Restriction analysis of the DNA of aleutian mink disease virus strain P-1

The II-1 strain of the Aleutian disease virus (ADV-II-1) was isolated from experimentally infected mink organs. The viral particles were isolated having 23 to 24 nm in diameter with the buoyant density of the virions in CsCl gradient being 1.41 g.ml-1. The single stranded ADV DNA extracted from the purified virus particles had the molecular mass about 1.4 . 10(6) (4800 bases). The double-stranded replicative form of ADV DNA has been synthesized in vitro with the use of a large "Klenow" fragment of DNA-polymerase I. A restriction endonuclease map of ADV-II-1 DNA has been constructed with the use of in vitro synthesized double-stranded DNA.     

Intermediate in adenovirus type 2 replication.

Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with S1 endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 10-6 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min.     

Composition and Size of Shope Fibroma Virus Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from purified virions of Shope fibroma virus (SFV) (by using DNA from Microccocus lysodeikticus as marker) had a buoyant density of 1.6996 +/- 0.0003 g/ml), hence a guanine plus cytosine (G + C) content of 40.4 +/- 0.3%, which is close to the G + C content of the DNA of susceptible rabbit cells (40.9 +/- 0.4%) and different from that of vaccinia virus DNA (35.5 +/- 0.4%). For the determination of the molecular weight of DNA, SFV and vaccinia purified virions, treated with Pronase and detergent, were cosedimented in sucrose density gradients. Results showed that SFV-DNA has a molecular weight of about 153 x 10(6) daltons. By electron microscopy, only one molecule corresponding to this value was observed (its length was 80.3 mum). The others had a median size of 49.8 mum +/- 0.9.     

Characterization of the molecular components in kinetoplast- mitochondrial DNA of Trypanosoma equiperdum. Comparative study of the dyskinetoplastic and wild strains

The structure of the kinetoplast DNA of Trypanosoma equiperdum has been studied and compared to the structure of the circular mitochondrial DNA extracted from a dyskinetoplastic strain of T. equiperdum. In T. equiperdum wild type, the kinetoplast DNA constitutes approximately 6% of the total cellular DNA and is composed of approximately 3,000 supercoiled minicircles of 6.4 x 10(5) daltons and approximately 50 circular supercoiled molecules of 15.4 x 10(6) daltons topologically interlocked; The buoyant density in CsCl of the minicircles is 1.691 g/cm 3. The large circles have a buoyant density of 1.684 g/cm 3, are homogeneous in size and are selectively cleaved by several restriction endonucleases which do not cleave the minicircles. The cleavage sites of six different restriction endonucleases have been mapped on the large circle. The minicircles are cleaved by two other restriction endonucleases, and their cleavage sites have been mapped. The mitochondrial DNA extracted from the dyskinetoplastic strain of T. equiperdum represents 7% of the total DNA of the cell and is composed of supercoiled circles, heterogeneous in size, and topologically associated in catenated oligomers. Its buoyant density in CsCl is 1.688 g/cm 3. These molecules are not cleaved by any of the eight restriction endonucleases tested. The reassociation kinetics of in vitro labeled kDNA minicircles and large circles has been studied. The results indicate that the minicircles as well as the large circles are homogeneous in sequence and that the circular DNA of the dyskinetoplastic strain has no sequence in common with the kDNA of the wild strain.     

DNA of a Human Hepatitis B Virus Candidate

Particles containing DNA polymerase (Dane particles) were purified from the plasma of chronic carriers of hepatitis B antigen. After a DNA polymerase reaction with purified Dane particle preparations treated with Nonidet P-40 detergent, Dane particle core structures containing radioactive DNA product were isolated by sedimentation in a sucrose density gradient. The radioactive DNA was extracted with sodium dodecyl sulfate and isolated by band sedimentation in a preformed CsCl gradient. Examination of the radioactive DNA band by electron microscopy revealed exclusively circular double-stranded DNA molecules approximately 0.78 mum in length. Identical circular molecules were observed when DNA was isolated by a similar procedure from particles that had not undergone a DNA polymerase reaction. The molecules were completely degraded by DNase 1. When Dane particle core structures were treated with DNase 1 before DNA extraction, only 0.78-mum circular DNA molecules were detected. Without DNase treatment of core structures, linear molecules with lengths between 0.5 and 12 mum, in addition to the 0.78-mum circles were found. These results suggest that the 0.78-mum circular molecules were in a protected position within Dane particle cores and the linear molecules were not within core structures. Length measurements on 225 circular molecules revealed a mean length of 0.78 +/- 0.09 mum which would correspond to a molecular weight of around 1.6 x 10(6). The circular molecules probably serve as primer-template for the DNA polymerase reaction carried out by Dane particle cores. Thermal denaturation and buoyant density measurements on the Dane particle DNA polymerase reaction product revealed a guanosine plus cytosine content of 48 to 49%.     

