Ultraviolet inactivation of Epstein--Barr virus-induced nuclear antigen (EBNA), DNA and IgM synthesis in human B lymphocytes

The B-lymphotropic papovavirus (LPV) isolated originally from African green monkey (AGM) B-lymphoblasts has been adapted to growth in the human Epstein-Barr virus (EBV)-negative B-lymphoma line BJA-B by repeated passages within these cells. After 11 passages of virus approximately 20–25% of the cells were infectable within a 6 day period. Except for an abortive infection of another EBV-negative human B-lymphoma line, Ramos, and the EBV-positive Raji line of Burkitt lymphoma origin, cells of all other human lines of lymphatic origin tested thus far proved to be refractory to this infection. Viral DNA isolated from the BJA-B adapted strain was compared to that from the original AGM isolate. Only minor modifications were demonstrable in the HaeIII cleavage pattern of both isolates. A BJA-B subline persistently infected with LPV was established after cocultivation of BJA-B cells with LPV-producing AGM lymphoblasts. About 0.5–15% of these cells synthesized viral antigens at various intervals of cultivation. Viral DNA isolated from these cells after 12 months of persistent infection revealed a remarkable heterogeneity in size and in the cleavage pattern when analyzed with restriction endonucleases. Molecular cloning of this DNA in pBR 322 plasmid underlined this heterogeneity. The majority of clones obtained revealed deletions which preferentially mapped within the D-F fragments of the HaeIII map.     

Infection of mouse lymphocytes by Epstein-Barr virus. II. Stimulation of cellular DNA synthesis by EBV in the absence of EBNA induction and cell transformation

Epstein-Barr virus (EBV) of the transforming and nontransforming strains induced transient stimulation of cellular DNA synthesis during lytic infection of normal mouse lymphocytes. In contrast to human B lymphocytes, the action of both EBV strains in mouse cells was additive. The nontransforming P3HR-1 virus had no cytotoxic effect on mouse lymphocytes. The EBV-infected, stimulated mouse lymphocytes did not express EB virus-determined nuclear antigen and do not grow into immortalized cell lines. The cells expressed EBV-determined early and virus capsid antigens. These results suggest that the stimulation of cellular DNA synthesis by EBV is independent of EBNA synthesis and cell transformation.     

Effect of n-butyrate on cellular and viral DNA synthesis in cells latently infected with Epstein-Barr virus

Cellular DNA synthesis and Epstein-Barr virus (EBV)-specific DNA synthesis was examined after n-butyrate treatment of the producer line P3HR-1. Butyrate was found to effectively inhibit cellular DNA synthesis. This was followed by activation of the viral cycle as seen by marked increase in the expression of the EBV early antigen (EA), viral capsid antigen, and EBV-specific DNA synthesis. In spite of efficient induction of EA in the nonproducer Raji line, there was no increase in EBV-specific DNA synthesis above the level of the untreated control. It is likely that the cellular inhibitory effects of n-butyrate are responsible for the activation of EBV in producer lines and that different cellular controls, not affected by n-butyrate, are responsible for keeping the virus latent in nonproducer cells.     

The synthesis and structure of visna virus DNA.

The synthesis of proviral DNA of visna virus was measured at various intervals after inoculation of sheep cell cultures at multiplicities of 0.3, 1, and 10 PFU/cell. The DNA from the infected cells was fractionated by the Hirt procedure into low (Hirt supernatant) and high (Hirt precipitate) molecular weight DNAs, and each fraction was quantitated for infectivity by plaque assay using the calcium phosphate transfection technique of Graham and Van Der Eb (1973). The appearance of infectious DNA in the Hirt supernatant (low molecular weight viral DNA) was biphasic at all multiplicities, apparently reflecting two rounds of synthesis of this DNA. The amount of infectious DNA in the Hirt precipitate fraction increased with time, reaching maximum levels in all cultures at the time of peak virus production. Hirt supernatant DNA consisted predominantly of molecules of molecular weight 6 × 10⁶, which appeared to be present as double-stranded linear molecules. Infectious DNA in the Hirt precipitate, in contrast, had a molecular weight of 166 x 106, suggesting its association with cellular sequences. The network test strongly suggested that the viral sequences were covalently linked with or integrated into the cellular DNA.     

