Inhibitors acting on Nucleic Acid Synthesis in an Oncogenic RNA Virus

IN infection with an oncogenic RNA virus, synthesis of viral RNA seems to be catalysed by an RNA dependent DNA polymerase in the host cell^1–4. Several specific inhibitors of viral DNA polymerases have been found^5–7 and Spiegelman⁸ has shown that the activity of viral enzymes depends strongly on the chemical composition of the template. We report here first a new highly specific poison of the Rauscher murine leukaemia virus (RMLV) DNA polymerases; second, several inactivators of the RNA and DNA template involved in the RMLV enzyme systems; and third, the action of actinomycin D on viral DNA polymerases and on host DNA/RNA polymerase. The results are discussed with respect to the influence of actinomycin D on virus multiplication.     

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m⁵C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m⁵C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.     

Distribution of circular single‐stranded DNA viruses associated with benthic amphipods of genus Diporeia in the Laurentian Great Lakes

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Diel,seasonal, and depth-related variability of viruses and dissolved DNA in the northern Adriatic Sea

The depth-dependent, seasonal, and diel variability of virus numbers, dissolved DNA (D-DNA), and other microbial parameters was investigated in the northern Adriatic Sea. During periods of water stratification, we found higher virus abundances and virus/bacterium ratios (VBRs) as well as a larger variability of D-DNA concentrations at the thermocline, probably as a result of higher microbial biomass. At the two investigated stations, virus densities were highest in summer and autumn (up to 9.5 × 10¹⁰ 1^–1) and lowest in winter (< 10⁹ 1^–1); D-DNA concentrations were highest in summer and lowest in winter. The VBR as well as an estimated proportion of viral DNA on total D-DNA showed a strong seasonal variability. VBR averaged 15.0 (range, 0.9–89.1), and the percentage of viral DNA in total D-DNA averaged 18.3% (range, 0.1–96.1%). An estimation of the percentage of bacteria lysed by viruses, based on 2-h sample intervals in situ, ranged from 39.6 to 212.2% d^–1 in 5 m and from 19.9 to 157.2% d^–1 in 22 m. The estimated contribution of virus-mediated bacterial DNA release to the D-DNA pool ranged from 32.9 to 161% d^–1 in 5 m and from 10.3 to 74.2% d^–1 in 22 m. Multiple regression analysis and the diel dynamics of microbial parameters indicate that viral lysis occasionally could be more important in regulating bacterial abundances than grazing by heterotrophic nanoflagellates. Correspondence to: M.G. Weinbauer     

Separation of Epstein-Barr Virus DNA from Large Chromosomal DNA in Non-virus-producing Cells

AT least four established human lymphocyte cell lines, one that originates from a Burkitt's lymphoma and the others from normal persons, contain Epstein-Barr virus (EBV) genome¹. These cells show no viral antigens by immunofluorescence tests nor do they produce virus particles. We are examining one of the four cell lines, Raji (cells from a Burkitt's lymphoma), in more detail. The DNA isolated from purified Raji chromosomes contains as much virus genome as the DNA extracted from whole cells (65 genome equivalents per cell)¹. The viral DNA therefore seems to be in the chromosomes. This result, however, does not necessarily indicate that the viral DNA is physically integrated into chromosomal DNA. The following experiments suggest that the EBV DNA in Raji cells is not covalently linked to the large chromosomal DNA, although the number of viral genomes per cell remains constant during passage. The results do not, however, exclude the possibility that small fragments of cell DNA are bonded to the viral DNA. The data also indicate that EBV DNA in Raji cells exists in strands of complete or nearly complete size.     

Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.     

Spiroplasma citri Virus SpV1: Characterization of Viral Sequences Present in the Spiroplasmal Host Chromosome

Spiroplasma citri virus SpV1-R8A2B is a naked, rod-shaped virus with a circular, single-stranded DNA genome of 8273 bp. SpV1-related sequences were detected in the chromosomal DNA of all S. citri strains tested. Southern blot hybridization analyses revealed that several copies of most, if not all, SpV1 ORFs are present in the chromosome of S. citri strain R8A2. For further study of the integrated viral sequences, a genomic DNA library of S. citri R8A2 was constructed, and two cloned chromosomal DNA fragments containing SpV1 viral sequences were studied by comparison with the free viral genome of SpV1-R8A2B. One fragment seems to contain a full-length viral genome, while the other contains only parts of the viral genome. In both fragments, the left and right ends of viral sequences consist of 31-bp inverted repeat sequences, those which are facing each other at nucleotide 4737 in the circular viral genome. In addition, both fragments contain the SpV1-ORF3, encoding a putative transposase, immediately upstream of the right repeat. These data suggest that the SpV1-ORF3 and the repeat sequences could be parts of an IS-like element of chromosomal origin.     

