Characterization of Nuclear Polyhedrosis Virus DNAs

The nuclear polyhedrosis virus DNAs characterized and compared in this study consist of the singly-enveloped nucleocapsids (SNPV) of Trichoplusia ni and the bundles of nucleocapsids common to a single envelope (MNPV) from Spodoptera frugiperda and Rachiplusia ou. The SNPV and MNPV DNAs are very similar in hydrodynamic properties and molecular weights. In addition, the NPV DNAs are similar in size to those extracted from the granulosis viruses that infect T. ni and S. frugiperda. As isolated from purified virus or directly from occluded virus, the nuclear polyhedrosis virus DNAs consist of a mixture of about 20 to 30% double-stranded covalently closed molecules and approximately 60% relaxed circles, with less than 10% in linear duplex form. The molecular weights of all nuclear polyhedrosis virus DNAs as compared in this study are slightly smaller than those of T4 bacteriophage DNA and perhaps slightly smaller than those of the granulosis virus DNAs. The best estimates of these molecular weights by neutral sucrose sedimentation for the nuclear polyhedrosis viruses range from 90 to 100 x 10(6) relative to a size of 108 x 10(6) for T4 DNA. The base compositions of the nuclear polyhedrosis viruses that infect T. ni and S. frugiperda are compared with the respective insect host DNAs.     

Identification, Mapping, and In Vitro Translation of Campoletis sonorensis Virus mRNAs from Parasitized Heliothis virescens Larvae

Expression of Campoletis sonorensis virus (CsV) in parasitized Heliothis virescens larvae was investigated by Northern blot analysis of poly(A)⁺ mRNAs isolated from H. virescens larvae at various times after parasitization by C. sonorensis. At least 12 CsV mRNAs were detected in parasitized H. virescens larvae. Injection of nonparasitized H. virescens larvae with purified CsV resulted in a pattern of viral mRNAs similar to that observed in naturally parasitized larvae. With CsV DNA restriction fragments which contained expressed sequences, individual CsV mRNAs were mapped to the superhelical DNAs of the viral genome. Two gene-specific probes, which consisted of cloned S1 nuclease-protected restriction fragments, each hybridized to several CsV superhelical DNAs, suggesting that some CsV genes may be shared on several superhelical DNAs. Cloned restriction fragments containing sequences which flank the expressed sequences also hybridized to numerous CsV superhelical DNAs. Some CsV proteins were identified by in vitro translation of hybrid-selected CsV mRNAs.     

The use of Haemophilus gallinarum DNA-relaxing enzyme to investigate the relationship between the number of superhelical turns and the molecular weight in a negatively twisted DNA.

Covalently closed-circular, superhelical DNAs, including viral DNAs, bacterial plasmid DNAs, and bacteriophage replicative-form DNA, were treated with a small amount of Haemophilus gallinarum DNA-relaxing enzyme to generate incompletely relaxed DNA molecules. Each sample consisted of a set of closed-circular DNA molecules differing by one turn in their number of superhelical turns. The DNA samples were analyzed by agarose gel electrophoresis under conditions such that the electrophoretic mobility was a function of the number of turns. The numbers of superhelical turns (at 37 degrees C in 20 mM Tris-HCl (pH 7.5)-5 mM MgCl2) in the DNAs of pSC101 (5.8 megadaltons), Colicin E1 (4.2 megadaltons), pMR4 (4.0 megadaltons; recombinant between pBR322 and lambda DNA fragment), phi X174 replicative-form (RF) I, Simian virus 40 (SV40), and polyoma virus (3.4--3.6 megadaltons each), and lambda dv021 (2.05 megadaltons) were estimated to be 36, 27, 23--24, 20--21, 20--21, 20--21, and 11--13, respectively. It appears that the number of superhelical turns is mainly a function of the molecular weight of the DNA, at least in the substrates tested here.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

Measurement of the Sequence Complexity of Cloned Moloney Murine Leukemia Virus 60 to 70S RNA: Evidence for a Haploid Genome

The sequence complexity of the 60-70S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C(r)t((1/2)), was compared with the C(r)t((1/2)) values for annealing of poliovirus 35S RNA (2.6 x 10(6) molecular weight) with poliovirus-specific DNA and Sindbis virus 42S RNA (4.3 x 10(6) molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35S RNA subunits of M-MuLV 60-70S RNA has a different base sequence.     

