RNA from unique and repetitive DNA sequences ofHerpesvirus saimiri

Herpesvirus saimiri DNA contains repetitive DNA at its termini that comprises about 30% of the virion DNA. After labeling late in infection, cytoplasmic RNA derived from these repetitive sequences was found to be below our limits of detection. Less than 1%, if any, of the viral-specific cytoplasmic poly(A)-containing RNAs contain sequences hybridizable to the isolated repetitive DNA.     

Inhibition by acyclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information

Thymidine kinase-deficient LM cells (LMTK^?-) biochemically transformed to the TK⁺ phenotype with herpes simplex virus genetic information showed an increased uptake of and ability to phosphorylate the acyclic nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine, acyclo-Guo). In growth inhibition studies the TK⁺ transformants were much more sensitive to inhibition with acyclovir than the untransformed cells (13- to 90-fold more sensitive). The synthesis of DNA in the transformed cells was significantly reduced by acyclovir treatment, whereas acyclovir had little effect on the DNA synthesis of the untransformed cells. Alkaline sucrose gradient sedimentation analysis of cellular DNA synthesized in the presence of acyclovir showed that, in contrast to untreated untransformed cells, the DNA newly synthesized by transformed cells was considerably smaller in size. In pulse-chase experiments the small fragments of DNA synthesized in the presence of acyclo-Guo were not chased to high molecular weight DNA. Finally, acyclo-Guo was shown to be incorporated terminally at 3′-ends of growing DNA chains in replicating cells.     

The integrated SV40 genome in permissive transformed monkey cells

The organization of the viral DNA sequences in three clonal lines of SV40-transformed permissive African Green monkey kidney cells was investigated by digesting the cellular DNA with restriction endonucleases and hybridizing the specific DNA fragments with labeled SV40 DNA. The genome of each transformed line contained a single insert of viral sequences located at a position which differed from line to line. No evidence for extrachromosomal SV40 DNA was obtained. Restriction mapping of the inserted SV40 DNA indicated that integration occurred without gross tandem duplication and that an intact early region was retained. Alterations were observed in the late region of the SV40 inserts. Although these alterations were not identical in the three transformed clonal populations, the HaeII restriction endonuclease recognition site at 0.83 SV40 map unit was absent in two of the three lines.     

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.     

Identification of a 39,000-dalton protein in cells transformed by the FBJ murine osteosarcoma virus

Two components of the FBJ murine osteosarcoma virus complex have been isolated separately in tissue culture; the FBJ murine leukemia virus (FBJ-MLV) by dilution and the FBJ murine sarcoma virus (FBJ-MSV) by the establishment of nonproducer transformed rat cells. Analysis of these cells using MLV antisera indicated that there were no new proteins related to viral structural proteins specifically associated with the presence of the FBJ-MSV genome. The FBJ-MSV nonproducer cells were used to induce tumors in syngeneic and allogeneic F₁ rats. Sera from tumor-bearing rats were examined for activity against FBJ-MSV-specific antigens. A number of sera were found to precipitate a 39,000-dalton protein, p39, from several producer and nonproducer FBJ-MSV transformed rodent cells, but not from cells transformed by other strains of MSV or cells infected with MLV. Precipitation of p39 was not blocked by the presence of excess viral proteins, indicating that p39 is not related to the viral structural proteins. This conclusion was confirmed by methionine tryptic peptide analysis which showed that the fingerprint of p39 was distinct from those of the viral gag or env gene proteins. The data demonstrate the presence of a unique antigenic protein, unrelated to the MLV proteins, in FBJ-MSV transformed cells.     

Adenovirus type 12 T antigen-related surface antigen detected by immunofluorescence on the membrane of transformed and infected cells

THA cells, derived from a hamster tumor induced by adenovirus type 12 (Ad12), were analyzed by immunofluorescence for the presence of virus-specific surface antigens. S antigen is a surface antigen detected on suspended living cells by a serum collected from hamsters immunized with Ad12. Besides S antigen, a new surface antigen (surface-T) was observed in formaldehyde-fixed cells treated with anti-T serum. Anti-T serum is not reactive against S antigen in living cells and anti-S serum did not detect surface-T antigen. The membrane location of surface-T antigen was confirmed with a Staphylococcus aureus binding assay. Surface-T antigen seems specific for Ad12, as it is not observed in hamster embryo cells, and is not recognized by antisera against SV40- or polyoma-T antigens nor by antiserum against hamster embryonic antigens. Surface-T antigen is also seen in infected KB cells but not in abortively infected RK13 cells. Anti-P serum, raised against an extract of infected RK13 cells, failed to detect surface-T antigen. Experiments with adsorbed anti-T and anti-S sera confirmed the specificity of surface-T antigen and its distinctiveness from S antigen.     

