Metabolic activation of 9([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human lymphoblastoid cell lines infected with Epstein-Barr virus.

9-([2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) is more potent and has a more prolonged inhibitory effect against Epstein-Barr virus (EBV) in vitro than does acyclovir (ACV). To assess the mechanism of this difference, we first compared the extent of phosphorylation of the two drugs in superinfected Raji cells. BW B759U is phosphorylated to levels 100-fold higher than is ACV. In addition, lower levels of phosphorylation of BW B759U and ACV were observed in uninfected Raji cells. Studies on the kinetics of formation of BW B759U triphosphate in superinfected Raji cells indicated that drug-phosphorylating activity was detected as early as 3 h after superinfection; this activity was steadily maintained for the first 7 h, followed by a burst of activity between 7 and 10 h and a doubling of phosphorylation between 10 and 25 h. During the superinfection cycle, the pool sizes of deoxyribonucleoside and ribonucleoside triphosphates were increased and reached their maxima at 10 h after infection. The maximal amount of triphosphorylated drug in a virus producer cell, P3HR-1 (LS), was obtained at 21 h after drug treatment. During long-term drug treatment, approximately 44 and 77% reduction in EBV genome copies per cell was observed on days 3 and 7, respectively. In a separate experiment, after treatment of P3HR-1 (LS) cells with BW B759U for 36 h, 4.2 pmol of BW B759U triphosphate per 10(6) cells was achieved. After the cells were released into drug-free medium, drug triphosphate was rapidly decreased to 11% of the original level in 1 day. Thereafter, the decrease was slow but steady, down to 0.22 pmol/10(6) P3HR-1 cells by 5 days. We calculated that 0.22 pmol of BW B759U triphosphate per 10(6) cells represents a cellular concentration of 0.22 microM, which is theoretically enough to inhibit EBV replication. This is based upon a comparison with the 50% effective dose of BW B759U (0.05 microM) for inhibition of genome replication and a Ki of 0.08 microM for BW B759U triphosphate inhibition of EBV DNA polymerase.     

Effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus DNA replication.

The effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus (EBV) DNA replication in the lymphoblastoid cell lines P3HR-1 and Raji is reported. Acyclovir at a concentration of 100 microM completely inhibited EBV DNA synthesis in superinfected Raji cells, but did not inhibit DNA synthesis in mock-infected cells. The number of EBV genome equivalents per cell in the virus-producing cell line P3HR-1 was significantly reduced by acyclovir, whereas the number of latent EBV genomes in Raji cells was not affected by the drug. In situ cytohybridization performed on untreated P3HR-1 cultures revealed the presence of relatively large amounts of EBV DNA in 15 to 20% of the cells. After a 100 microM drug treatment, no P3HR-1 cells contained levels of EBV DNA detectable by in situ cytohybridization. Indirect immunofluorescence studies demonstrated that during treatment with 100 microM acyclovir for 7 days, the percentage of P3HR-1 cells expressing viral capsid antigen was reduced. The EBV DNA remaining in P3HR-1 cells after treatment with 100 microM acyclovir (approximately 14 genomes per cell) had the properties of covalently closed circular DNA with an average molecular weight of 108 X 10(6), as determined by contour length measurements.     

Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious virus particles in superinfected Raji cells.

Human lymphoblastoid Raji cells, which do not produce virus, supported replication of Epstein-Barr virus (EBV) upon superinfection. Early antigen, viral capsid antigen, and virions were produced in Raji cells superinfected with EBV. Viral DNA replicated under complete inhibition of host cell DNA synthesis to the extent that a few micrograms of EBV DNA were recovered from 107 superinfected Raji cells, corresponding to 5,000 viral genomes/cell. Homology of the synthesized viral DNA to parental EBV DNA was more than 90%. Virions produced by the Raji cells contained a 55S DNA but failed to induce early antigen, viral capsid antigen, and viral DNA synthesis after a second superinfection of Raji cells.     