Synthesis of Avian Oncornavirus DNA in Infected Chicken Cells

The intracellular synthesis and integration of viral DNA (vDNA) into the host cell genome was studied in cultured chicken embryo fibroblasts infected with avian sarcoma or leukemia viruses. The newly synthesized vDNA was detected by hybridization with 70S viral RNA. Extraction of infected cell DNA by the selective procedure of Hirt resulted in the enrichment of newly synthesized vDNA in the low molecular weight supernatant fraction while leaving the bulk of cellular DNA containing integrated vDNA in the high molecular weight pellet fraction. This approach led to detection of intracellular vDNA synthesis within 1 h after infection and to vDNA integration into cellular DNA within 24 h. There was a several-fold increase in the vDNA content of infected cells during the initial phase of virus infection. But only a part of this newly synthesized vDNA appeared to become covalently linked with high molecular weight cellular DNA. Most of the remaining unintegrated vDNA gradually disappeared. The sedimentation profiles of minimally sheared cellular DNA in alkaline sucrose velocity gradients suggest that vDNA is synthesized as free linear molecules of approximately 3 x 10(6) daltons which subsequently are covalently linked to host cell DNA.     

Construction and isolation of a transforming murine retrovirus containing the src gene of Rous sarcoma virus.

Recombinant murine retroviruses containing the src gene of the avian retrovirus Rous sarcoma virus were isolated. Such viruses were isolated from cells after transfection with DNAs in which the src gene was inserted into the genome of the amphotropic murine retrovirus 4070A. The isolated viruses had functional gag and pol genes, but they were all env defective since the src gene was inserted in the middle of the env gene coding region. Infectious transforming virus could be isolated only from cells transfected with DNA constructions in which the src gene was in the same polarity as that of a long terminal repeat of the amphotropic viral genome. These recombinant viruses encoded a pp60src protein with a molecular weight similar to that of the Schmidt-Ruppin strain of Rous sarcoma virus. In addition, the src protein(s) of these recombinant viruses was as active as protein kinases in the immune complex protein kinase assay. Intravenous injection of helper-independent Moloney and Friend murine leukemia virus pseudotypes of the src recombinant viruses into 6-week-old NIH Swiss mice resulted in the appearance of splenic foci within 2 weeks, splenomegaly and, later after infection (8 to 10 weeks), anemia. Infectious transforming virus could be recovered from the spleens of diseased animals. Such viruses encoded pp60src but not p21ras or mink cell focus-forming virus-related glycoproteins.     

Cleavage of Circular, Superhelical Simian Virus 40 DNA to a Linear Duplex by S1 Nuclease

S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.     

Distribution and Expression in Mammals of Genes Related to an Endogenous Type C RNA Virus of Odocoileus hemionus

An endogenous type C virus recently isolated from the Columbian black-tailed deer (Odocoileus hemionus) was used as a molecular probe to study the distribution of virus-related nucleotide sequences in cellular DNAs of mammalian species. By DNA-DNA hybridization, the most extensive homology was demonstrated between the viral complementary DNA and cellular DNA isolated from Odocoileus species. DNAs of representatives of other genera within the same family, Cervidae, were partially related to the virus, consistent with the phylogenetic relationship of these species to Odocoileus. O. hemionus viral sequences were also detected within cellular DNAs of members of a more distantly related artiodactyl family, Bovidae. These findings suggest the genetic transmission of type C viral genes within cervids and bovids for a period of at least 25 to 30 million years. There was no detectable nucleotide sequence homology between O. hemionus virus and representatives of other major groups of mammalian type C viruses. These results indicate that despite the known antigenic relatedness of mammalian type C viruses, the O. hemionus virus has diverged sufficiently to be considered the prototype of a separate group. By radioimmunological techniques, it was possible to detect and partially purify, from normal tissues of cervid species, antigens related to the major structural protein of the O. hemionus virus. The present findings, that O. hemionus virus has been genetically transmitted for millions of years and yet has maintained the ability to be expressed as infectious virus, argue for positive evolutionary selective pressures for the maintenance of type C viral genes.     

Isolation, some properties and heterogeneous nature of DNA-topoisomerase (relaxing enzyme) from Ehrlich ascites carcinoma cells

DNA-topoisomerase catalyzing the conversion of a superhelical circular covalently closed DNA molecule into a super-helix free circular molecule, was isolated from mouse Ehrlich ascites carcinoma cells and purified 209-fold. The optimal conditions for the action and stability of the enzyme were elaborated. Using polyacrylamide gel electrophoresis under non-denaturating conditions as well as in the presence of Na-DS the heterogeneity of purified DNA-topoisomerase was established. This heterogeneity implies the presence of three active forms of the enzyme with Mr of 97 000, 81 000 and 69 000, respectively. Using one-dimensional fingerprint method and limited proteolysis with Staphylococcus aureus protease, it was demonstrated that the low molecular weight enzyme forms are products of limited proteolysis of the highest molecular weight form of DNA-topoisomerase.     

