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At least 1,495 records · Page 83Linked to original sources
PubMed2003

The simian virus 40 minor structural protein Vp3, but not Vp2, is essential for infectious virion formation.

The SV40 capsid is composed of pentameric capsomeres of the major structural protein Vp1. The two minor structural proteins, Vp2 and Vp3, interact with the capsid. Here, the roles of Vp2 and Vp3 were explored during the course of SV40 infection. Start codons of Vp2, Vp3, or both Vp2 and Vp3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40DeltaVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40DeltaVp3 and SV40 DeltaVp2/Vp3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, Vp3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length Vp3 appears to be in virus-cell interactions at post-packaging steps of a permissive infection.

Animals↗
PubMed1980

In vitro recombination of bacteriophage T7 DNA damaged by UV radiation.

A system capable of in vitro packaging of exogenous bacteriophage T7 DNA has been used to monitor the biological activity of DNA replicated in vitro. This system has been used to follow the effects of UV radiation on in vitro replication and recombination. During the in vitro replication process, a considerable exchange of genetic information occurs between T7 DNA molecules present in the reaction mixture. This in vitro recombination is reflected in the genotype of the T7 phage produced after in vitro encapsulation; depending on the genetic markers selected, recombinants can comprise nearly 20% of the total phage production. When UV-irradiated DNA is incubated in this system, the amount of in vitro synthesis is reduced and the total amount of viable phage produced after in vitro packaging is diminished. In vitro recombination rates are also lower when the participating DNA molecules have been exposed to UV. However, biochemical and genetic measurements confirmed that there is little or no transfer of pyrimidine dimers from irradiated DNA into undamaged molecules.

Cell-Free System↗
PubMed2005

VariScan: Analysis of evolutionary patterns from large-scale DNA sequence polymorphism data.

SUMMARY: VeriScan is a software package for the analysis of DNA sequence polymorphisms at the whole genome scale. Among other features, the software (1) can conduct many population genetic analyses; (2) incorporates a multiresolution wavelet transform-based method that allows capturing relevant information from DNA polymorphism data; (3) facilitates the visualization of the results in the most commonly used genome browsers.

Algorithms↗
PubMed2001

Coupling between replication and packaging of flavivirus RNA: evidence derived from the use of DNA-based full-length cDNA clones of Kunjin virus.

In order to study whether flavivirus RNA packaging is dependent on RNA replication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN1dGDD, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-length Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. As expected, transfection of pKUN1 plasmid DNA into BHK cells resulted in the recovery of secreted infectious Kunjin virions. Transfection of pKUN1dGDD DNA into BHK cells, however, did not result in the recovery of any secreted virus particles containing encapsidated dGDD RNA, despite an apparent accumulation of this RNA in cells demonstrated by Northern blot analysis and its efficient translation demonstrated by detection of correctly processed labeled structural proteins (at least prM and E) both in cells and in the culture fluid using coimmunoprecipitation analysis with anti-E antibodies. In contrast, when dGDD RNA was produced even in much smaller amounts in pKUN1dGDD DNA-transfected repBHK cells (where it was replicated via complementation), it was packaged into secreted virus particles. Thus, packaging of defective Kunjin virus RNA could occur only when it was replicated. Our results with genome-length Kunjin virus RNA and the results with poliovirus replicon RNA (C. I. Nugent et al., J. Virol. 73:427-435, 1999), both demonstrating the necessity for the RNA to be replicated before it can be packaged, strongly suggest the existence of a common mechanism for minimizing amplification and transmission of defective RNAs among the quasispecies in positive-strand RNA viruses. This mechanism may thus help alleviate the high-copy error rate of RNA-dependent RNA polymerases.

Animals↗
PubMed1993

The cos region of lactococcal bacteriophage phi LC3.