Inhibitory effect of 1-beta-D-arabinofuranosylthymine on synthesis of Epstein-Barr virus

The effect of 1-beta-D-arabinofuranosylthymine (ara-T) on cell growth and synthesis of Epstein-Barr virus (EBV) in human lymphoblastoid cell lines was determined. The growth of P3HR-1 cells was not inhibited by 1 microgram of the drug per ml; however, infectious virus production was strongly inhibited and was accompanied by decreased expression of early antigen (EA) and viral capsid antigen (VCA). The ability of 12-O-tetradecanoylphorbol-13-acetate or n-butyric acid to induce synthesis of VCA, but not EA, in P3HR-1 cells was inhibited by ara-T. Similarly, VCA synthesis but not EA synthesis was inhibited by ara-T in Jijoye cells superinfected with the P3HR-1 strain of EBV. The results suggest that ara-T has a specific inhibitory action against EBV replication.     

Amplification of Epstein-Barr virus (EBV) DNA by superinfection with a strain of EBV derived from nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) from a nasopharyngeal carcinoma (NPC) hybrid cell line (NPC-KT) lacking defective viral DNA molecules superinfected Raji cells and induced EBV early antigens (EA), as did virus from P3HR-1 cells, which contained defective molecules. The EBV polypeptides induced by NPC-KT appeared to be identical to those induced by P3HR-1 virus. The ability of NPC-KT virus to induce EA was enhanced more than 10-fold by treatment of superinfected cells with dimethyl sulfoxide; however, dimethyl sulfoxide treatment did not enhance superinfection by P3HR-1 virus. After infection, DNA synthesis of both the superinfecting NPC-KT virus and the resident Raji viral genome was induced. In addition to amplified Raji EBV episomal DNA, a fused terminal fragment of NPC-KT viral DNA was detected. The detection of fused terminal DNA fragments suggests that the superinfecting virion DNA either circularizes or polymerizes after superinfection and is possibly amplified through circular or concatenated replicative intermediates.     

Qualitative and quantitative analyses of Epstein-Barr virus early antigen diffuse component by western blotting enzyme-linked immunosorbent assay with a monoclonal antibody.

We report the use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers. The kinetics of synthesis of EA-D in P3HR-1, B95-8, and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. Surprisingly, no expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching a peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay was employed to identify polypeptides representing EA-D. A family of four polypeptides with molecular weights of 46,000 (46K protein), 49,000, 52,000, and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8, and Ramos/AW cells and in superinfected Raji cells. In addition, two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. However, none of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos, and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, we employed the enzyme-linked immunosorbent assay and immunoblot dot methods by using one of the purified EA-D components to construct a standard curve. Depending upon the cell lines, it was estimated that ca. 1 to 3% (determined by the enzyme-linked immunosorbent assay) and 0.8 to 1.6% (determined by immunoblot dot) of total proteins from maximally induced cells were EA-D. These results suggest that differential expression of EA-D polypeptides could be of importance in the diagnosis of state of EBV infection.     

Synthesis of Epstein-Barr virus DNA in vitro: effects of phosphonoacetic acid, N-ethylmaleimide, and ATP.

Nuclei from superinfected Raji cells synthesized Epstein-Barr Virus (EBV) DNA in vitro in the absence of cell DNA synthesis. The synthesis of EBV DNA in vitro was inhibited by phosphonoacetic acid and N-ethylmaleimide, and maximum synthesis was achieved in the absence of an ATP-regenerating system. Nuclei from mock-infected cells required an ATP-regenerating system for maximum DNA synthesis.     

Inhibition of Epstein-Barr virus replication by a benzimidazole L-riboside: novel antiviral mechanism of 5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole.

Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. D-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5, 6-dichloro-2-bromo-1-beta-D-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to the L-riboside 1263W94 [5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1. 1 microM, compared with 10 microM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D.     