Purification and characterization of deoxythymidine kinase (dTK) induced in dTK- 3T3 mouse cells by equine herpesvirus type 1 (EHV-1).

Infection of mouse 3T3 cells deficient in cytosol deoxythymidine kinase (dTK^?) with the Kentucky A strain of EHV-1 resulted in a 20- to 30-fold increase in cytosol dTK activity. The EHV-1-induced dTK was partially purified from such cells by affinity chromatography on deoxythymidine (dT)-Sepharose and characterized by electrophoretic, enzymatic, and immunological criteria. The purified EHV-1 dTK migrated in polyacrylamide gels with an R_f of 0.25 and sedimented in glycerol gradients with an S value of 5.2, corresponding to a molecular weight of 85,000. Deoxycytidine could not serve as an alternative substrate for the viral-induced enzyme. A rabbit antiserum prepared against EHV-1-infected horse cells neutralized the viral-induced dTK activity purified from infected mouse (dTK^? 3T3) cells, but not the kinase activities from uninfected 3T3 cells. EHV-1 dTK differed from 3T3 cytosol dTK also in the greater resistance of the viral enzyme to feedback inhibition by dTTP and dCTP, its inhibition by arabinosylthymine, and its ability to utilize a variety of nucleoside triphosphates as phosphate donors. The purified EHV-1 kinase could be distinguished from 3T3 mitochondrial dTK by electrophoretic mobility and inhibition by anti-(EHV-1) serum. These results lend further support to the hypothesis that the dTK induced by EHV-1 is coded by the virus genome.     

State of viral DNA in BK virus-transformed rabbit cells

Semipermissive rabbit liver, brain, and kidney cells were transformed by BK virus (BKV). All cells of the three resulting cell lines produced BKV T antigen. Viral DNA was detected by DNA-DNA reassociation kinetics and by blot-transfer hybridization. Hybridization patterns were different for the three lines, indicating a different mode of integration for each line. In addition to integrated viral DNA, two of the lines contained also free viral DNA sequences, which in one case were defective viral genomes. Absence or splitting of particular regions of the viral genome was not a necessary condition for the maintenance of the transformed state. HindIII-generated segment A, which contains all the sequences coding for the late viral proteins, was absent (in an intact form) in the only line from which virus could not be rescued.     

RNA-nascent DNA complexes in Ehrlich ascites tumor cells in vivo

When mice bearing Ehrlich ascites tumors were given a single intraperitoneal injection of [5-³H]uridine, the ascites cells incroporated radioactivity into DNA for 4 hr, while incorporation into total cellular RNA ceased after 20–30 min. Nascent DNA, isolated by Cs₂SO₄ isopycnic centrifugation 30 min, 4 hr or 50 hr after [5-³H]uridine injection contained RNA fragments. Treatments with hydroxyurea, an inhibiton hibitor of DNA synthesis, reduced the incorporation of [5-³H]uridine into the DNA-RNA complex to 43% of controls. Treatment with actinomycin D, an inhibitor of DNA-dependent RNA synthesis, abolished the incorporation of [5-³H]uridine into the DNA-RNA complex. Finally, when DNA was pulse-labeled with [¹⁴C]deoxythymidine and then denatured by heat-treatment, it banded in a region with a higher density than that of reference DNA. However, if treated with alkali, it banded at the density of reference DNA, again suggesting the presence of RNA fragments in nascent DNA. The length of the RNA chains in this DNA-RNA complex was estimated to be in the order of 70 nucleotides.     

Asymmetric replication of hepatitis B virus DNA in human liver: demonstration of cytoplasmic minus-strand DNA by blot analyses and in situ hybridization

In situ and blot hybridization techniques have been used with strand- and region-specific probes to characterize the forms of hepatitis B virus (HBV) DNA in the liver of a patient with chronic active hepatitis B. The hepatocytes contain a heterogeneous population of rapidly migrating DNA species in the 0.5-1.4 kb position that are localized predominantly in the cytoplasm and are of minus-strand polarity. The findings indicate that the replication is asymmetric, with separate pathways for plus- and minus-strand synthesis of HBV DNA; that viral DNA synthesis is initiated at a site near the nick in the minus strand of virion DNA; and that actively replicating forms of HBV DNA can be identified at the cellular level by in situ hybridization.     