Macromolecular synthesis in cells infected by frog virus 3. VIII. The nucleus is a site of frog virus 3 DNA and RNA synthesis

The replication of frog virus 3 (FV 3) had been thought to occur exclusively in the cytoplasm. However, we recently reported that FV 3 did not replicate in enucleated or ultraviolet light (uv)-irradiated cells nor was viral DNA or RNA synthesized [Goorha, R., Willis, D. B., and Granoff, A. (1977).]. To elucidate the role of the nucleus in FV 3 replication further, we studied viral DNA and RNA synthesis in infected cells by electron microscopic autoradiography and by biochemical techniques. The results show that a fraction of FV 3 DNA is synthesized in the nucleus and then transported into the cytoplasm. The same is true for a fraction of FV 3-specific RNA. We have characterized the DNA molecules in these cell compartments. Both nuclear and cytoplasmic DNA appeared between 1 and 2 hr after infection. The molecular weight of newly synthesized nuclear viral DNA, as measured by sedimentation in alkaline sucrose gradients, did not exceed 8 × 10⁶ regardless of the labeling period. In contrast, the cytoplasmic fraction contained only DNA sedimenting at a rate expected for a single strand of the viral genome (50 × 10⁶ daltons). Pulse-chase experiments provided evidence for a precursor-product relationship between the nuclear and cytoplasmic DNA: About 35% of the nuclear DNA was chased into the cytoplasm and was genome size at the end of a 4-hr chase period. Nuclear viral DNA synthesis was more sensitive to cycloheximide, which inhibits initiation of DNA synthesis by inhibiting viral protein synthesis, than was cytoplasmic viral DNA synthesis. These data provide strong evidence for the nucleus as a site of FV 3 DNA and RNA synthesis and support the conclusion that FV 3 DNA synthesis is initiated in the nucleus and completed in the cytoplasm.     

Effect of temperature-sensitive, replication-defective mutations on RNA synthesis of cowpea chlorotic mottle virus

The RNA synthesis of replication-deficient, temperature-sensitive mutants of cowpea chlorotic mottle virus (CCMV) with mutations on either RNA 1 or RNA 3 was examined in temperature shift experiments. Viral RNA synthesis by the mutants at 25 degrees was similar to that of wild-type CCMV, but upon shift to 35 degrees , synthesis of mutant virus RNAs declined over a 16-hr period in contrast to continued synthesis by wild-type CCMV. Continued RNA synthesis, though at a reduced level, by the mutants during the initial periods following the shift to 35 degrees demonstrated that the enzymes involved in viral RNA synthesis of the mutants continued activity after the shift to the nonpermissive temperature. There was no clear correlation between a specific, defect in viral RNA synthesis and whether the mutation occurred on either RNA 1 or RNA 3. New replicative complexes appeared not to be produced at 35 degrees suggesting that gene products from both RNA 1 and RNA 3 may function coordinately in the replication complex. One mutant produced a reduced ratio of RNA 3 that was exaggerated upon shift to the restrictive temperature.     

Macromolecular synthesis in cells infected by frog virus 3. X. Inhibition of cellular protein synthesis by heat-inactivated virus

Heat-inactivated frog virus 3 (ΔFV3) inhibits host cell protein synthesis without interfering with protein synthesis directed by active FV3. An examination of various parameters of cellular protein synthesis after exposure of cells to ΔFV3 revealed the following. (i) The rate of inhibition was multiplicity dependent. (ii) There was a rapid disaggregation of both free and membrane-bound polysomes. (iii) Rates of protein chain elongation remained unaltered. (iv) There was neither a detectable breakdown nor modification of cellular mRNAs; the mRNAs isolated from ΔFV3-treated cells could be translated in vitro and the pattern of the translational products appeared identical to that of mRNAs from untreated cells. These data are consistent with the conclusion that inhibition of host cell protein synthesis by ΔFV3 is the result of a selective effect on a step(s) essential for the initiation of translation of cellular mRNAs.     

Effect of dexamethasone on expression of endogenous mouse mammary tumor virus sequences in BALB/c tumor cell lines.