The nucleotide sequence of the DNA regions flanking the cohesive end site of the temperate lactococcal bacteriophage phi LC3 was determined. Four pairs of sequence repeats of 8 to 13 base-pairs in length were revealed close to the cohesive ends. These repeats possibly represent signal sequences involved in phage DNA maturation and packaging. The start of an open reading frame was localized only 42 nucleotides from the left end of the phi LC3 phage double-stranded region, implying that transcription probably proceeds across the cos site. Compilation of the characteristics of the DNA termini of bacteriophages with complementary cohesive DNA ends suggests that phages of Gram-negative bacteria generally carry 5'-protruding single-strands whereas those of Gram-positive hosts carry 3'-protruding ends.

Bacteriophages↗
PubMedDate not supplied

Interactions between DNA and coat protein in the structure and assembly of filamentous bacteriophage fd.

Bacteriophage fd is a class I filamentous virus (others are M13 and f1) that comprises a circular, single-stranded DNA molecule enclosed in a cylindrical protein sheath to form a flexible particle approximately 890 nm long and 7 nm in diameter. The viral DNA contains 6,408 nucleotides incorporating 10 genes, and the protein sheath is composed of about 2,700 major coat protein subunits in a shingled helical array, the symmetry of which is defined by a fivefold rotational axis combined with a twofold screw axis of pitch 3.2 nm. The DNA extends throughout the length of the particle but is not base-paired and has a symmetry different from that of the protein helix. How the DNA is packed remains unclear but the number (2.4) of nucleotides packaged per major coat protein subunit is certainly not integral, in contrast with, say, the packaging of RNA in tobacco mosaic virus. The coat protein subunit is 50 amino-acid residues in length and, in the virus particle, adopts a largely alpha-helical conformation, with the long axis of the helix aligned close to the long axis of the filament. This protein is arranged with its negatively charged N-terminal region on the outside of the filament and its positively charged C-terminal region on the inside abutting the DNA. We report here that positive charge on one of the four lysine side chains in the latter region has a direct effect on DNA packaging, because when this charge is absent, elongated particles are produced with lengths that can be correlated with the residual positive charge in the C-terminal region of the coat protein subunit.

Amino Acid Sequence↗
PubMed1994

Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos.

A novel approach to study the higher level packaging of specific DNA sequences has been developed by coupling high-resolution fluorescence hybridization with biochemical fractionation to remove histones and distend DNA loops to form morphologically reproducible nuclear "halos." Results demonstrate consistent differences in the organization of specific sequences, and further suggest a relationship to functional activity. Pulse-incorporated bromodeoxyuridine representing nascent replicating DNA localized with the base of the chromatin loops in discrete clustered patterns characteristic of intact cells, whereas at increasing chase times, the replicated DNA was consistently found further out on the extended region of the halo. Fluorescence hybridization to unique loci for four transcriptionally inactive sequences produced long strings of signal extending out onto the DNA halo or "loop," whereas four transcriptionally active sequences remained tightly condensed as single spots within the residual nucleus. In contrast, in non-extracted cells, all sequences studied typically remained condensed as single spots of fluorescence signal. Interestingly, two transcriptionally active, tandemly repeated gene clusters exhibited strikingly different packaging by this assay. Analysis of specific genes in single cells during the cell cycle revealed changes in packaging between S-phase and non S-phase cells, and further suggested a dramatic difference in the structural associations in mitotic and interphase chromatin. These results are consistent with and suggestive of a loop domain organization of chromatin packaging involving both stable and transient structural associations, and provide precedent for an approach whereby different biochemical fractionation methods may be used to unravel various aspects of the complex higher-level organization of the genome.

Cell Cycle↗
PubMed2000

Bacteriophage and host mutants causing the rolling-circle lambda DNA replication early after infection.

There are two modes of bacteriophage lambda DNA replication during its lytic development in Escherichia coli cells. The circle-to-circle (theta) replication predominates at early stages of the phage growth, whereas rolling-circle (sigma) replication occurs late after infection to produce long concatemers that serve as substrates for packaging of lambda DNA into phage proheads. The mechanism regulating the switch from theta to sigma replication remains unknown. Our previous genetic studies indicated that the bacteriophage lambda Pts1piA66 mutant cannot replicate at 43 degrees C in the wild-type E. coli host, but it can replicate in the dnaA46(ts) mutant. Density shift experiments suggested that the parental DNA molecules of the infecting phage enter sigma replication. Here, using electron microscopy, we demonstrate that as soon as 5 min after infection of the dnaA46(ts) mutant by the lambdaPts1piA66 phage at 43 degrees C, the sigma replication intermediates are highly predominant over theta replication intermediates, contrary to the wild-type conditions (wild-type bacteria infected with the lambdaP(+) phage). The initiation of replication of the lambdaPts1piA66 mutant at 43 degrees C was strongly inhibited in the dnaA(+) host, as demonstrated by electron microscopy and by pulse-labeling of the phage-derived plasmid replicon. Implications for the mechanism of the regulation of the switch from theta to sigma replication mode are discussed.