Fate of Unintegrated Viral DNA in Fv-1 Permissive and Resistant Mouse Cells Infected with Murine Leukemia Virus

We have found that levels of unintegrated linear viral DNA were nearly identical in several Fv-1 resistant cell lines, whereas levels of closed circular viral DNA are markedly reduced in these resistant cells, to the same extent as virus production (P. Jolicoeur and E. Rassart, J. Virol. 33:183-195, 1980). To determine the fate of linear viral DNA made in resistant cells we performed pulse-chase experiments, labeling viral DNA with 5-bromodeoxyuridine and following it with a thymidine chase. 5-Bromodeoxyuridine-labeled viral DNA (HH) recovered by banding on cesium chloride gradients was sedimented on neutral sucrose density gradients or separated by the agarose gel-DNA transfer procedure and detected by hybridization with complementary DNA. Levels of linear viral DNA made in Fv-1^b/b (JLS-V9 and SIM.R) and Fv-1^n/n (NIH/3T3 and SIM) cells were found to decrease during the chase period at about the same rate in permissive and nonpermissive conditions, indicating that linear viral DNA is not specifically degraded in Fv-1 resistant cells. Levels of the two species of closed circular viral DNA made in Fv-1 permissive cells increased relative to the levels of linear DNA during the chase period. This confirmed the precursor-product relationship between linear DNA and the two species of circular DNA. In Fv-1 resistant cells, this apparent conversion of linear viral DNA into circular forms was not seen, and no supercoiled viral DNA could be detected. To determine whether the transport of linear viral DNA from the cytoplasm into the nucleus was prevented by the Fv-1 gene product, SIM.R cells were fractionated into cytoplasmic and nuclear fractions, and viral DNA was detected in each fraction by the agarose gel-DNA transfer procedure. Levels of linear viral DNA were nearly identical in both cytoplasmic and nuclear fractions of permissive or resistant cells. Circular viral DNA could be detected in the nuclear fraction of permissive cells, but not in that of resistant cells. A pulse-chase experiment was also performed with SIM.R cells. During the thymidine chase period, linear viral DNA was seen to accumulate in nuclei of both permissive and resistant cells, whereas supercoiled viral DNA accumulated only in nuclei of permissive cells. These results indicate that the Fv-1 gene product does not interfere with the transport of linear viral DNA into the nucleus. Our data also suggest that the Fv-1 restriction does not operate through a degradation process. Therefore, the Fv-1 gene product could either block the circularization of linear viral DNA directly or promote the synthesis of a faulty linear viral DNA whose defect (yet undetected) would prevent its circularization.     

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

Co-dependence of HTLV-1 p12 and p8 Functions in Virus Persistence

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4⁺ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.     

ISOLATION AND CHARACTERIZATION OF A DNA VIRUS INFECTING FELDMANNIA SIMPLEX (PHAEOPHYCEAE)1

We report on the isolation and characterization of a virus that is formed in modified zoidangia of the marine brown alga Feldmannia simplex (Crouan) Hamel (Ectocarpales, Phaeophyceae). Isolated virus particles had a buoyant density of about 1.35 g·mL^?1 in CsCl equilibrium gradients. They contained one major polypeptide (MW = 55,000) and at least six additional polypeptides (MW = 15,000–120,000). Four of these proteins were glycosylated. The viral genome consisted of double-stranded DNA and formed two freely migrating fractions in pulsed-field-gel electrophoresis, namely linear DNA with a size of 220 kilobase pairs, and fragments of 10–60 kilobase pairs. However, electron microscopic examination revealed that a substantial fraction of the viral DNA occurred as closed circles. We suggest that the viral DNA in native particles is circular but tends to break at random sites during the preparation.     

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP⁺) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP⁺ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP⁺ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.     