Anatomy of herpes simplex virus DNA. III. Characterization of defective DNA molecules and biological properties of virus populations containing them.

We have characterized the virus progeny and its DNA from plaque-purified and undiluted passages of herpes simplex virus 1 in HEp-2 cells. Secifically, (i) infectious virus yields declined progressively in passages 1 through 10 and gradually increased at passages 11 through 14. The yields correlated with PFU/particle ratios. (ii) In cells infected with virus from passages 6 through 10, there was an overproduction of an early viral polypeptide (no. 4) and a delay in the synthesis of late viral proteins. In addition, the virus in these passages interfered with the replication of a nondefective marker virus. Cells infected with passage 14 virus produced normal amounts of polypeptide 4 and, moreover, this virus showed minimal interfering capacity. (iii) In addition to DNA of density 1.726 g/cm-3, which was the sole component present in viral progeny of passage 0, passages 6 through 14 contained one additional species (p 1.732) and in some instances (passages 6 and 10) also DNA of an intermediate buoyant density. The ratio of p 1.732 to p 1.726 DNA increased to a maximum of 4 in passages 6 through 9 and gradually decreased to 1 in passages 10 through 14. (iv) p 1.732 DNA cannot be differentiated from p 1.726 DNA with respect to size; however, it has no Hin III restriction enzyme cleavage sites and yields only predominantly two kinds of fragments with molecular weights of 5.1 x 10-6 and 5.4 x 10-6 upon digestion with EcoRI enzyme. (v) Partial denaturation profiles of purified p 1.732 DNA from passage 14 revealed the presence of two types of tandemly repeated units corresponding roughly in size to the EcoRI fragments and situated in different molecules. (vi) In addition to the two kinds of p 1.732 molecules consisting of tandem repaeat units of different sizes, other evidence for the diversity of defective DNA molecules emerged from comparisons of specific infectivity and interfering capacity of the progeny from various passages. The data suggest that some of the particles with DNA of normal buoyant density (1.726) must also be defective since the capacity to interfere and to produce an excess of polypeptide 4 did not appear to be proportional to the amount of high-buoyant-density defective DNA. The data suggest that defective interfering particles are replaced by defective particles with diminished capacity to interfere and that more than one species of defective DNA molecules evolves on serial preparation of HSV.     

Comparison of JC and BK Human Papovaviruses with Simian Virus 40: Restriction Endonuclease Digestion and Gel Electrophoresis of Resultant Fragments

JC virus was found to have a buoyant density of 1.20 g/cm(3) in linear sucrose-D(2)O and 1.35 g/cm(3) in cesium chloride isopycnic gradients. DNA extracted either from JC-infected cultures or from gradient-purified virions occupied a dense position relative to linear DNA in cesium chloride/ethidium bromide gradients, and the circular configuration of the extracted DNA was confirmed by electron microscopy, with a measured molecular weight of 2.93 x 10(6). DNA from BK virus was similarly prepared and compared to JC and to an SV40 DNA standard by digestion with restriction endonuclease preparations from Haemophilus influenzae, Haemophilus parainfluenzae, and Escherichia coli. Digests were electrophoretically analyzed on gradient polyacrylamide slab gels or agarose gels, and the three viruses were found to have distinctly different cleavage patterns by this form of analysis: JC and BK viruses were almost entirely different from SV40 and significantly different from each other. Thus, JC and BK human papovaviruses appear to be discrete new members of the papovavirus group, rather than SV40 variants.     