Bacterial Proteins↗
PubMed1993

The genetics of the Luria-Latarjet effect in bacteriophage T4: evidence for the involvement of multiple DNA repair pathways.

The Luria-Latarjet effect is an increase in resistance of a virus to DNA damage during infection of a host. It has often been assumed to involve recombinational repair, but this has never been demonstrated experimentally. Using nine bacteriophage (phage) T4 mutants, I present evidence indicating that, for phage T4, the Luria-Latarjet effect is due to three repair pathways-excision repair, post-replication-recombinational-repair (PRRR) and multiplicity reactivation (MR) (a second form of recombinational repair). The results also show that the Luria-Latarjet effect develops in two stages. The first stage starts soon after infection. Damage which occurs during the first stage can be repaired by excision repair or PRRR. The second stage appears to start after the first round of DNA replication is complete. DNA damage which occurs during this stage can apparently be repaired by MR as well as the other two repair pathways. The results of this study support the hypothesis that recombinational repair has been selected to ensure that the progeny phage genomes which are packaged have minimum DNA damage. Since other viruses which infect bacterial, animal and plant cells show a Luria-Latarjet effect similar to that in phage T4, the conclusions from this study may have wide applicability.

Bacteriophage T4↗
PubMed1993

Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants.

The herpes simplex virus type 1 UL28 gene contains a 785-amino-acid open reading frame that codes for an essential protein. Studies with temperature-sensitive mutants which map to the UL28 gene indicate that the UL28 gene product (ICP18.5) is required for packaging of viral DNA and for expression of viral glycoproteins on the surface of infected cells (C. Addison, F. J. Rixon, and V. G. Preston, J. Gen. Virol. 71:2377-2384, 1990; B. A. Pancake, D. P. Aschman, and P. A. Schaffer, J. Virol. 47:568-585, 1983). In this study, we describe the isolation of two UL28 deletion mutants that were constructed and propagated in Vero cells transformed with the UL28 gene. The mutants, gCB and gC delta 7B, contained deletions of 1,881 and 537 bp, respectively, in the UL28 gene. Although the mutants synthesize viral DNA, they fail to form plaques or produce infectious virus in cells that do not express the UL28 gene. Transmission electron microscopy and Southern blot analysis demonstrated that both mutants are defective in cleavage and encapsidation of viral DNA. Analysis by cell surface immunofluorescence showed that the UL28 gene is not required for expression of viral glycoproteins on the surface of infected cells. A rabbit polyclonal antiserum was made against an Escherichia coli-expressed Cro-UL28 fusion protein. This antibody reacted with an infected-cell protein having an apparent molecular mass of 87 kDa. The 87-kDa protein was first detected at 6 h postinfection and was expressed as late as 24 h postinfection. No detectable UL28 protein was synthesized in gCB- or gC delta 7B-infected Vero cells.

Animals↗
PubMed1999

Molecular genetic analysis of herpesviruses and their potential use as vectors for gene therapy applications.

Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses. Some of these cells include neuronal cells and B-lymphocytes infected with herpes simplex virus (HSV) and Epstein-Barr virus (EBV), respectively as well as human T-lymphocytes with herpesvirus saimiri. These large DNA viruses have the potential to deliver fragments of human heterologous DNA > 150 kbp to specific cells. Additionally, EBV recombinants can maintain these large pieces of DNA in the infected B-cells as episomal DNA. The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 150 kbp and to infect cells that can stably maintain DNA as episomes. Moreover, other herpesvirus vector systems can be utilized as a source for the propagation of specific DNA fragments of interest, including wild-type genes to correct genetic defects in human cells. Other herpesvirus systems include defective infectious single cycle (DISC-HSV) mutants of HSV, which are now undergoing trials for the treatment of genital herpesvirus infections and certain types of cancers. The use of herpesviruses as an agent to deliver heterologous gene products has the potential to make a significant difference to the development of therapeutic approaches to human diseases.