Effects of salts on internal DNA pressure and mechanical properties of phage capsids

Based on atomic force microscopy nanoindentation measurements of phage λ, we previously proposed a minimal model describing the effect of water hydrating DNA that strengthens viral capsids against external deformation at wild-type DNA packing density. Here, we report proof of this model by testing the prediction that DNA hydration forces can be dramatically decreased by addition of multivalent ions (Mg²⁺ and Sp⁴⁺). These results are explained using a DNA hydration model without adjustable parameters. The model also predicts the stiffness of other DNA-filled capsids, which we confirm using bacteriophage ?29 and herpes simplex virus type 1 particles.     

Restricted infectivity of ecotropic type C retroviruses in mouse teratocarcinoma cells: Studies on viral DNA intermediates

Replication of Gross strain N-tropic type C retrovirus was markedly restricted in a pluripotential undifferentiated embryonal cell line (PCC₄) of murine teratocarcinoma, whereas the same virus could cause productive infection in a myoblast-derived differentiated line (PCD₁) of the same tumor origin. To investigate the restriction mechanism, we compared the initial viral DNA formation in these two cell lines. Analyses by means of a modified Hirt extraction procedure and a modified Southern gel transfer method indicated that PCC₄ and PCD₁ cells supported the synthesis of viral DNA intermediates after inoculation of the Gross virus. In both cells, a linear DNA duplex (form III viral DNA) appeared at 4 hr, reached a maximal level at 8–9 hr, and declined rapidly thereafter, while two closed-circular supercoiled DNA duplexes (form I viral DNA) showed their appearance, increase and decline in the 8–24 hr period. During the period from 34 to 78 hr after virus inoculation, another burst of viral DNA synthesis occurred in PCD₁ cells, presumably due to secondary virus infection, while at this period both form III and form I viral DNAs became undetectable in PCC₄ cells. The Hirt supernatant DNAs prepared from PCD₁ and PCC₄ cells 10 hr after virus inoculation were equally infectious for NIH3T3 cells in a DNA transfection assay. Both PCD₁ and PCC₄ cells were very poor recipients for DNA transfection, although one positive result with PCD₁ cells might suggest a difference between the two cell types in this aspect. These results indicate that restriction of type C retrovirus in undifferentated embryonl carcinoma cells occurs at a step subsequent to formation and maturation of viral DNA intermediates.     

RNA N6-methyladenosine modification suppresses replication of rice black streaked dwarf virus and is associated with virus persistence in its insect vector

N⁶ methylation of adenosine (m⁶A) was recently discovered to play a role in regulating the life cycle of various viruses by modifying viral and host RNAs. However, different studies on m⁶A effects on the same or different viruses have revealed contradictory roles for m⁶A in the viral life cycle. In this study, we sought to define the role of m⁶A on infection by rice black streaked dwarf virus (RBSDV), a double-stranded RNA virus, of its vector small brown planthopper (SBPH). Infection by RBSDV decreased the level of m⁶A in midgut cells of SBPHs. We then cloned two genes (LsMETTL3 and LsMETTL14) that encode m⁶A RNA methyltransferase in SBPHs. After interference with expression of the two genes, the titre of RBSDV in the midgut cells of SBPHs increased significantly, suggesting that m⁶A levels were negatively correlated with virus replication. More importantly, our results revealed that m⁶A modification might be the epigenetic mechanism that regulates RBSDV replication in its insect vector and maintains a certain virus threshold required for persistent transmission.     

New archaeal viruses discovered by metagenomic analysis of viral communities in enrichment cultures

Viruses infecting hyperthermophilic archaea of the phylum Crenarchaeota display enormous morphological and genetic diversity, and are classified into 12 families. Eight of these families include only one or two species, indicating sparse sampling of the crenarchaeal virus diversity. In an attempt to expand the crenarchaeal virome, we explored virus diversity in the acidic, hot spring Umi Jigoku in Beppu, Japan. Environmental samples were used to establish enrichment cultures under conditions favouring virus replication. The host diversity in the enrichment cultures was restricted to members of the order Sulfolobales. Metagenomic sequencing of the viral communities yielded seven complete or near-complete double-stranded DNA virus genomes. Six of these genomes could be attributed to polyhedral and filamentous viruses that were observed by electron microscopy in the enrichment cultures. Two icosahedral viruses represented species in the family Portogloboviridae. Among the filamentous viruses, two were identified as new species in the families Rudiviridae and Lipothrixviridae, whereas two other formed a group seemingly distinct from the known virus genera. No particle morphotype could be unequivocally assigned to the seventh viral genome, which apparently represents a new virus type. Our results suggest that filamentous viruses are globally distributed and are prevalent virus types in extreme geothermal environments.