Studies on the activity of DNase associated with the replication of the Epstein-Barr virus

A new deoxyribonuclease (DNase) was found to be induced in Epstein-Barr virus (EBV)-superinfected Raji cells or iododeoxyuridine (IUdR)-induced D98/HR-1 cells. The induced DNase has a unique electrophoretic mobility as well as immunospecificity.     

Effects of arabinofuranosylthymine on Epstein-Barr virus replication

The effect of 1-β-d-arabinofuranosylthymine (AraT) on Epstein-Barr virus (EBV) replication was studied using the producer lymphoblastoid cell line P3HR-1. Virus-specific early and late capsid antigens were induced with a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) alone, or in combination with sodium butyrate. The expression of VCA was completely inhibited by the analog after chemical induction of these cells, whereas no effect on early antigen production or on the increase of an EBV-specific DNA polymerase activity was observed. The activation of P3HR-1 TK^? cells by TPA-sodium butyrate induced high stimulation of [³H]thymidine incorporation into the viral DNA, which was entirely inhibited by the analog, as demonstrated by CsCl gradient analysis. The work reported here strongly suggests that AraT is an efficient inhibitor of EBV replication, acting during the period of initiation of viral DNA replication.     

Analysis of early and late Epstein-Barr virus associated polypeptides by immunoprecipitation.

The Epstein-Barr virus-producing cell lines P3HR-1 and B95-8 and the nonproducer cell lines Raji clone No. 7 and NC37 were induced to viral antigen synthesis by the tumor promoter TPA and then analyzed by immunoprecipitation with human sera for early and late virus-associated polypeptides. After labeling of producer cells for a 4-day period with [³⁵S]methionine, two polypeptides with molecular weights of 140,000 and 150,000 were identified reacting predominately with virus capsid antigen (VCA⁺) sera. Analysis of purified Epstein-Barr virus demonstrated that the 140,000 polypeptide presumably represents an envelope protein while the 150,000 polypeptide is a nucleocapsid protein. In 4-hr radioactively labeled producer cells an additional polypeptide with a molecular weight of 130,000 was found to be immunoreactive with VCA⁺ sera. Immunoprecipitation of [³⁵S]methionine-labeled cell extracts from nonproducer cells resulted in the specific precipitation of two polypeptides with molecular weights of 85,000 and 35,000 which most likely represent early EBV-associated proteins. Producer cells exhibit three additional apparently early EBV-associated polypeptides with molecular weights of 120,000, 18,000, and 16,000. None of these polypeptides could be detected in EBV genome-negative Ramos cells after TPA treatment.     

Effects of 12-O-tetradecanoyl-phorbol-13-acetate on cell proliferation and Epstein-Barr virus DNA replication

The amino acid sequence at the cleavage site of the fowl plague virus hemagglutinin has been analyzed. The amino terminus of HA₂ is formed by glycine-343. The carboxy terminus of HA₁ is serine-336 to which occasionally parts of the basic intervening peptide are still attached. The data indicate that the fowl plague virus hemagglutinin like other influenza hemagglutinins is cleaved by the sequential action of a trypsin-like endopeptidase and carboxypeptidase B. There is evidence, however, that the endopeptidases involved in the cleavage of both types of hemagglutinins differ in their specificities.     

Protease activity associated with the capsule protein of Estigmene acres granulosis virus

The polypeptide composition of a granulosis virus of Estigmene acrea was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enveloped nucleocapsids are made up of 24 polypeptides with molecular weights ranging from 11,000 to 98,000. Virus capsule protein contains protease activity which degrades capsule protein from 30,000 to 25,000 through two intermediates of 29,000 and 27,000. The putative endoprotease is active at alkaline pH, sensitive to heat and diethylpyrocarbonate inhibition, and resistant to 10(-3) M phenylmethylsulfonyl fluoride. No alteration in the electrophoretic pattern of viral structural proteins was detected in virus preparations containing endoproteolytic activity. A low level of alkaline exoprotease activity associated with capsule protein was also detected.     