Flow cytometric evaluation of antiviral agents against human herpesvirus 6

Antiviral activities of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV), penciclovir (9-[4-hydroxy-3-(hydroxymethyl) butyl] guanine, PCV), ganciclovir ([9-(1,3-dihydroxy-2-propoxy) methyl] guanine, GCV), and foscarnet (phosphonoformic acid, PFA) were determined against Human Herpesvirus 6 (HHV-6) by flow cytometric technique. The technique is based on the detection of gp116 antigen expression in virus infected cells. Susceptibility was defined in terms of drug concentration which reduced the number of cells expressing HHV-6 gp116 antigen with a mean fluorescent intensity (MFI) by 50% as compared to virus infected untreated cells. GCV was found to be most effective against HHV-6 followed by PFA, PCV and ACV. For HHV-6A, the mean 50% inhibitory concentrations (IC50) of GCV and PFA were found to be 3.4 microM and 34.7 microM respectively, whereas the IC50 of ACV and PCV were found to be 53.7 microM and 37.9 microM respectively. For HHV-6B, the IC50 of GCV and PFA were found to be 5.7 microM and 71.4 microM respectively, whereas the IC50 of ACV and PCV were found to be 119.0 microM and 77.8 microM respectively. Flow cytometry is a valuable technique for the evaluation of antiviral compounds against viruses including HHV-6.     

Phosphorylation of acyclovir [9-(2-hydroxyethoxymethyl)guanine] in Epstein-Barr virus-infected lymphoblastoid cell lines.

The extent of phosphorylation of 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]) in fresh peripheral leukocytes, in Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines, and in herpes simplex virus type 1-infected lymphoblastoid (P3HR-1) and monkey kidney (Vero) cells was determined by high-pressure liquid chromatography, Mono-, di-, and triphosphorylated derivatives of [8-14C]ACV were detected at low levels at various times after superinfection of Raji cells with EBV. The extent of phosphorylation appeared to be related to the concentration of ACV in the medium. Small amounts of ACV mono-, di-, and triphosphates were formed in fresh peripheral leukocyte preparations from EBV- seropositive and -seronegative donors. Comparable ACV monophosphate levels were detected in EBV-negative BJAB and the EBV-positive BJAB/GC cell lines; however, no di- or triphosphate derivatives were detected. Comparable ACV-monophosphate levels were detected in both P3HR-1 and HSV-infected P3HR-1 cell lines; however, larger amounts of ACV di- and triphosphorylated derivatives were detected in the HSV-infected P3HR-1 cells. ACV was converted to the triphosphate to a greater extent in HSV-infected Vero cells than in mock-infected Vero cells or in HSV-infected P3HR-1 cells. ACV or its phosphorylated derivatives were converted to guanine nucleotides to a greater extent in lymphoblastoid cells than in fibroblasts (Vero). In conclusion, neither the productive replication of EBV nor the presence of latent viral DNA is required for ACV monophosphate formation in B lymphoblastoid cells. ACV triphosphate, however, was detected only in cells infected productively with EBV.     

Activation of latent Epstein-Barr virus genomes: selective stimulation of synthesis of chromosomal proteins by a tumor promoter.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a potent inducer of Epstein-Barr virus (EBV) gene expression. The optimal conditions for maximum activation of latent EBV genomes by TPA were determined. Although TPA is able to induce replication of EBV genomes in P3HR-1 cells in all phases of growth, the greatest increase in viral genome copies per cell (15-fold above the control level) occurred in nonproliferating cells as opposed to cells growing exponentially (6-fold above the control level). The synthesis of chromosomal proteins in nonproliferating cells under the conditions that induce maximum activation of latent virus genomes by TPA was studied. Selective stimulation in chromosomal protein synthesis accompanied the increase in EBV genomes in P3HR-1 cells despite an overall reduction in total cellular protein synthesis. Comparison of the chromosomal proteins from TPA-induced P3HR-1 cells and from superinfected Raji cells revealed comigrating chromosomal polypeptides of 145K, 140K, 135K, 110K, 85K, and 55K that are presumably EBV associated. The selective stimulation of synthesis of these chromosomal proteins in TPA-treated P3HR-1 cells was closely associated with the activation of latent EBV genomes.     

Phosphorylation of the antiviral precursor 9-(1,3-dihydroxy-2-propoxymethyl)guanine monophosphate by guanylate kinase isozymes

