Isolation and characterization of entomopox virions from virus-containing inclusions of Amsacta moorei (Lepidoptera: Arctiidae)

Preparations of Amsacta moorei entomopox virions were obtained from virus-containing inclusions (VCI) by using a carbonate-thioglycolate solution (pH 10.7–11.5). The virions possessed a uniform coat (“halo”) surrounding the viral envelope and exhibited an RNA polymerase activity. The “halo” could be removed by prolonged exposure to the carbonate-thioglycolate solution. Virions obtained by this treatment, however, possessed low infectivity and no detectable RNA polymerase activity. Removal of the “halo” by trypsin resulted in virions which possessed RNA polymerase activity and relatively high infectivity.Preparations of particles with and without the “halo” were similar in percent DNA, protein per OD₂₆₀, number of particles per OD₂₆₀, and RNA polymerase activity. Particles without the “halo,” however, were less dense (1.262 g/cm³) in CsCl than those with the “halo” (1.282 g/cm³) and 15–45 times more infective.Parallel studies of “nonhaloed” Amsacta virions (trypsin-treated) and vaccinia virions showed that both viruses contained similar amounts of protein per OD₂₆₀, but Amsacta virions contained only 36% of the DNA found in vaccinia.     

A comparison of the DNA of the "R" and "T" strains of mosquito iridescent virus.

The buoyant density (1.7135 g/cm³), percentage GC (53.9%), and melting temperature (T_m = 76.4°) of the DNA of “regular” mosquito iridescent virus (RMIV) and of “turquoise” mosquito iridescent virus (TMIV) are similar although the molecular weights of the two DNAs are different; 243.3 × 10⁶ and 286.7 × 10⁶, respectively. Analyses show that RMIV contains two identical duplex DNA molecules which are about 15% smaller than the single duplex DNA molecule of TMIV. Reassociation studies show that RMIV and TMIV contain about 17 and 30% repetitious DNA, respectively. Homology studies show the two DNAs to be 100% homologous in their DNA nucleotide sequences. We conclude that the DNA of RMIV and TMIV contain identical sequences and that the portion of the TMIV-DNA molecule accounting for the higher molecular weight is composed of repeated sequences common to both strains.     

Relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA)

The relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) has been investigated by immunofluorescence (cocapping) and radiolabeling. In neuraminidase-treated and untreated thymocytes there are two groups of glycoproteins which bind roughly equivalent amounts of PNA. One group also carries all the detectable receptors for HPA, the other binds only PNA. Binding inhibition experiments suggest that PNA and HPA receptors are in close proximity on the shared glycoproteins. The same two groups of receptors are present on 35–10% of neuraminidase-treated spleen lymphoid cells, mainly immunoglobulin (Ig)-negative lymphocytes. Almost all B cells have only PNA-specific receptors. Five–12% of the untreated spleen cells appreciably bind PNA and only a few bind HPA. Solubilized glycoproteins specific for PNA or HPA were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major PNA-specific radioiodinated glycoproteins of neuraminidase-treated thymocytes, as isolated by affinity chromatography, consist of the 185-kDa and 195-kDa components of the T200 antigen and of two (diffuse) components of about 140 and 120–125 kDa. All these molecules also bind to HPA-Sepharose, with the exception of the 185 kDa component, which is probably the main constituent of the “pure” PNA receptors on the intact thymocytes. In gels directly labeled with radioactive lectins, the only band strongly labeled by PNA and HPA is the diffuse 140-kDa band. The band at 120 kDa is well labeled by PNA, but all the other components are weakly labeled. The mobility of the 140- and 120-kDa bands depends strongly on neuraminidase-treatment. These bands cannot be detected in gels of untreated thymocytes, but a major HPA-and PNA-specific band of lower molecular weight can be labeled after treating the gels with neuraminidase. The factors determining the differences in labeling pattern obtained by different methods as well as the nature of PNA and HPA binding sites are discussed. The same major PNA- and HPA-binding glycoproteins (apart from minor differences) are present on neuraminidase-treated Ig-negative spleen lymphocytes. The major PNA-binding protein of B lymphocytes appears to correspond to the 225-kDa (“B220”) antigen specific for these cells.     