Cell Line↗
PubMed2003

Motifs in nucleic acids: molecular mechanics restraints for base pairing and base stacking.

In building and refining nucleic acid structures, it is often desirable to enforce particular base pairing and/or base stacking interactions. Energy-based modeling programs with classical molecular mechanics force fields do not lend themselves to the easy imposition of penalty terms corresponding to such restraints, because the requirement that two bases lie in or near the same plane (pairing) or that they lie in parallel planes (stacking) cannot be easily expressed in terms of traditional interactions involving two atoms (bonds), three atoms (angles), or four atoms (torsions). Here we derive expressions that define a collection of pseudobonds and pseudoangles through which molecular mechanics restraints for base pairing and stacking can be imposed. We have implemented these restraints into the JUMNA package for modeling DNA and RNA structures. JUMNA scripts can specify base pairing with a variety of standard geometries (Watson-Crick, Hoogsteen, wobble, etc.), or with user-defined geometries; they can also specify stacking arrangements. We have also implemented "soft-core" functions to modify van der Waals and electrostatic interactions to avoid steric conflicts in particularly difficult refinements where two backbones need to pass through one another. Test cases are presented to show the utility of the method. The restraints could be adapted for implementation in other molecular mechanics packages.

Base Pairing↗
PubMed1993

Herpes simplex virus 1 alkaline nuclease is required for efficient egress of capsids from the nucleus.

We previously described the isolation of AN-1, a null mutant of the HSV-1 alkaline nuclease gene, which is able to synthesize near wild-type levels of viral DNA and late viral proteins under nonpermissive growth conditions; these results lead us to conclude that the alkaline nuclease is not essential for viral DNA synthesis (Weller, S. K., Seghatoleslami, R. M., Shao, L., Rowse, D., and Carmichael, E. P., 1990, J. Gen. Virol. 71, 2941-2952). AN-1 was found to be deficient in the production of infectious virions suggesting that the nuclease may play a role in processing or packaging of viral DNA into infectious virions. In this report we demonstrate that the defect is distinct from that observed in other late HSV-1 mutants which make but fail to process viral DNA under nonpermissive growth conditions. Following restriction enzyme digestion, specific terminal fragments are observed in DNA from AN-1-infected Vero cells, indicating that specific cleavage has occurred; moreover, the efficiency of cleavage is at near wild-type levels. Also in contrast to cells infected with previously described late mutants, DNase I or staphylococcal nuclease resistant DNA is observed in these cells further indicating that encapsidation has occurred. Three lines of evidence suggest, however, that maturation of DNA-containing AN-1 capsids is defective in some ways. First, in contrast to wild-type, very small amounts of protected DNA is detected in cytoplasmic extracts from AN-1-infected cells. Second, very few if any mature, DNA-containing C capsids are observed in the cytoplasm when analyzed by electron microscopy or sucrose gradient sedimentation. Finally, analysis of nuclei by sucrose gradient sedimentation indicates an elevated ratio of A to B capsids. These data indicate that AN-1 may be defective for the production of capsids competent to mature into the cytoplasm. Possible models for the nature of the defect in AN-1 will be discussed.

Animals↗
PubMed1999

Structure of the 13-fold symmetric portal protein of bacteriophage SPP1.

We have determined the three-dimensional structure of bacteriophage SPP1 portal protein (gp6) using electron microscopy at liquid-helium temperatures and angular reconstitution. The 13-fold symmetric gp6 oligomer is a turbine-shaped structure with three distinct regions: a conical stem with a central channel; the turbine wings region; and a fringe of small 'tentacles' at the end of the channel exposed to the viral head interior. The tentacle region appears flexible and may be associated with a particular function - sensing when the correct amount of DNA has been packaged. The three-dimensional structure of the gp6 SizA mutant, which packages a smaller chromosome, reveals significant differences in that region.