Episomal Viral DNA in a Herpesvirus saimiri-Transformed Lymphoid Cell Line

The lymphoid cell line #1670 has been derived from the infiltrated spleen of a tumor-bearing marmoset monkey infected with Herpesvirus saimiri. The cells contain both types of H. saimiri DNA, unique light (L-) DNA (36% cytosine plus guanine) and repetitive heavy (H-) DNA (71% cytosine plus guanine), without producing infectious virus. Viral DNA was found to persist in these cells as nonintegrated circular DNA molecules. Closed circular superhelical viral DNA molecules were isolated by three subsequent centrifugation steps: (i) isopycnic centrifugation in CsCl, (ii) sedimentation through glycerol gradients, and (iii) equilibrium centrifugation in CsCl-ethidium bromide. The isolated circles had a molecular weight of 131.5 +/- 3.6 x 10(6). This is significantly higher than the molecular weight of linear DNA molecules isolated from purified H. saimiri virions (about 100 x 10(6)). Partial denaturation mapping of circular molecules from #1670 lymphoid cells showed uniform arrangement of H- and L-DNA sequences in all circles. All denatured molecules contained two L-DNA regions (molecular weights of 54.0 +/- 1.8 x 10(6) and 31.5 +/- 1.3 x 10(6)) and two H-DNA regions (molecular weight of 25.6 +/- 1.9 x 10(6) and 20.0 +/- 0.8 x 10(6)) of constant length. Maps of both L-regions suggested that the sequences of the shorter L-DNA region were a subset of those of the longer region. The sequences of both L-regions had the same orientation. Circular molecules from H. saimiri-transformed lymphoid cell line #1670 appeared to represent defective genomes, containing only 75% of the genetic information present in L-DNA of H. saimiri virions.     

Characterization of a New Type of Human Papillomavirus That Causes Skin Warts

A human papillomavirus (HPV) was isolated from the lesions of a patient (ML) bearing numerous hand common warts. This virus was compared with the well-characterized HPV found in typical plantar warts (plantar HPV). ML and plantar HPV DNAs have similar molecular weights (5.26 x 10(6) and 5.23 x 10(6), respectively) but were shown to be different by restriction enzyme analysis. When the cleavage products of both DNAs by endonuclease EcoRI, BamI, HpaI, or Hind were analyzed by electron microscopy, one, two, one, and four fragments were detected for ML HPV DNA instead of the two, one, two, and six fragments, respectively, detected for plantar HPV DNA. In contrast to plantar HPV DNA, a high proportion of ML HPV DNA molecules were resistant to these restriction enzymes. Most, if not all, of the molecules were either resistant to BamI and sensitive to EcoRI or sensitive to BamI and resistant to EcoRI. After denaturation and renaturation of the cleavage products of ML HPV DNA by a mixture of the two enzymes, the circular "heteroduplexes" formed showed one to three heterology loops corresponding to about 4 to 8% of the genome length. No sequence homology was detected between ML and plantar HPV DNAs by cRNA-DNA filter hybridization, by measuring the reassociation kinetics of an iodinated plantar HPV DNA in the presence of a 25-fold excess of ML HPV DNA, or by the heteroduplex technique. The two viruses had distinct electrophoretic polypeptide patterns and showed no antigenic cross-reaction by immunodiffusion or immunofluorescence techniques. Preliminary cRNA-DNA hybridization experiments, using viral DNAs from single or pooled plantar or hand warts, suggest that hand common warts are associated with viruses similar or related to ML HPV. The existence of at least two distinct types of HPVs that cause skin warts was demonstrated; they were provisionally called HPV type 1 and HPV type 2, with plantar HPV and ML HPV as prototypical viruses, respectively.     

Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line.