Tyrosine-specific protein kinase activity associated with p105 of avian sarcoma virus PRCII

A protein kinase activity was found to be associated with the transformation-specific polyprotein (p105) of avian sarcoma virus PRCII. The kinase was detected in immune complexes with antisera reactive with the gag sequences of p105. Addition of [γ-³²P]ATP to these complexes resulted in phosphorylation of p105 and, with some sera, phosphorylation of immunoglobulin heavy chain. Phosphoamino acid analysis showed that phosphotyrosine was the major product of the in vitro kinase reaction and the major phosphoamino acid of p105 extracted from ³²P-labeled cells. The same tryptic peptides of p105 were found to be labeled by the in vitro kinase reaction and by ³²P labeling of PRCII-transformed cells. These phosphopeptides were not found in Pr76^gag Thus, like Rous sarcoma virus, PRCII has an associated tyrosine-specific protein kinase which may be responsible for its transforming activity.     

Epstein-Barr virus induction by a serum factor. I. Induction and cooperation with additional inducers

Serum contains a factor (i) that can induce synthesis of early antigens (EA) of Epstein-Barr virus (EBV) in EBV genome-positive human lymphoblastoid cells and (ii) that cooperates with the inducers TPA, IUdR n-BA, and anti-IgM. When compared to induction by inducer alone, this cooperation is characterized by (i) a faster rate of appearance of EA-positive cells, (ii) a much higher percentage of cells that finally express EA, (iii) a dramatic influence on the dose-response relationship between inducer and EA induction. In vitro, the factor requires treatment by a pH shock to gain maximal activity. The factor may play an important role in the activation of latent viral information and may have other physiological functions.     

Early DNA-binding polypeptides of Epstein-Barr virus

The kinetics of Epstein-Barr virus (EBV)-specific protein synthesis has been studied after induction of P3HR-1 cells with n-butyrate. The majority of the EBV polypeptides became detectable on SDS-polyacrylamide gel electrophoresis between 15 and 18 hr after addition of the drug. Overall viral protein synthesis was at maximum after about 24 hr. Some polypeptides appeared significantly earlier (152K, 55K, 51K) while others were synthesized during a limited period of the cycle only (65K). At least four of the polypeptides detected (152K, 134K, 55K, 51K) bound to DNA cellulose columns. The EBV specificity of the polypeptides was confirmed by immunoprecipitation and by two-dimensional gel analysis. The 152K polypeptide had a pI around 8 whereas the other polypeptides varied between basic and acidic pI. The DNA binding proteins isolated by DNA cellulose chromatography represent a subclass of the virus-specific early proteins that can be immunoprecipitated from the total cell extract.     

Recovery of Epstein-Barr virus from nonproducer neonatal human lymphoid cell transformants.

Lymphoid cell lines (LCL) were established by infection of two batches of human umbilical cord lymphocytes with low multiplicities of the B95-8 strain of Epstein-Barr virus. Three of the 17 lines released minute amounts of transforming virus. The rest did not, nor did they make capsid antigen. However virus could be regularly recovered by lethal X-irradiation of transformed cells followed by cocultivation with primary human umbilical cord leukocytes. By this technique transforming activity could be identified in 15 of the 17 lines. These data indicate that these nonproducer human neonatal cell transformants established by EBV infection in vitro possess sufficient genetic information to code for production of biologically active mature virions. X rays alone failed to cause a detectable increase in the number of cells with capsid antigen or to enhance extracellular virus production. EBV-positive human serum blocked rescue if it was added during the first 2–4 hr after cocultivation, but not thereafter. Transforming virus could be recovered from X-rayed cells which were immediately thereafter lysed by freezing and thawing. These results suggest that recovery of virus following X-ray and cocultivation is not due to activation of the intracellular virus genome. Rather, it is likely that the method detects small numbers of virions which are cell associated. While transforming virus could regularly be rescued from lymphoblastoid cell lines resulting from in vitro transformation, attempts to rescue virus from Raji or EBV-converted BJAB cells were unsuccessful. This discrepancy suggests differences in genome complexity or in genome-cell interactions in different types of EBV-transformed cells.     

Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate.

n-Butyrate was found to increase the number of virus producer cells to a dramatic extent in the Epstein-Barr virus (EBV)-carrying P3HR-1 and B95-8 lines. Induction was also seen in the nonproducer Raji and the low producer Daudi lines, but at a mucch lower level. The virus containing supernatant of the butyrate treated P3HR-1 cells induced preferentially EBNA in EBV-negative Ramos target cells, whereas the spontaneously produced virus induced predominantly EA in Raji indicator cells. This suggests a possible difference in the biological properties of the butyrate induced vs the prototype virus. In addition to providing a convenient method to obtain a high yield of viral-DNA and virus antigen-producing cells in the severely restricted EBV system, the findings raise interesting questions on the mechanism of EBV induction, and its possible relationship to the known differentiation inducing ability of n-butyrate.     

Isolation of two DNA-binding proteins from the intracellular replication complex of vaccinia virus.

Two DNA-binding proteins, designated FP11 and FP14, have been purified from the viral replication complex which forms in the cytoplasm of HeLa cells during productive infection with vaccina virus. Polypeptide FP11 has an apparent molecular weight of 34,000 (by SDS-PAGE) and binds strongly to denatured DNA-cellulose columns, eluting as a narrow peak centered at 0.25 M NaCl. This polypeptide represents 30–40% of the [³H]lysine but only 17% of the [3H]methionine which can be incorporated into the complex. Polypeptide FP14 has an apparent molecular weight of 28,000 (by SDS-PAGE) and elutes from denatured DNA-cellulose as a relatively broad peak over the range 0.45–0.80 M NaCl. FP14 accounts for approximately 10% of incorporated radioactivity, regardless of whether lysine or methionine is employed as the radioactive precursor.     

Effects of cytochalasin B on herpes simplex virus type 1 replication.

The replication of herpes simplex virus type 1 (HSV-1) was studied in human embryonic lung cells cultured in the presence of varying concentrations of cytochalasin B. At a concentration of 50 μg/ml, the drug decreased the incorporation of [³H]glucosamine and [³H]fucose into acid-insoluble material by 96 and 92%, respectively, while allowing the incorporation of [¹⁴C]amino acids to proceed at a near-normal level. Analysis of [¹⁴C]amino acid-labeled HSV-induced polypeptides by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed that as the concentration of cytochalasin B increased, a progressive shift was observed from the major envelope glycopeptide of the virus to a new, presumably nonglycosylated, component designated CB92 (MW 92,000). Concomitant with the appearance of CB92 was a failure of all major virus-induced glycopeptides to incorporate [³H]glucosamine and [³H]fucose. At 50 μg/ml, the drug also decreased HSV-1 infectivity by 96%, although only a 49% reduction of physical particles was observed at this concentration.     

Initiation and termination of vaccinia virus DNA replication

The initiation and termination sites of replication of vaccinia DNA has been studied by determining the radioactivity in restriction fragments of the pulse-labeled newly synthesized molecules. The results indicate a bimodal distribution of radioactivity in the molecules completed during a pulse shorter than the completion time of synthesis, implying termination at both ends. The symmetry of replication was studied by hybridization of an intermediate in replication (34 S) to the isolated strands of virion DNA followed by restriction analysis. The frequency of hybridization of the fragments to the light or heavy strand shows a unimodal distribution of radioactivity indicating asymmetric replication. Together, these observations suggest that initiation and termination sites are located at both ends of the chromosome. Changes observed in the conformation of parental DNA at the time of replication, such as high degree of single-strandness, are compatible with strand displacement during synthesis.     