BALB/c mammary tumor cell lines which contain only endogenous murine mammary tumor virus (MMTV) sequences respond to dexamethasone (DXS) treatment with minimal (approximately 2-fold) increases in MMTV RNA. This is in marked contrast to the 10? to 20-fold increases observed with cell lines harboring exogenous MMTV variants. Comparison of hybridization results obtained with two complementary DNA probes representative of either the entire MMTV RNA genome or its poly(A)-adjacent sequences suggests that the DXS response of BALB/c lines is also qualitatively different from that of exogenous MMTV-producer cell lines. Thermal stability studies suggested a 2–3% divergence between the RNA sequences of endogenous BALB/c and exogenous C3H viruses, with the 3′-end of the viral RNA appearing to be conserved relative to the rest of the genome.     

Inhibition of cellular protein synthesis by vaccinia virus surface tubules

The infection of HEp-2 cells with vaccinia virus results in a rapid and selective inhibition of cellular protein synthesis. This effect appears to be due to a structural component(s) of the infecting virion. Experiments investigating the nature of the inhibitory principle showed that a vaccinia virion component, the surface tubule (ST), inhibits protein synthesis in HEp-2 cells without affecting either RNA or DNA synthesis. Furthermore, ST added to a rabbit reticulocyte cell-free system inhibited the incorporation of radiolabeled amino acids into acid-insoluble protein. ST inhibitory activity was destroyed by heat (56° for 60 min) and by prior incubation with specific anti-ST serum. A decrease in the polyribosome complement of cells exposed to purified ST, with a concomitant increase in the free ribosome pool, suggests that the main effect of tubules on cellular protein synthesis occurs at the level of polypeptide chain initiation.     

The isolation of SV40 tsA/deletion, double mutants and the induction of host DNA synthesis

Simian virus 40 mutants were constructed that contain both a tsA mutation leading to temperature sensitivity of the 92K T-antigen, and deletions of 20–200 base pairs leading to a loss in the expression of the 20K t-antigen. As expected, these mutants were temperature sensitive for viral growth and viral DNA replication in lytically infected cells. At nonpermissive temperatures, the ts/deletion mutants stimulated the incorporation of nucleosides into host DNA as did the tsA mutant alone. This induction of incorporation by the tsA mutants resulted from semiconservative DNA replication, not repair synthesis. At 200 μg/ml caffeine the induction of host DNA by A209 was inhibited by 30 to 50%, whereas induction by the ts/deletion mutants was abolished.     

Simian varicella virus DNA shares homology with human varicella-zoster virus DNA

Delta herpesvirus (DHV) and Medical Lake Macaque (MLM) virus are cell-associated simian herpesviruses that cause varicella-like disease in nonhuman primates, and are antigenically related to human varicella-zoster virus (VZV). The results of studies designed to determine if homology exists between the DNA of DHV and MLM and the DNA of VZV are reported here. Southern blot hybridizations conducted at Tm-20 degrees did not detect DNA homology between the VZV and simian varicella virus genomes. However, under conditions of lower stringency (Tm-36 degrees and Tm-43 degrees), VZV DNA probes hybridized to specific HindIII fragments within DNA isolated from simian varicella virus-infected cells. Under similar hybridization conditions, DNA homology was not detected between VZV DNA and herpes simplex virus DNA. Further studies using cloned VZV DNA HindIII fragments as probes suggested that the homology between VZV DNA and DHV DNA is distributed across the viral genomes. These results demonstrate that the genomes of VZV and simian varicella virus share regions of conserved nucleotide sequences, and indicate a close evolutionary relationship between VZV and simian varicella viruses. In addition, the studies show that the DHV and MLM strains of simian varicella virus are more closely related to each other than to human VZV.     

Induction of host DNA synthesis and DNA polymerase by DNA-negative temperature-sensitive mutants of human cytomegalovirus.