Guanylate kinase was purified from human erythrocytes by affinity chromatography using GMP-agarose, and the four isozymes which are present were separated by chromatofocusing. The kinetic properties of each isozyme were analyzed with respect to the natural substrates GMP and dGMP, and the 5'-monophosphate derivatives of the antiviral nucleoside analogs 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and 9-(2-hydroxyethoxymethyl)guanine (ACV, Acyclovir). The analysis of substrate kinetics yielded Km values for DHPG 5'-monophosphate which were similar with all isozymes (42-54 microM), and about 3-fold higher than the Km values obtained for GMP. Km values obtained with ACV 5'-monophosphate were 10-20-fold higher than the GMP values and varied nearly 4-fold among isozymes (209-753 microM). GMP produced the highest enzyme velocities with all isozymes, followed by dGMP, DHPG 5'-monophosphate, and ACV 5'-monophosphate, in that order. Differences in maximal velocities among isozymes were generally small. DHPG 5'-monophosphate inhibited the isozymes by a simple competitive mechanism with respect to GMP. In contrast, ACV 5'-monophosphate acted as an apparent hyperbolic mixed-type inhibitor. Similar patterns of inhibition were obtained with all isozymes. It is probable that differences is the reactivity of DHPG 5'-monophosphate and ACV 5'-monophosphate with individual guanylate kinase isozymes do not contribute significantly to differences in their antiviral effects.     

Sedimentation characteristics of newly synthesized Epstein-Barr viral DNA in superinfected cells.

Replicating Epstein-Barr virus (EBV) DNA molecules isolated from superinfected Raji cells were shown to consist of 80S to 65S and 58S (mature) molecules Pulse-chase experiments showed that radioactive label of DNAS molecules with the larger sedimentation coefficients was partially chased into 58S labeled forms. Formation of large concatemers of viral DNA could not be detected at any time after superinfection. The continuous presence of the 65S viral DNA intermediate throughout the replicative cycle combined with the observed inhibition of EBV DNA synthesis by addition of nontoxic levels of ethidium bromide to the superinfected cell culture led us to propose that EBV replication proceeds via a relaxed circular DNA intermediate.     

Evidence for an essential role of long chain acyl-CoA synthetase in animal cell proliferation. Inhibition of long chain acyl-CoA synthetase by triacsins caused inhibition of Raji cell proliferation

Triacsins A, B, C, and D are new inhibitors of long chain acyl-CoA synthetase (EC 6.2.1.3) and possess different inhibitory potencies against the enzyme (Tomoda, H., Igarashi, K., and Omura, S. (1987) Biochim. Biophys. Acta 921, 595-598). Acyl-CoA synthetase activity in the membrane fraction of Raji cells was also inhibited by triacsins. The same hierarchy of inhibitory potency as that against the enzyme from other sources, triacsin C greater than triacsin A much greater than triacsin D greater than or equal to triacsin B, was observed. When Raji cells were cultivated in the presence of triacsins, cell proliferation was inhibited in a dose-dependent fashion. The drug concentrations required for 50% inhibition of cell growth at day 2 were calculated to be 1.8 microM for triacsin A, much greater than 20 microM for triacsin B, 1.0 microM for triacsin C, and much greater than 15 microM for triacsin D, demonstrating a hierarchy for inhibitory potency of triacsins similar to that against the acyl-CoA synthetase activity. To understand the role of long chain acyl-CoA synthetase in animal cells, the effect of triacsins on the lipid metabolism of Raji cells was studied. When intact Raji cells were incubated with [14C]oleate in the presence of individual triacsins, the incorporation of [14C]oleate into each of the lipid fractions such as phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol was inhibited to an analogous extent. A common hierarchy, triacsin C greater than triacsin A much greater than triacsin D greater than triacsin B, was shown for the inhibition in each synthesis of the three lipids, which was identical with that for acyl-CoA synthetase. These findings indicate that the inhibition of acyl-CoA synthetase is well correlated with the inhibition of lipid synthesis. Taken together, the data strongly suggest that the inhibition of acyl-CoA synthetase by triacsins leads to the inhibition of lipid synthesis and eventually to the inhibition of proliferation of Raji cells.     

Acyclovir transport into human erythrocytes

The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.     

Differential effects of acyclovir and 9-(1,3-dihydroxy-2-propoxymethyl)guanine on herpes simplex virus and Epstein-Barr virus in a dually infected human lymphoblastoid cell line.

We investigated the effects of acyclovir and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) on a lymphoblastoid cell line dually infected with Epstein-Barr virus and herpes simplex virus (HSV) type 1. The numbers of Epstein-Barr virus genomes were reduced during 70 days of treatment with either drug. Both drugs suppressed HSV replication in a dose-related manner. In the continued presence of the drugs, HSV developed resistance, rapidly to acyclovir and much more slowly to 30 microM DHPG. Analysis of HSV glycoprotein C production and viral DNA showed that treatment with 100 microM DHPG eliminated HSV production, curing the cell line of HSV persistent infection.     