Physical Heterogeneity Among Bacillus subtilis Deoxyribonucleic Acid Molecules Carrying Particular Genetic Markers

In a Bacillus subtilis deoxyribonucleic acid (DNA) preparation, extracted and purified by the Marmur procedure, the DNA molecules carrying a particular marker are heterogeneous with respect to molecular weight, buoyant density, and thermal stability. This finding constitutes evidence against unique points of breakage during DNA isolation. The variation in buoyant density suggests a local compositional heterogeneity in the chromosomal region of certain markers. The variation in molecular weight provides an explanation for the results of certain transformation experiments that are otherwise poorly understood. An example of such a result is the observation that acridine orange increases the efficiency of differential thermal inactivation of markers. An explanation of this phenomenon is suggested by the demonstration that acridine orange can decrease the natural intramarker heterogeneity in melting behavior.     

Caspase cleavage of the nonstructural protein NS1 mediates replication of Aleutian mink disease parvovirus

Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD downward arrow S) and NS1:285 (DQTD downward arrow S). Replication of ADV containing either of these mutations was reduced 10(3)- to 10(4)-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.     

Anatomy of Herpes Simplex Virus DNA. VIII. Properties of the Replicating DNA

This paper concerns the properties of herpes simplex virus 1 DNA replicating in HEp-2 and human embryonic lung cells. The results were as follows. (i) Only a small fraction of input viral DNA entered the replicative pool. The bulk of the input viral DNA cosedimented with marker viral DNA and did not appear to be degraded or dissociated into L and S components. (ii) Nascent DNA sedimented faster and banded at a higher density than that of mature viral DNA extracted from virions. Pulse-chase experiments indicated that nascent DNA acquires the sedimentation rate and buoyant density of viral DNA within 30 to 40 min after its synthesis. (iii) Electron microscopic studies indicated that the DNA extracted from cells replicating viral DNA and banding at the density of viral DNA contained: (a) linear, full-size molecules with internal gaps and single-stranded regions at termini; (b) molecules with lariats, consisting of a linear segment up to 2x the size of mature DNA and a ring ranging from 0.5 x 10(6) to 100 x 10(6) in molecular weight, showing continuous and discontinuous forks; (c) circular, double-stranded molecules, both full-size and multiples of 18 x 10(6) in molecular weight, but without forks or loops; (d) molecules showing "eye" and "D" loops at or near one end of the DNA; (e) large, tangled masses of DNA, similar to those observed for T4 and pseudorabies virus replicating DNAs, containing loops and continuous and discontinuous forks. The electron micrographs are consistent with the hypothesis that the single-stranded ends on the DNA anneal to form a hairpin, that the DNA synthesis is initiated at or near that end and proceeds bidirectionally to form a lariat, and that resulting progeny derived by semiconservative replication are "head-to-head" and "tail-to-tail" dimers.     

Size and Composition of Marek''s Disease Virus Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from purified nucleocapsids of Marek's disease herpesvirus (MDV) was cosedimented with T4 and with herpes simplex virus (HSV) DNA in neutral sucrose density gradients and with T4 DNA in alkaline sucrose density gradients. These experiments indicated that the intact MDV DNA had a sedimentation constant of 56S corresponding to a molecular weight of 1.2 x 10(8) daltons. In the alkaline gradients, the largest and most prominent band contains a DNA sedimenting at 70S corresponding to 6.0 x 10(7) daltons in molecular weight. The DNA is therefore double-stranded and not cross-linked. Isopycnic sedimentation of the MDV DNA molecules with SPO1, Micrococcus lysodeikticus, and HSV DNA gave a density of 1.705 g/cm(3) corresponding to 46 guanine plus cytosine moles per cent. Lastly, in hybridization tests the DNA hybridized with RNA of infected cells but not with that of uninfected cells supporting the conclusion that it is viral.     

Recombinational-type transfer of viral DNA during bacteriophage 2C replication in Bacillus subtilis.