Four DNA-negative temperature-sensitive (ts) mutants of human cytomegalovirus, belonging to different complementation groups, were studied for their ability to induce cell DNA synthesis and DNA polymerase in permissive human embryo lung (HEL) and nonpermissive rabbit lung (RL) cells. These is mutants stimulated host cell DNA synthesis in HEL and RL cells and DNA polymerase activity in HEL and RL cells at permissive (33.5°) and nonpermissive temperatures (39.5°). Salt stimulation of induced DNA polymerase activity was used to distinguish between virus and cell DNA polymerase from HEL cells. DNA polymerase activity was stimulated by 100 mM (NH₄)₂SO₄ at either 33.5 or 39.5° in cultures infected with three of the mutants (ts 9, is 153, and ts 155). However, DNA polymerase activity was not stimulated by 100 mM (NH₄)₂SO₄, in cultures infected with one of the ts mutants (ts 13). These data suggest that at least four cistrons control the synthesis of virus DNA and that virus DNA synthesis is not required for the induction of cell DNA synthesis.     

Inhibition of host cellular ribonucleic acid synthesis by glycoprotein of mumps virus

After infection with mumps virus, cellular ribonucleic acid synthesis of a murine lymphoma cell line, EL4, was appreciably depressed. The inactivation of viral infectivity by ultraviolet irradiation or the treatment of cells with mouse interferon did not abolish the inhibiting effect, suggesting that virus replication is not required for the depressed RNA synthesis. Envelope glycoproteins isolated from disrupted mumps virus caused inhibition of cellular RNA synthesis. The addition of low concentrations of specific antibody enhanced the inhibitory effect, probably through the formation of aggregates of glycoproteins. On the contrary, the glycoproteins showed no effect on RNA synthesis in the presence of cytochalasin D.     

Biogenesis of poxviruses: Preliminary characterization of conditional lethal mutants of vaccinia virus defective in DNA synthesis

Characterization of six is mutants of the DNA^? phenotype of vaccinia 1HD-W is described. Complementation analysis revealed that four of these map into different complementation groups while one was able to complement, albeit inefficiently, with only one among the other ts mutants tested. Judging by the rate of viral DNA synthesis it became evident that all DNA^? mutants except ts 6389 became more “leaky” for DNA synthesis at the restrictive temperature in single-cycle infection if adsorption was carried out at 4° when compared with values obtained after initiation of infection at 39.5°. This suggested that these mutants are defective in some function required very early in the virus life cycle. This notion was further supported by the observation that multiple rounds of viral DNA synthesis could occur at 39.5° provided that the temperature was raised after initiating the infection at a lower temperature. Employing density-shift experiments coupled with analysis of the size of replicative intermediates by means of alkaline sucrose gradients, it was found that progeny DNA synthesized by all but ts 6389 was indistinguishable from wild-type DNA. By contrast, DNA synthesis in ts 6389-infected cytoplasm was is throughout the period of viral DNA replication, regardless of the time when temperature was shifted up, relegating this mutant to the “fast-stop” category. This idea is supported by evidence that all of the DNA^? isolates except ts 6389 are deficient at 39.5° in some of the virus-specified DNA-binding proteins. Therefore, ts 6389 is the only isolate at hand with the characteristics expected of a mutant with a defective protein related to the growing fork of the vaccinia DNA replication apparatus.     

Cell cycle dependence of synthesis of unintegrated viral DNA in mouse cells newly infected with murine leukemia virus

We have studied Moloney murine leukemia virus (MuLV) replication in newly infected NIH/3T3 cells brought to a stationary phase by serum depletion. Progeny viruses were markedly decreased under these conditions. Studies of the early phase of the virus cycle by the Southern blot hybridization procedure revealed that levels of unintegrated linear double-stranded and supercoiled viral DNAs were decreased in quiescent NIH/3T3 cells as compared to levels detected in serum-replenished cells. When serum was added to quiescent cells up to 48 hr postinfection, we could detect an increase of viral DNA, suggesting the presence of a stable intermediate encoding viral information. In order to characterize this intermediate, stationary cells were labeled with BrdU at the time of serum addition so that substituted viral DNA molecules made under serum stimulation could be separated on CsCl gradients from those made under serum depletion. The analysis of this experiment revealed that upon serum addition, the majority of viral DNA was fully substituted (HH), indicating that it must have been synthesized from an RNA template. Also, an important part of viral DNA made after serum addition had an intermediate density (HL), suggesting that incomplete molecules made in quiescent cells were completed after serum addition. Our results clearly show that host factors are required for synthesis of viral DNA in NIH/3T3 cells newly infected with MuLV.     