Ribonucleotides in newly synthesized DNA of herpes simplex virus

Newly synthesized DNA of herpes simplex virus type 1 (HSV-1), obtained from primary rabbit kidney cells pulse-labeled with [³H]thymidine or [³H]uridine at 6 hr postinfection, was purified by two cycles of centrifugation in CsCl density gradients. These intracellular viral DNA preparations hybridized specifically with homologous HSV-1 DNA but not with host cell DNA or E. coli DNA. Upon denaturation by alkali, the [³H]thymidine-labeled HSV-1 DNA cleaved to smaller pieces. The alkali-labile material in the viral DNA was identified as ribonucleotides on the basis of the following observations: (1) When labeled with [³H]uridine for short periods, the labeled viral “DNA” was susceptible to RNase and NaOH, and all the radioactivity was confined to the nucleoside [³H]uridine; however, upon longer labeling periods (up to 20 hr), the [³H]uridine-labeled viral “DNA” became more susceptible to DNase, as most but not all of the [³H]uridine was converted to deoxyribonucleosides. (2) Denaturation of [³H]uridine-labeled double-stranded HSV-1 “DNA” (?_Cs2SO4 = 1.45 g/cm³) by heat shifted the buoyant density to single-stranded DNA region (?_Cs2SO4 = 1.48?1.50 g/cm³) but not to single-stranded RNA region; however, treatment with hot NaOH considerably reduced the radioactivity of this “DNA.” Treatment with DNase, but not with pronase, shifted the buoyant density to the heavier RNA region of the gradient. Heat-denatured DNA but not the native DNA was susceptible to single-strand specific nuclease S1.     

Identification of single-stranded DNA fragments of bacteriophage T5

Five fractions containing single-stranded fragments of bacteriophage T5 DNA were isolated by preparative agarose gel electrophoresis (bands I through V). The composition of these fractions and the arrangement of the fragments in the double-stranded T5 DNA molecule were determined, using DNA-DNA and DNA-RNA hybridization techniques and microscopic visualization of specific DNA-DNA reassociation products. The data confirm the previously proposed overall structure of T5 DNA and suggest the frequent occurrence of a further strand interruption in the pre-early section of the DNA. It was also found that only 20% of the molecules in band II are fragment IIb and more than 90% of the molecules in band V are fragment Va [fragments defined by Hayward, G. S., and Smith, M. G., J. Mol. Biol.63, 383–395, 397–407 (1972); Bujard, H., and Hendrickson, H. W., Europ. J. Biochem.33, 517–528 (1973)].     

Partial characterization of herpes simplex virus type 2 (HSV-2)-specific poly(A)+ RNA by hybridization to EcoRI-generated HSV-2 DNA fragments.

Polyadenylated RNA appearing in polyribosomes from Herpes simplex virus type 2 (HSV-2)-infected cells has been isolated at different stages of infection. The amounts of virus-specific RNA hybridizing to individual EcoRI fragments of HSV-2 DNA have been detected. Templates coding for “early” virus-specific RNA are widely dispersed within the HSV-2 genome in the short and long segment since each of the EcoRI fragments showed hybridization with “early” isolated poly(A)+ RNA. The virus-specific fraction of labeled poly(A)+ RNA which hybridized to each of the EcoRI fragments increased with 1-β-d-arabinofuranosylcytosine-treated (Ara-C) poly(A)+ RNA as well as with “late” poly(A)+ RNA. Poly(A)+ RNA from infected, Ara-C-treated cells contained virus-specific RNA which hybridized to each of the EcoRI fragments. We found that the poly(A)+ RNA, specific for one EcoRI fragment, varied with the stage of infection, comparing it to other fragment-specific poly(A)+ RNAs.     

Analysis by restriction enzyme cleavage of human varicella-zoster virus DNAs.

The DNAs from three varicella virus isolates and two herpes zoster virus isolates were digested with either Eco R_I or Hin D III site-specific endonucleases. The restriction patterns of the DNAs obtained from the five isolates were then compared following separation of the specific fragments by electrophoresis through 0.5% agarose gels. The number and mobilities of all DNA bands were found to be indistinguishable for all five isolates. Further analysis of Eco R_I-digested varicella-zoster virus DNA revealed the presence of both molar and submolar DNA fragments as found previously for other human herpesvirus DNAs.     