Bacillus Phages↗
PubMed1969

Effect of nalidixic acid on deoxyribonucleic acid synthesis in bacteriophage SPO1-infected Bacillus subtilis.

The effect of nalidixic acid on deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis cells infected with bacteriophage SPO1 was studied. Nalidixic acid had little inhibitory effect on SPO1 DNA synthesis at concentrations that drastically inhibited B. subtilis DNA synthesis. Inhibition of DNA synthesis, appropriate to the concentration used, was imposed within 1 min after addition of nalidixic acid, suggesting that it acts directly on DNA synthesis in both infected and uninfected cells. The SPO1 DNA synthesized in the presence of high concentrations of nalidixic acid had a density characteristic of normal SPO1 DNA and was packaged into viable progeny phage particles, but its rate of synthesis was reduced and bacterial lysis was delayed.

Adenine↗
PubMed2005

Identification, subviral localization, and functional characterization of the pseudorabies virus UL17 protein.

Homologs of the UL17 gene of the alphaherpesvirus herpes simplex virus 1 (HSV-1) are conserved in all three subfamilies of herpesviruses. However, only the HSV-1 protein has so far been characterized in any detail. To analyze UL17 of pseudorabies virus (PrV) the complete 597-amino-acid protein was expressed in Escherichia coli and used for rabbit immunization. The antiserum recognized a 64-kDa protein in PrV-infected cell lysates and purified virions, identifying PrV UL17 as a structural virion component. In indirect immunofluorescence analyses of PrV-infected cells the protein was predominantly found in the nucleus. In electron microscopic studies after immunogold labeling of negatively stained purified virion preparations, UL17-specific label was detected on single, mostly damaged capsids, whereas complete virions and the majority of capsids were free of label. In ultrathin sections of infected cells, label was primarily found dispersed around scaffold-containing B-capsids, whereas on DNA-filled C-capsids it was located in the center. Empty intranuclear A-capsids were free of label, as were extracellular capsid-less L-particles. Functional characterization of PrV-DeltaUL17F, a deletion mutant lacking codons 23 to 444, demonstrated that cleavage of viral DNA into unit-length genomes was inhibited in the absence of UL17. In electron microscopic analyses of PrV-DeltaUL17F-infected RK13 cells, DNA-containing capsids were not detected, while numerous capsidless L-particles were observed. In summary, our data indicate that the PrV UL17 protein is an internal nucleocapsid protein necessary for DNA cleavage and packaging but suggest that the protein is not a prominent part of the tegument.

Animals↗
PubMed2002

Molecular dynamics simulation of a DNA containing a single strand break.

Molecular dynamics simulations were performed for a dodecamer DNA containing a single strand break (SSB), which has been represented by a 3'-OH deoxyribose and 5'-OH phosphate in the middle of the strand. Molecular force field parameters of the 5'-OH phosphate region were determined from an ab initio calculation at the HF/6-31G level using the program package GAMESS. The DNA was placed in a periodic boundary box with water molecules and Na+ counter-ions to produce a neutralised system. After minimisation, the system was heated to 300 K, equilibrated and a production run at constant NTP was executed for 1 ns using AMBER 4.1. Snapshots of the SSB-containing DNA and a detailed analysis of the equilibrated average structure revealed surprisingly small conformational changes compared to normal DNA. However, dynamic properties calculated using the essential dynamics method showed some features that may be important for the recognition of this damage by repair enzymes.

Base Pairing↗
PubMed1976

Altered cotransduction frequencies exhibited by HT-mutants of Salmonella-phage P22.

Phage P22-mutants with increased transduction ability (HT-mutants) show in comparison to wild type 22, different frequencies of contransduction for markers on two different transducing fragments of the Salmonella chromosome. The data are interpreted as indicating that host DNA to be packaged is cut by HT-mutants at sites different from those cut by wild-type phage, due to an altered specificity of the nuclease responsible for this step.

DNA, Bacterial↗