Inhibition of SV40-induced cellular DNA synthesis by microinjection of monoclonal antibodies

The region of the SV40 large T-antigen molecule recognized by a panel of monoclonal antibodies has been determined using hybrid Adeno-SV40 viruses, and manual microinjection of cloned deletion mutants. In addition, an investigation was made of how monoclonal antibodies microinjected into the nucleus can affect the ability of the T-antigen coding gene to stimulate cell DNA synthesis. The monoclonal antibody Pab 14, that recognized the -COOH terminal half of large T, was comicroinjected into quiescent cells together with plasmid pCl-1. This plasmid contains only that part of the T-antigen coding gene that extends from nucleotide residue 120, counterclockwise to nucleotide residue 4002, and makes a truncated T antigen 33,000 in molecular weight and missing the last 435 amino acids on the -COOH terminal side. Monoclonal antibody Pab 14 did not inhibit the stimulation of cellular DNA synthesis caused by microinjection of pCl-1, although it did inhibit cell DNA synthesis induced by microinjection of pSV2G, a recombinant plasmid that contains the entire T-antigen coding gene of SV40.     

A transfection assay for transformation by feline sarcoma virus proviral DNA

We report that fibroblast lines derived from mouse (NIH 3T3) and mink (CCL-64) can be transformed via transfection with the total cellular DNA extracted from cells transformed with feline sarcoma virus. Replication of helper virus is not required in the recipient cell. Transformation of recipient cells is observed when the donor DNA is extracted from either virus producer or nonproducer transformed cell lines. A transforming virus can be rescued from nonproducer transformed recipients upon superinfection with a replication competent helper virus.     

Inhibition of SV40 replication by 5-azacytidine: effect on DNA synthesis and conformation.

SV40 DNA synthesis was inhibited by approximately 50% following a 3-hr treatment with 100 μg/ml 5-azacytidine (5-AzaCR). Cesium chloride-ethidium bromide analysis of SV40 DNA from drug-treated infected cultures demonstrated that SV40 DNA I banded at a higher buoyant density and therefore had a decreased ability to bind ethidium bromide. Sedimentation analysis in sucrose gradients containing various concentrations of ethidium bromide indicated that these molecules were deficient in superhelical terms. Treatment of infected cultures with 5-AzaCR was shown to inhibit protein synthesis which preceeded the inhibition of DNA synthesis. It is concluded that inhibition of SV40 DNA synthesis and the conformational alterations in DNA I result from the inhibition of protein synthesis.     

Activation of mouse retrovirus by herpes simplex virus type 1 cloned DNA fragments

Cloned DNA fragments from herpes simplex virus (HSV) type 1 (strain Patton) were tested for activation of endogenous mouse retrovirus in BALB/3T3 cells. Activation within the L region of HSV-1 DNA was observed with the ～3.4-kilobase pair (kbp) BamHI fragment which contains the virus thymidine kinase (TK) gene, and the ～5.3-kbp EcoRI L fragment. Activation by the TK-containing BamHI fragment was abrogated by digestion with EcoRI. Activation within the S region of HSV-1 DNA was observed with the ～15.2-kbp EcoRI H ragment and the ～8.4-kbp Eco RI/HindIII H/G fragment. Assaying for retrovirus activation serves as an additional parameter for mapping biological functions within the HSV genome.     

Evidence for control of herpes simplex virus mutagenesis by the viral DNA polymerase

We have measured the effect of mutations associated with the DNA polymerase of herpes simplex virus type 1 on the production of spontaneous mutants resistant to bromodeoxyuridine. Bromodeoxyuridine-resistant mutants contain mutations in the viral thymidine kinase gene. The conditional lethal mutation in strain C7, which prevents growth at 39° and is located in the DNA polymerase gene, reduced slightly the yields of bromodeoxyuridine-resistant progeny compared to the parent virus. In contrast, a temperature-resistant derivative of C7, ts⁺C, produced levels of resistant progeny 11-fold greater than normal. Evidence is presented that the ts⁺CO derivative is not a true revertant of C7, but instead contains a second site mutation which suppresses the C7 temperature-sensitive mutation. This suppressor mutation appears closely linked to the original C7 mutation. Possible mechanisms for the elevated production of bromodeoxyuridine-resistant progeny by ts⁺C are discussed.