Viral nucleic acid sequences in transformed cells: IV. A study of the sequences of adenovirus 5 DNA and RNA in four lines of Adenovirus 5-transformed rodent cells using specific fragments of the viral genome

Specific fragments of Adenovirus 5 DNA were produced by digestion of intact, ³²P-labeled viral DNA with restriction endonucleases Eco R1 and and Hpa 1. The kinetics of renaturation of each fragment and of complete Adenovirus 5 DNA were measured in the presence of DNA extracted from four lines of Adenovirus 5-transformed rodent cells and from nontransformed control cells. All four transformed cell lines contained sequences homologous to the Hpa 1 fragment comprising the left 4% of the viral genome, but varied in the other Adenovirus 5 DNA sequences which were present: three lines of transformed cells contain segments of DNA extending from the left hand end to points 35, 40, and 12% along the viral genome and carry no other Adenovirus 5 DNA sequences. The fourth line also contains sequences homologous to the left half of the viral genome, but these could not be precisely defined. Therefore, the gene(s) encoded by the left end of Adenovirus 5 DNA must specify any viral gene functions expressed in transformed cells.Separated strands of the three Eco R1 fragments and certain Hpa 1 fragments of ³²P-labeled Adenovirus 5 DNA were hybridized with unlabeled, cytoplasmic RNA extracted from each of the four transformed cell lines. In each case, about 10% only of the r strand sequences of the largest Eco R1 fragment were complementary to transformed cell RNA. These sequences have been mapped to the left end of the viral genome using Hpa 1 fragment strands. The same sequences are shown to be expressed as mRNA during the early phase of an Adenovirus 5 lytic infection.     

Replication of vaccinia DNA in mouse L cells. IV. Protein synthesis and viral DNA replication

The requirement for protein synthesis during vaccinia DNA replication in mouse L cells was investigated. Within the first 30 min after reversal of a hydroxyurea (HU) block, viral DNA replication was not affected in cells treated with cycloheximide (100 μg/ml) to inhibit protein synthesis. During this period the intermediates in DNA replication detected, the rate of chain elongation, and the accumulation of crosslinked viral DNA molecules were all identical to those observed in vaccinia-infected cells not treated with cycloheximide. Thereafter, DNA replication, as measured by incorporation of [³H]thymidine, was inhibited in cycloheximide-treated infected cells (>90%, 2 hr post-HU reversal). Inhibition of viral DNA synthesis was further demonstrated by the sparse appearance and failure of cytoplasmic viral factories to increase in size after HU reversal, when protein synthesis was inhibitied. Density labeling of replicating viral DNA molecules with bromodeoxyuridine and analysis of equilibrium density centrifugation in CsCl showed that hybrid moelcules (hl, ? = 1.77 g/ml) accumulated in cycloheximide-treated cells. The hybrid molecules were not converted to “heavy” viral DNA (hh, ? = 1.825 g/ml), as was observed to occur during viral DNA replication in cells continuously synthesizing protein. The results of these experiments showed that after an initial round of viral DNA replication was completed, new protein synthesis was required to initiate additional rounds of viral DNA replication. The dissociation of viral DNA molecules, synthesized after HU reversal, from cytoplasmic DNA complexes was inhibited by cycloheximide but not rifampin. Continuous protein synthesis, apparently to permit expression of a “late” viral function, not related to viral assembly is required for release of the newly replicated viral genomes from complexes.     

Characterization of human cytomegalovirus-induced DNA polymerase and the associated 3'-to-5', exonuclease

A DNA polymerase activity induced by human cytomegalovirus (HCMV) was separated from host cell DNA polymerase and purified by phosphocellulose and DNA-cellulose column chromatography. The DNA polymerase activity was strongly inhibited by phosphonoacetic acid, aphidicolin, araATP, and N-ethylmaleimide, but it was resistant to 2',3'-dideoxyTTP. The sensitivity of HCMV-induced DNA polymerase to these reagents resembles that of host cell DNA polymerase alpha. However, HCMV-induced DNA polymerase activity was stimulated several fold by 100 mM ammonium sulfate, by which DNA polymerase alpha activity was strongly inhibited. Furthermore, it was found that a 3'-to-5' exonuclease activity was tightly associated with the HCMV-induced DNA polymerase. The exonuclease was also stimulated by ammonium sulfate, was inhibited by phosphoacetic acid, and it preferred single-stranded DNA as a substrate. The results suggest that the 3'-to-5' exonuclease may play a role in proofreading in the polymerization process as an integral part of the HCMV-induced DNA polymerase.