A non-integrated form of Epstein-Barr virus DNA was purified from the Burkitt lymphoma-derived human lymphoid cell line Raji by CsCl density gradient centrifugation and neutral glycerol gradient centrifugation. This intracellular form of the virus DNA sediments at a rate typical of a covalently closed circular DNA molecule of the size of the virus genome in both neutral and alkaline solution. Treatment with low doses of X-rays leads to a discontinuous conversion of the molecules to a form with the sedimentation properties of open circular DNA (a circular duplex molecule containing one or more single-strand breaks). The direct observation of large circular DNA molecules by electron microscopy further confirms the covalently closed circular duplex structure of part of the intracellular viral DNA. Such circular molecules were not detected in corresponding DNA fractions from Epstein-Barr virus-negative human lymphoid cell lines. In ethidium bromide/CsCl density gradient centrifugation experiments, the purified non-integrated virus DNA behaves as twisted, covalently closed DNA circles with the same initial superhelix density as polyoma virus DNA. The latter additional purification technique permits the isolation of intracellular Epstein-Barr virus DNA in > 90% pure form from non-producer cells. The molecular weight of the circular virus DNA from Raji cells, determined by contour length measurements, is the same within experimental error as that of the linear DNA from virus particles.     

Variation in p30-related proteins in gross virus-induced tumor cell lines derived from H-2 congenic mice

The distribution of DNA topoisomers in intracellular simian virus 40 DNA was analyzed by gel electrophoresis. The results suggested that DNA extracted from 70S chromatin had a different superhelical density distribution as compared with the DNA obtained from virions or virion assembly intermediates. The heterogeneity of simian virus 40 viral DNA superhelical density at a late time after infection was partly due to increased virion production and partly due to the intrinsic heterogeneity of the superhelical density of DNA extracted from virions. Using two-dimensional gel electrophoretic analysis we also showed that simian virus 40 DNA templates used for DNA replication have a higher average superhelical density than the bulk of intracellular viral DNA.     

Comparison of the restriction endonuclease maps of unintegrated proviral DNAs from four xenotropic murine leukemia viruses.

From purified linear and superhelical DNAs, the restriction endonuclease maps of four xenotropic murine leukemia virus DNAs from NFS, NZB, BALB/c, and AKR mice were determined with ten restriction endonucleases. Each xenotropic proviral DNA was found to be a unique restriction endonuclease map, with differences in the gag, pol, env, and terminal repeated sequence regions. However, type-specific SacI and EcoRI sites in the env region were identical in all four xenotropic murine leukemia virus DNAs and were not found in ecotropic murine leukemia virus DNA. Comparison of the xenotropic murine leukemia virus DNA maps with maps of ecotropic murine leukemia virus DNA showed that the pol and terminal repeated sequence regions were highly conserved. Other similarities in ecotropic and some xenotropic viral DNAs suggest common origins.     

The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity

Systems for gel electrophoresis in the presence of one of the intercalative unwinding ligands, ethidium or chloroquine, have been developed which permit the resolution of highly supercoiled closed circular DNA molecules differing by unit values of the topological winding number, alpha. All native closed circular DNAs examined, including the viral and intracellular forms of SV40 and polyoma DNA, bacterial plasmid DNAs, and the double stranded closed circular DNA genome of the marine bacteriophage, PM2, are more heterogeneous with respect to the number of superhelical turns present than are the thermal distributions observed in the limit products of the action of nicking-closing (N-C) enzyme on the respective DNAs. In the cases of SV40 and polyoma, where it has been shown that the supercoiling is a combined consequence of the binding of the four nucleosomal histones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of the distributions within the form I DNAs poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution of 21. If it is assumed that all nucleosomes have identical structures, and that the DNA within a nucleosome is not free to rotate, the native DNA would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in N-C enzyme relaxed SV40 and polyoma DNAs.The absolute number of superhelical turns (at 37 degrees C in 0.2 M NaCl) in virion polyoma DNA has been determined to be 26 +/- 1, which is the same value obtained for virion SV40 DNA. This is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as SV40 DNA. In addition, the distributions within the virion and intracellular form I DNAs of both SV40 and polyoma were found to be indistinguishable.Images     

AMP-dependent DNA relaxation catalyzed by DNA ligase occurs by a nicking-closing mechanism.

In the presence of AMP and Mg2+, a covalently closed duplex DNA containing negative superhelical turns was treated with DNA ligase isolated from bacteriophage T4-infected E. coli. This resulted in the gradual and not sudden loss of superhelical turns as for example in the case of type I DNA topoisomerase. All DNA products remain covalently closed. Since T4 enzyme-mediated DNA relaxation is inhibited by both pyrophosphate and by ATP this suggests that DNA relaxing and DNA joining activities probably coincide. EDTA addition in the presence of a large excess of enzyme, induces the formation of nicked DNA products while protein denaturing treatments are not very effective. Our observations might suggest an involvement of the relaxing activity of DNA ligase during the ligation process.