Structure of the cauliflower mosaic virus genome. II. Variation in DNA structure and sequence between isolates

The DNA genomes of four different cauliflower mosaic virus (CaMV) isolates (Cabbage B-Davis, Cabbage B-John Innes, CM4–184, and Australian) have been mapped using the restriction endonucleases, EcoRI, SalGI and BamHI, and S₁ nuclease. The differences between maps of the isolates indicate that one region of the CaMV genome is subject to considerable change while the rest is more highly conserved. EcoRI digestion patterns of the DNA from five other CaMV isolates correspond to two of the described isolates, Cabbage B-Davis and Cabbage B-John Innes. Minor DNA fragments (in less than molar amounts) appearing in restriction patterns of CaMV DNAs could be attributed to: (i) the presence of linear DNA molecules which appear to arise by double-strand breaks at specific points in the circular genome, (ii) heterogeneity within some of the viral ioslates, indicated by the appearance of a faint, underlying DNA restriction pattern characteristic of another isolate type, and (iii) the occurrence of a few “difficult” restriction sites in the genomes of some isolates.     

The base composition of entomopoxvirus DNA

Equilibrium centrifugation of entomopoxvirus DNAs from Lepidoptera (Amsacta moorei, Euxoa auxiliaris), Coleoptera (Othnonius batesi), and Diptera [Chironomus near decorus ( = attenuatus)], ans vertebrate proxvirus DNA (vaccinia, IHD-D) in cesium chloride gradients gave buoyant density values of 1.690 g/ml for vaccinia DNA and 1.676 to 1.681 g/ml for the entomopoxvirus DNAs. The molar fraction of guanine and cytosine (G+C) calculated from the buoyant density of entomopoxvirus DNA was 30 to 48% lower than the G+C values for vaccinia DNA. Thermal denaturation studies (T_m) of entomopoxvirus DNA confirmed the low G+C values and established that these DNAs were double-stranded in vitro. DNA base ratios calculated on the basis of buoyant density and T_m indicate that there are no abnormal bases present in the entomopoxvirus genome.     

Biogenesis of vaccinia: specific inhibition of rapidly labeled host DNA in vaccinia inoculated cells.

Because separation of the bulk and nascent (i.e., rapidly labeled) DNA fractions of HeLa cells could be effected satisfactorily it became possible to ascertain the effects of inoculating uv-irradiated vaccinia virus on in vivo host DNA synthesis. In both control and infected cells, labeled 4s fragments, formed during 1-min pulses with [³H]Tdr, existed as single- or double-stranded molecules covalently linked to short stretches of RNA. During the chase period, most of the rapidly labeled DNA within uninfected cells became associated with the bulk DNA component but failed to do so within inoculated cells. Host DNA ligase activity was unaffected by the infection, and there was a rapid breakdown of the 4s DNA fragments, leading to the conclusion that arrest of HeLa nuclear DNA synthesis by vaccinia is, most probably, due to the in vivo hydrolysis of nascent DNA. The present data further substantiate previous studies from this laboratory demonstrating the hydrolysis of in vitro synthesized nascent DNA by a DNAse activity originating from the virus core.     

In situ detection of DNA-binding proteins in herpes simplex virus type 1-infected cells

An in situ assay for detecting DNA-binding proteins in herpes simplex virus type 1 (HSV-1)-infected cells is described. Seventeen HSV-induced DNA-binding species were visible with nicked, double-stranded DNA as a substrate, while fourteen virus-induced DNA-binding fractions were present in gels containing nuclease-treated, single-stranded DNA. The effects of HSV on cellular DNA-binding protein expression could also be seen. The resolution of DNA-binding fractions was dependent upon the type of DNA substrate utilized, high salt extraction of DNA-binding components and their physical separation from infected cell DNAs, dialysis of the high salt and the length of DNase treatment of gels following electrophoresis.     