The Bacillus subtilis phage 2C contains one molecule of double-stranded DNA of about 100 x 10(6) daltons in which thymine is replaced by hydroxymethyluracil; the two strands have different buoyant densities. Parental DNA, labeled with either [3H]uracil of [32P]phosphate, was quite effectively transferred to offspring phage, and the efficiency of transfer was the same for the two strands. Labeled nucleotide compositions of the H and L strands from parental and progeny virions were very close. These data exclude a degradation of the infecting DNA and reutilization of nucleotides. Upon infection of light unlabeled cells with heavy radioactive viruses, no DNA with either heavy or hybrid density was extracted from offspring phage. Instead, an heterogeneous population of DNA molecules of densities ranging from that of almost hybrid to that of fully light species was obtained. Shear degradation of such progeny DNA to fragments of decreasing molecular weight produced a progressive shift to the density of hybrid molecules. Denaturation of sheared DNA segments caused the appearance of labeled and heavy single-stranded segments. These findings indicate that 2C DNA replicates semiconservatively and then undergoes extensive genetic recombination with newly formed viral DNA molecules within the vegatative pool, thus mimicking a dispersive transfer of the infecting viral genome. The pieces of transferred parental DNA have an average size of 10 x 10(6) daltons.     

Analysis of Aleutian disease virus infection in vitro and in vivo: demonstration of Aleutian disease virus DNA in tissues of infected mink.

Aleutian disease virus (ADV) infection was analyzed in vivo and in vitro to compare virus replication in cell culture and in mink. Initial experiments compared cultures of Crandell feline kidney (CRFK) cells infected with the avirulent ADV-G strain or the highly virulent Utah I ADV. The number of ADV-infected cells was estimated by calculating the percentage of cells displaying ADV antigen by immunofluorescence (IFA), and several parameters of infection were determined. Infected cells contained large quantities of viral DNA (more than 10(5) genomes per infected cell) as estimated by dot-blot DNA-DNA hybridization, and much of the viral DNA, when analyzed by Southern blot hybridization, was found to be of a 4.8-kilobase-pair duplex monomeric replicative form (DM DNA). Furthermore, the cultures contained 7 to 67 fluorescence-forming units (FFU) per infected cell, and the ADV genome per FFU ratio ranged between 2 X 10(3) and 164 X 10(3). Finally, the pattern of viral antigen detected by IFA was characteristically nuclear, although cytoplasmic fluorescence was often found in the same cells. Because no difference was noted between the two virus strains when cultures containing similar numbers of infected cells were compared, it seemed that both viruses behaved similarly in infected cell culture. These data were used as a basis for the analysis of infection of mink by virulent Utah I ADV. Ten days after infection, the highest levels of viral DNA were detected in spleen (373 genomes per cell), mesenteric lymph node (MLN; 750 genomes per cell), and liver (373 genomes per cell). In marked contrast to infected CRFK cells, the predominant species of ADV DNA in all tissues was single-stranded virion DNA; however, 4.8-kilobase-pair DM DNA was found in MLN and spleen. This observation suggested that MLN and spleen were sites of virus replication, but that the DNA found in liver reflected sequestration of virus produced elsewhere. A final set of experiments examined MLN taken from nine mink 10 days after Utah I ADV infection. All of the nodes contained ADV DNA (46 to 750 genomes per cell), and although single-stranded virion DNA was always the most abundant species, DM DNA was observed. All of the lymph nodes contained virus infectious for CRFK cells, but when the genome per FFU ratio was calculated, virus from the lymph nodes required almost 1,000 times more genomes to produce an FFU than did virus prepared from infected cell cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Further characterization of intracytoplasmic A particle-associated DNA.

A DNA species with buoyant densities greater than mouse cellular DNA was found associated with intracytoplasmic A particles (CAP) isolated from mouse mammary tumor virus-infected mouse mammary tumors and mouse Leydig cell tumors which produce CAP but no complete mouse mammary tumor virus virions. This DNA species was absent in identically prepared tissue fractions from tumors which did not contain CAP. Treatment of CAP-associated DNA with pancreatic RNase A did not alter the buoyant density although a reduction in apparent molecular weight (broadening of the DNA band at equilibrium) was observed upon analytical equilibrium sedimentation in CsCl. The molecular weight of untreated CAP-associated DNA was estimated to range from 0.8 x 10(6) to 3.1 x 10(6). Base composition analysis showed CAP-DNA to possess an approximate guanine plus cytosine content of 38%. Ninety percent of CAP-associated DNA eluted as single-stranded molecules upon hydroxyapatite column chromatography, a characteristic that accounts in part for its higher buoyant density in neutral CsCl compared to native double-stranded mouse DNA. In two preparations, CAP-DNA had a sedimentation coefficient of 7 to 8S.     