Esh avian sarcoma virus codes for a gag-linked transformation-specific protein with an associated protein kinase activity

Esh sarcoma virus, initially isolated from a spontaneous tumor of a chicken, transforms fibroblasts in vitro and induces fibrosarcomas in vivo. It is defective for replication, and infectious viral stocks consist of a mixture of a sarcomagenic virus (ESV) and an a avian leukosis virus of subgroup A (EAV) which serves as helper. Cloned stocks of infectious ESV contain two RNA components of M_r, 3 and 1.5 × 10⁶, respectively, as determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The component of M_r 1.5 × 10⁶ appears to be the genome of defective ESV, since it is not detected in preparations of the helper virus EAV. The size of the ESV genome suggests major deletions of replicative genes, and ESV-transformed nonproducer cells fail to express functional translation products of the gag, pol, and env genes. ESV-transformed producer and nonproducer clones also do not express pp60^src but contain a gag-related protein of M_r 80,000 (p80). Two-dimensional analyses of the [³⁵S]methionine-labeled tryptic peptides of p80 indicate that this protein contains part of the sequences of gag-p19 covalently linked to additional sequences unrelated to gag, pol, and env gene products. These ESV-specific sequences are also unrelated to pp60^src and to gag-linked polyproteins found in cells transformed by defective avian sarcoma viruses PRCII and Fujinami or defective leukemia viruses AEV, MC29, and MH2. P80 is phosphorylated in vivo at two major sites, one involving phosphoserine and the other phosphotyrosine residues. Immunoprecipitates containing ESV-p80 are associated with a protein kinase activity that is specific for tyrosine residues of several acceptor molecules including p80 itself, rabbit immunoglobulin H chain of the immune complex and exogenously added α-casein. p80 is phosphorylated in vitro at the same tyrosine site as in vivo suggesting that the enzyme activity detected in vitro is of physiological significance. The p80-associated protein kinase activity is strictly dependent on the presence of Mg²⁺ or Mn²⁺ but was found independent of known effectors of cellular protein kinases Ca²⁺, cAMP, or cGMP.     

Transformation of lymphocytes by Epstein-Barr virus requires only one-fourth of the viral genome

Inactivation studies were performed to measure the target sizes of three Epstein-Barr virus (EBV) -associated functions: (i) transformation of lymphocytes; (ii) stimulation of cell DNA synthesis in lymphocytes; and (iii) induction of the EBV nuclear antigen (EBNA) expression in lymphocytes. The target size of the plaque-forming function of herpes simplex virus type 1 (HSV-1) was studied in parallel and was used as a standard for comparison with the measured target sizes. When ionizing irradiation was used as an inactivating agent, the target size of EBV transforming function was found to be as large as that determined for the plaque-forming function of HSV-1. A similar size was measured for the EBV function required to stimulate cell DNA synthesis. In contrast, the target size of the viral function needed for induction of EBNA expression was found to be only 35% of that observed for HSV-1 plaque-forming function. Inactivation studies were also performed using the chemical mutagen, N-acetoxyacetylaminofluorene. The target size measured using this agent for the transforming function of EBV was about 25% of that determined for plaque-forming function of HSV-1. This result indicates that only a portion of the viral DNA encodes information necessary for the initiation and maintenance of transformation by EBV.     

Infection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus II. The viral DNA and the kinetics of its replication

The kinetics of replication of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA in Spodoptera frugiperda cells in culture were studied. Viral DNA replication started at about 5 hr postinfection, the rate of viral DNA replication reached a maximum at about 18 hr postinfection and thereafter decreased. Parental viral DNA could be detected within the first hour postinfection in the total intracellular DNA by the Southern technique. There was no evidence for the occurrence of AcNPV DNA sequences which became covalently linked to cellular DNA between 1 and 3 hr post-infection in the productive cycle. The AcNPV DNA appeared as a covalently closed circular molecule of about 92 x 10(6) daltons. The AcNPV DNA did not seem to be methylated, at least there were no 5'-CmCGG3' sequences detectable in this DNA. Restriction enzyme analysis of viral DNA preparations derived from several single plaque isolates of AcNPV as well as of DNA from different virus stocks revealed a certain heterogeneity of the AcNPV DNA.