therapeutic gene and a selectable marker gene in the same AAV vector efficiently, we developed a novel dicistronic AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marker in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3' end of the luciferase gene (Luc) under the control of the human cytomegalovirus (CMV) promoter. This dicistronic gene conferred blasticidin S-resistance to 293 cells besides luciferase activity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to 3.6 kilobases (kb) (including promoter/enhancer elements) as well as a selectable marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vector described here is useful for expressing many kinds of cDNA besides a selectable marker.

Animals↗
PubMed2003

Relationships between fuselloviruses infecting the extremely thermophilic archaeon Sulfolobus: SSV1 and SSV2.

The fusellovirus SSV2 from an Icelandic Sulfolobus strain was isolated, characterized and its complete genomic sequence determined. SSV2 is very similar in morphology, replication, genome size and number of open reading frames (ORFs) to the type virus of the family, SSV1 from Japan, except in its high level of uninduced virus production. The nucleotide sequences are, however, only 55% identical to each other, much less than related bacteriophage, related animal viruses and the rudiviruses of Sulfolobus, SIRV1 and SIRV2. Nevertheless the genome architecture is very similar between the two viruses, indicating that despite this genomic dissimilarity the virus genomes are mostly homologous. Unlike SSV1, the sequence of SSV2 indicates integration into a glycyl tRNA gene and is completely missing a DNA packaging gene. There is a unique, perfectly tandemly directly repeated sequence of 62 nucleotides in SSV2 that has no similarity to known sequences or structures. By comparison to the SSV2 genome, an integrated partial fusellovirus genome was found in the Sulfolobus solfataricus P2 genome further confirming the dynamism of the Sulfolobus genome. Clustering of cysteine codon containing ORFs both in SSV1 and SSV2 indicates that these Fuselloviridae arose from a genome fusion event.

Fuselloviridae↗
PubMed2003

An elastic network model of HK97 capsid maturation.

The structure of the capsid of bacteriophage HK97 has been solved at various stages of maturity by crystallography and cryo-electron microscopy, and has been reported previously in the literature. Typically the capsid assembles through polymerization and maturation processes. Maturation is composed of proteolytic cleavages to the precursor capsid (called Prohead II), expansion triggered by DNA packaging (in which the largest conformational changes of the capsid appear), and covalent cross-links of neighboring subunits to create the mature capsid called Head II. We apply a coarse-grained elastic network interpolation (ENI) to generate a feasible pathway for conformational change from Prohead II to Head II. The icosahedral symmetry of the capsid structure offers a significant computational advantage because it is not necessary to consider the whole capsid structure but only an asymmetric unit consisting of one hexamer plus an additional subunit from an adjacent pentamer. We also analyze normal modes of the capsid structure using an elastic network model which is also subject to symmetry constraints. Using our model, we can visualize the smooth evolution of capsid expansion and revisit in more detail several interesting geometric changes recognized in early experimental works such as rigid body motion of two compact domains (A and P) with two refolding extensions (N-arm and E-loop) and track the approach of the two particular residues associated with isopeptide bonds that make hexagonal cross-links in Head II. The feasibility of the predicted pathway is also supported by the results of our normal mode analysis.

Bacteriophages↗
PubMed1999

A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme.

The elongator complex is a major component of the RNA polymerase II (RNAPII) holoenzyme responsible for transcriptional elongation in yeast. Here we identify Elp3, the 60-kilodalton subunit of elongator/RNAPII holoenzyme, as a highly conserved histone acetyltransferase (HAT) capable of acetylating core histones in vitro. In vivo, ELP3 gene deletion confers typical elp phenotypes such as slow growth adaptation, slow gene activation, and temperature sensitivity. These results suggest a role for a novel, tightly RNAPII-associated HAT in transcription of DNA packaged in chromatin.

Acetyltransferases↗
PubMed2003

Multigene expression from a replicating adenovirus using native viral promoters.

We have developed a novel therapeutic gene delivery system for oncolytic adenoviruses that takes advantage of the endogenous gene expression machinery (promoters, splicing, polyadenylation signals) of the E3 transcription unit for gene delivery. In this work, we use two sites in the E3 region (6.7 K/gp19K and ADP sites) to demonstrate that (1) multiple therapeutic genes (MCP-3, TNFalpha) can be expressed from a single replicating Ad, (2) timing of expression of these therapeutic genes mimics that of the E3 region genes they replaced, (3) expression of the remaining genes in the complex E3 transcription unit is maintained, and (4) the multigene-expressing virus retains replication competence and ability to induce classical adenovirus cytopathic effects that parallel those of the parental adenovirus (ONYX-320). This system conserves the DNA packaging capacity of the size-constrained viral genome for therapeutic genes and can potentially be used to link therapeutic transgene expression to tumor-restricted viral replication. Potential clinical implications are discussed.

Adenoviridae↗