Characterization of the Kilham Rat Virus

Kilham rat virus (KRV) was found to grow in a rat nephroma cell line and to form plaques on secondary rat embryo monolayers. The virus was purified by enzymatic treatment and isopycnic cesium chloride sedimentation. KRV bands at a density of 1.41 g/cm(3) in cesium chloride. It contains about 26.5% deoxyribonucleic acid (DNA). The sedimentation coefficient S(20,w) in sucrose gradients was 122 corresponding to a molecular weight of 6.6 x 10(6) daltons. The reaction of formaldehyde with the KRV virion suggests that the DNA in situ is single-stranded. DNA extracted from KRV had a buoyant density of 1.715 g/cm(3) in cesium chloride. The S(20,w) was determined in sucrose gradients to be 16, and the molecular weight was calculated to be approximately 1.7 x 10(6) daltons. The base composition of the DNA is 26.7% adenine, 30.8% thymine, 20.0% guanine, and 22.5% cytosine. On the basis of its noncomplementary nucleotide ratio, melting curve, and the reaction with formaldehyde, the DNA of KRV is believed to be single-stranded.     

Analysis of Euglena gracilis chloroplast deoxyribonucleic acid with a restriction endonuclease, EcoRI.

Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoRI) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight of the circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosomal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.     

Density Heterogeneity of Simian Virus 40 Ribonucleic Acid Late After Infection of Permissive Cells

Ribonucleic acid (RNA) was isolated from CV-1 cells 44 hr after simian virus 40 (SV40) infection. The molecules containing SV40 base sequences were characterized with respect to their buoyant density distribution. The density of these molecules was compared to that of single- and double-stranded RNA synthesized by using SV40 DNA and Escherichia coli RNA polymerase. The results suggest that about 20% of the SV40 RNA in the infected cells consisted of partially double-stranded molecules.     

Multiplication of Polyoma Virus in Mouse-Hamster Somatic Hybrids: a Hybrid Cell Line Which Produces Viral Particles Containing Predominantly Host Deoxyribonucleic Acid

The multiplication of polyoma virus in a mouse-hamster (3T3 x BHK) somatic hybrid line (10A), which, although permissive for viral multiplication, produces very low amounts of virus, has been studied. In this cell line, the efficiency of productive infection is high, but the yield of infectious virus is on the order of 0.5% of that of 3T3 cells. The amount of viral deoxyribonucleic acid (DNA) synthesized by these cells upon infection is about 5% of that of 3T3 cells. An examination of the virus produced in hybrid 10A revealed that it was only one-tenth as infectious as the virus grown in 3T3. Although the viral DNA synthesized in the infected 10A cells is normal, the DNA extracted from purified virus grown in 10A consists of approximately 10% of normal, supercoiled polyoma DNA molecules and of approximately 90% linear DNA molecules with a sedimentation coefficient of 14 to 16S. These DNA molecules appear to be of cellular origin but contain a limited amount of viral DNA sequences. The host DNA-containing particles are not infectious but appear to possess some biological activity; they give rise to a weak complementation effect, and part of them are able to induce T-antigen synthesis. In addition, the host DNA present in these particles is predominantly that which has been synthesized after infection. The correlation between the block in viral DNA synthesis in this cell line and the abnormal encapsidation of host DNA is discussed.     

Deoxyribonucleic Acid Polymerase Activity Associated with Strain MC29 Tumor Virus

Chick embryo cells infected with strain MC29 tumor virus yielded progeny virus that contained detectable deoxyribonucleic acid (DNA) polymerase within the first 48 hr after infection. The noninfected culture fluids displayed no such enzyme activity when examined in an identical manner. Enzyme activity was greatly stimulated by adding DNA template to the reaction mixture and required all four deoxyribonucleoside triphosphates for full activity. When calf thymus DNA was used to direct synthesis, the DNA polymerase from the MC29 virus catalyzed the formation of DNA product having a higher buoyant density in CsCl. DNA product formed in the reaction directed by Micrococcus lysodeikticus DNA had the same buoyant density as the template DNA.