Nonradioactive, photobiotin-labelled DNA probes for routine diagnosis of viroids in plant extracts

Avocado sunblotch viroid (ASBV), coconut cadang cadang viroid (CCCV), chrysanthemum stunt viroid (CSV) and potato spindle tuber viroid (PSTV) were detected in plant extracts by dot-blot hybridization using nonradioactive photobiotin-labelled nucleic acid probes. Recombinant DNA probes, containing full-length monomer viroid inserts in the plasmid vectors pSP64 or pUC9, were biotinylated with photobiotin and used as sonicated double-stranded DNA fragments. Using fresh leaf material, a general method (suitably modified for avocado tissue) was developed for the rapid preparation of purified nucleic acid extracts. Plant extracts from a range of field samples were spotted onto nitrocellulose, subjected to hybridization and the biotin-labelled DNA bound to the target nucleic acid was detected with an avidin-alkaline phosphatase conjugate. Under the stated hybridization and washing conditions, each individual viroid probe was specific. Each viroid was readily detected with a sensitivity similar to that obtained with the same (or a like) probe labelled with 32P. Healthy plant extracts gave colourless spots.     

Evidence for TYMV-induced RNA and DNA synthesis in the nuclear fraction from infected Chinese cabbage leaves

Radioactively labeled nucleic acids prepared from the nuclear fraction from Chinese cabbage leaf tissue infected with turnip yellow mosaic virus (TYMV) contained two components not present in equivalent material from healthy leaves. One of these components had the properties expected for a virus-specific double-stranded RNA; the other contained DNA, with properties expected for DNA-RNA hybrids. In leaf tissue supplied with ³H-thymidine this DNA reached maximal labeling much more rapidly than the rest of the DNA.     

Complete genome sequence of enterobacteria phage 4MG,a new member of the subgroup “PVP-SE1-like phage” of the “rV5-like viruses”

A novel virulent enterobacteria phage, 4MG, which was isolated from soil near a sewer, belongs to the family Myoviridae, as it possesses an isometric head and a long contractile tail. The complete genome of 4MG consists of a double-stranded DNA with a length of 148,567 bp, a G + C content of 46.3 %, 271 open reading frames (ORFs), and 21 tRNAs. Bioinformatic analysis revealed that 4MG highly resembles “rV5-like viruses” but can be separated, together with Salmonella phage PVP-SE1 and Cronobacter sakazakii phage vB_CsaM_GAP31, as part of the subgroup “PVP-SE1-like phage”.     

Autographa californicaNuclear Polyhedrosis Virusp143Gene Product Is a DNA-Binding Protein

We have identified the protein product of theAutographa californicanuclear polyhedrosis virus (AcMNPV)p143gene by constructing a recombinant baculovirus overexpressing the gene product P143. The overexpressed protein exhibited a relative mobility of approximately 140 kDa and was stable for at least 12 hr after synthesis. Immunoblotting using a monoclonal antibody developed against the overexpressed protein identified a similar polypeptide in AcMNPV-infected cells which was detectable by 4 hr postinfection. P143 was present within infected cell nuclei at relatively constant amounts until at least 72 hr after infection, suggesting that P143 may perform other functions at late times after infection. P143, purified from infected cell nuclei by chromatography over hydroxylapatite and DNA cellulose, bound in a sequence-independent fashion to double-stranded but not to single-stranded DNA to form a ladder of retarded protein–DNA complexes. Together, these data are consistent with the essential role of P143 for viral DNA replication and suggest that P143 may function by direct binding to DNA.     

Thirty-one human adenovirus serotypes (Ad1-Ad31) form five groups (A-E) based upon DNA genome homologies.