Epstein-barr virus-specific RNA. II. Analysis of polyadenylated viral RNA in restringent, abortive, and prooductive infections.

The complexity and abundance of Epstein-Barr (EBV)-specific RNA in cell cultures restringently, abortively, and productively infected with EBV has been analyed by hybridization of the infected cell RNA with purified viral DNA. The data indicate the following. (i) Cultures containing productively infected cells contain viral RNA encoded by at least 45% of EBV DNA, and almost all of the species of viral RNA are present in the polyadenylated and polyribosomal RNA fractions. (ii) Restringently infected Namalwa and Raji cultures, which contain only intranuclear antigen, EBNA, and enhanced capacity for growth in vitro, contain EBV RNA encoded by at least 16 and 30% of the EBV DNA, respectively. The polyadenylated and polyribosomal RNA fractions of Raji and Namalwa cells are enriched for a class of EBV RNA encoded by approximately 5% of EBV DNA. The same EBV DNA sequences encode the polyadenylated and polyribosomal RNA of both Raji and Namalwa cells. (iii) After superinfection of Raji cultures with EBV (HR-1), the abortively infected cells contain RNA encoded by at least 41% of EBV DNA. The polyadenylated RNA of superinfected Raji cells is enriched for a class of EBV RNA encoded by approximately 20% of EBV HR-1 DNA. Summation hybridization experiments suggest that the polyadenylated RNA in superinfected Raji cells is encoded by the same DNA sequences as encode RNA present in Raji cells before superinfection, most of which is not polyadenylated. That the same EBV RNA sequences are present in the polyadenylated and polyribosomal fractions of two independently derived, restringently infected cell lines suggests that these RNAs may specify functions related to maintenance of the transformed state. The complexity of this class of RNA is adequate to specify a sequence of a least 5,000 amino acids. That only some RNA species are polyadenylated in restringent and abortive infection suggests that polyadenylation or whatever determines polyadenylation may play a role in the restricted expression of the EVB genome.     

Nucleosomal structure of Epstein-Barr virus DNA in transformed cell lines.

Micrococcal nuclease digestion was used to analyze Epstein-Barr virus (EBV) DNA structure in nuclei of transformed cells. Digests of virus-producing (P3HR-1), non-virus-producing (Raji), and superinfected Rajii cell nuclei were fractionated by electrophoresis on agarose gels, transferred to nitrocellulose, and hybridized to 32P-labeled EBV DNA. The viral DNA of Raji nuclei produced a series of bands on electrophoresis whose lengths were integral multiples of a unit size, which was the same as the repeat length of host DNA. Viral DNA in nuclei of P3HR-1 and superinfected Raji cells produced faintly visible bands superimposed on a smear of viral DNA which dominated the hybridization pattern. No differences were detected in the patterns when total DNA digests from Raji, P3HR-1, and an EBV DNA-negative cell line (U-698M) were analyzed by ethidium bromide staining or by hybridization with the use of 32P-labeled lymphoblastoid cell DNA as probe. We conclude that the EBV episomal DNA of Raji cells is folded into nucleosomes, whereas most of the viral DNA of P3HR-1 and superinfected Raji cells is not. This pattern of DNA organization differs signficantly from that in papova group viruses.     

Cocaine potentiates the switch between latency and replication of Epstein-Barr virus in Raji cells.

This paper shows that cocaine amplifies Epstein-Barr virus (EBV) reactivation in Raji cells. Its effect on early viral protein synthesis was maximal when it was added with 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus n-butyrate, but nil when added alone. The enhancing effect of cocaine on early replicative stages of latent EBV was associated with an increase of Ca(2+) mobilization induced by the drug and with an induction of cellular protein phosphorylation in chemicals and cocaine-treated Raji cells. Cocaine also acted synergistically with TPA and n-butyrate to induce Z Epstein-Barr replication activator (ZEBRA), a nuclear phosphoprotein responsible for the activation of early viral gene expression. These findings provide the first evidence that cocaine may represent an important co-factor in the reactivation of early stages of latent EBV infection.