The DNA homology relationships among 31 human adenovirus serotypes (Ad1–Ad31) were investigated by liquid-phase molecular hybridization, using in vitro labeled viral DNA as probe. Hybridizations were carried to 40 times the $\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and were assayed by batchwise chromatography on hydroxylapatite (HAP), and in some experiments by use of the more stringent S1 nuclease procedure. Five distinct DNA homology groups, A to E, were identified. DNAs of group A Ads (Ad12, 18, and 31) hybridized 48 to 69% with each other and 8 to 20% with DNAs of other serotypes (HAP). DNAs of group B Ads (Ad3, 7, 11, 14, 16, and 21) hybridized 89 to 94% (HAP; 81 to 89% by S1 nuclease) with each other and 9 to 20% (HAP; 8 to 15% by S1 nuclease) with DNAs of other types. DNAs of group C Ads (Ad1, 2, 5, and 6) hybridized 99 to 100% with each other and 10 to 16% with DNAs of other types (HAP). DNAs of group D Ads (Ad8–10, 13, 15, 17, 19, 20, and 22–30) hybridized 95 to 99% (HAP; 88 to 98% by S1 nuclease) with each other and 4 to 17% with DNAs of other types. Ad4 DNA hybridized to 4 to 23% (HAP; 3 to 22% by S1 nuclease) with DNAs of other types, and thus Ad4 is the only member of group E. Members within all groups except group A were closely related. Members within group A showed considerable heterology, and six isolates, classified as Ad12 by neutralization tests, were much more related to Ad31 than to Ad12 prototype Huie strain. These DNA homology groupings are consistent in the main with the properties of other “groupings” of human Ads, e.g., oncogenic groups (tumorigenicity in newborn hamsters), T-antigen groups, G + C content of viral DNA, hemagglutination groups, molecular characteristics of subviral particles and virion proteins (e.g., length of fiber), and human epidemiology and pathogenicity.     

The localization of virus-specific double-stranded RNA of cowpea mosaic virus in subcellular fractions of infected Vigna leaves

The fraction of a homogenate of cowpea mosaic virus (CPMV) infected leaves sedimenting at 1000g for 15 min was further divided by centrifugation on a discontinuous gradient consisting of layers of 60, 45, and 20% sucrose. It was possible to separate the nuclei and nuclear fragments from most of the chloroplasts.Hybridization with [³H]uridine labeled CPMV RNA and electron microscopy were used to detect CPMV-specific double-stranded RNA in different fractions of the gradient. By hybridization 70–90% of the double-stranded RNA was found in the so called chloroplast-fraction. By electron microscopy it was confirmed that double-stranded RNAs with lengths characteristic for double-stranded viral RNAs occurred predominantly in this chloroplast-fraction. The chloroplast-fraction also contained vesiculated membrane structures and amorphous electron-dense material, which are characteristic structures from CPMV-infected cells, suggesting that CPMV-specific double-stranded RNA might be associated with either or both of these structures rather than with the chloroplasts.     

The clinical importance of the nomenclature,evolution and taxonomy of human papillomaviruses

Human papillomaviruses (HPVs) are formally described by isolation of their circular double-stranded DNA genomes and establishment and comparison of the nucleotide sequence of these genomes. Alternatives such as serological diagnosis and maintenance of HPVs in culture are neither clinically useful nor consistently feasible. Novel HPV isolates have traditionally been described as “types”. The analysis of specific HPV types is of medical importance, because HPV types typically induce type-specific lesions, i.e. they may be specific for cutaneous or mucosal epithelia, or give rise to benign warts or malignant carcinomas. Recently, it was formally decided that papillomaviruses are a virus family separate from the polyomaviruses. Within the papillomavirus family, closely or remotely related types form species or genera. These formal agreements were important as they brought the taxonomy of papillomaviruses in line with that of other viruses, bacteria and higher organisms, although their impact on medical practice and terminology used in clinical studies is limited. Notably, however, HPV types that are closely related (i.e. form “species”) are associated with similar lesions. Confusion of the terms “type” and “subtype” should be avoided, as the latter term refers to some specific but rare taxonomic assemblages. In contrast to many RNA viruses, HPV types evolve very slowly, and diverged since the origin of humans only by about 2%. These divergent isolates are called “variants”. HPVs evolved together with humankind and Homo sapiens was never without HPVs, and consequently never without warts and cervical cancer. Variants of the same HPV type may have different pathogenicity and may account for part of the worldwide disparities in the occurrence of genital cancers.     

Pulsed-field gel electrophoretic analysis of Schizophyllum commune chromosomal DNA

Summary Six chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35–36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed.