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At least 1,369 records · Page 76Linked to original sources
PubMed2000

Combined use of the adenosine A(2A) antagonist KW-6002 with L-DOPA or with selective D1 or D2 dopamine agonists increases antiparkinsonian activity but not dyskinesia in MPTP-treated monkeys.

The novel selective adenosine A(2A) receptor antagonist KW-6002 improves motor disability in MPTP-treated parkinsonian marmosets without provoking dyskinesia. In this study we have investigated whether KW-6002 in combination with l-DOPA or selective D1 or D2 dopamine receptor agonists enhances antiparkinsonian activity in MPTP-treated common marmosets. Combination of KW-6002 with the selective dopamine D2 receptor agonist quinpirole or the D1 receptor agonist SKF80723 produced an additive improvement in motor disability. Coadministration of KW-6002 with a low dose of L-DOPA also produced an additive improvement in motor disability, and increased locomotor activity. The ability of KW-6002 to enhance antiparkinsonian activity was more marked with L-DOPA and quinpirole than with the D1 agonist. However, despite producing an enhanced antiparkinsonian response KW-6002 did not exacerbate L-DOPA-induced dyskinesia in MPTP-treated common marmosets previously primed to exhibit dyskinesia by prior exposure to L-DOPA. Selective adenosine A(2A) receptor antagonists, such as KW-6002, may be one means of reducing the dosage of L-DOPA used in treating Parkinson's disease and are potentially a novel approach to treating the illness both as monotherapy and in combination with dopaminergic drugs.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

Antiparkinsonian actions of ifenprodil in the MPTP-lesioned marmoset model of Parkinson's disease.

Dopamine-replacement strategies form the basis of most symptomatic treatments for Parkinson's disease. However, since long-term dopamine-replacement therapies are characterized by many side effects, most notably dyskinesia, the concept of a nondopaminergic therapy for Parkinson's disease has attracted great interest. To date, it has proved difficult to devise a nondopaminergic therapy with efficacy comparable to that of dopamine replacement. In animal models of Parkinson's disease, loss of striatal dopamine leads to enhanced excitation of striatal NR2B-containing NMDA receptors. This is responsible, in part at least, for generating parkinsonian symptoms. Here we demonstrate that, in the MPTP-lesioned marmoset, monotherapy with the NR2B-selective NMDA receptor antagonist, ifenprodil, administered de novo, has antiparkinsonian effects equivalent to those of l-DOPA (administered as its methyl ester form). In MPTP-lesioned marmosets, median mobility scores, following vehicle-treatment were 12.5/h (range 6-21), compared to 61/h (range 26-121) in normal, non-MPTP-lesioned animals. Following ifenprodil (10 mg/kg) treatment in MPTP-lesioned marmosets, the median mobility score was 66/h (range 34-93), and following l-DOPA (10 mg/kg i.p.) treatment 89/h (range 82-92). The data support the proposal that NR2B-selective NMDA receptor antagonists have potential as a nondopaminergic monotherapy for the treatment of parkinsonian symptoms when given de novo.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

The antioxidant ebselen prevents neurotoxicity and clinical symptoms in a primate model of Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), via its major metabolite 1-methyl-4-phenylpyridinium (MPP(+)), produces in primates including humans clinical, biochemical, and neuropathological changes similar to those which occur in idiopathic Parkinson's disease. Ebselen is an antioxidant drug with glutathione peroxidase-like activity and a proven neuroprotective action in stroke patients. Here we show that Ebselen, when administered before, during, and after MPTP injections, prevents both neuronal loss and clinical symptoms in a primate MPTP model of Parkinson's disease. Ebselen also prevents peroxide radical overproduction induced by serum withdrawal in cultured PC12 cells and hydroxyl radical generation induced by the mitochondrial toxin, MPP(+), in vivo in rat brain. Moreover, Ebselen inhibits MPP(+)-induced toxicity in PC12 cells, without interacting with the dopamine uptake system. Our results demonstrate that compounds which prevent mitochondrial dysfunction and free radical production may be useful as preventive treatment of Parkinson's disease.

1-Methyl-4-phenylpyridinium↗
PubMed2001

Coadministration of (-)-OSU6162 with l-DOPA normalizes preproenkephalin mRNA expression in the sensorimotor striatum of primates with unilateral 6-OHDA lesions.

The substituted phenylpiperidine (-)-OSU6162 is a novel modulator of the dopaminergic systems with low affinity for dopamine D(2) receptors and potent normalizing effects on l-DOPA-induced dyskinesias. We studied the effects of coadministration of (-)-OSU6162 with l-DOPA on the regulation of striatal preproenkephalin (PPE) and prodynorphin (PDyn) mRNA expression in the primate brain by in situ hybridization histochemistry. Common marmoset monkeys sustaining unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway received l-DOPA/carbidopa, l-DOPA/carbidopa plus (-)-OSU6162, or vehicle over 14 days. In vehicle-treated animals, PPE mRNA levels were markedly increased in the sensorimotor territory of the lesioned striatum. By contrast, a rather uniform lesion-induced reduction of PDyn mRNA levels was found in the vehicle group. Subchronic l-DOPA treatment induced a further increase in PPE mRNA expression in a number of sensorimotor and associative subregions of the denervated striatum. Coadministration of (-)-OSU6162 with l-DOPA partially reversed the lesion- and l-DOPA-induced elevation of PPE expression and, by affecting PPE mRNA expression differentially on the intact and lesioned striatum, markedly reduced the side-to-side difference in PPE mRNA expression. The effects on PPE mRNA expression were apparent throughout the rostrocaudal extent of the putamen and the dorsal portions of the caudate nucleus. l-DOPA treatment resulted in an enhancement in PDyn mRNA expression in all functional compartments of the striatum. Coadministration of (-)-OSU6162 had no apparent influence on these l-DOPA-induced changes in PDyn mRNA expression. The present results suggest that (-)-OSU6162 acts primarily by modifying striatal output via the indirect pathway.

Animals↗
PubMed2001

Mu- and delta-opioid receptor antagonists reduce levodopa-induced dyskinesia in the MPTP-lesioned primate model of Parkinson's disease.

Long-term treatment of Parkinson's disease with levodopa is complicated by the emergence of involuntary movements, known as levodopa-induced dyskinesia. It has been hypothesized that increased opioid transmission in striatal output pathways may be responsible for the generation of dyskinesia. In this study, we have investigated the effect of blockade of opioid peptide transmission on levodopa-induced dyskinesia in a primate model of Parkinson's disease-the MPTP-lesioned marmoset. Coadministration of nonselective and mu- or delta-subtype-selective opioid receptor antagonists with levodopa resulted in a significant decrease in dyskinesia. There was no attenuation of the anti-parkinsonian actions of levodopa. These data suggest that specific mu- or delta-opioid receptor antagonists might be applicable clinically in the treatment of levodopa-induced dyskinesia in Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2001

Antiparkinsonian action of a delta opioid agonist in rodent and primate models of Parkinson's disease.

The opioid peptides localized in striatal projection neurons are of great relevance to Parkinson's disease, not only as a consequence of their distribution, but also due to the pronounced changes in expression seen in Parkinson's disease. It has long been suspected that increased expression of enkephalin may represent one of the many mechanisms that compensate for dopamine (DA) depletion in Parkinson's disease. Here we demonstrate that a systemically delivered, selective delta opioid agonist (SNC80) has potent antiparkinsonian actions in both rat and primate models of Parkinson's disease. In rats treated with either the D2-preferring DA antagonist haloperidol (1 mg/kg) or the selective D1 antagonist SCH23390 (1 mg/kg), but not a combination of D1 and D2 antagonists, SNC80 (10 mg/kg) completely reversed the catalepsy induced by DA antagonists. In rats rendered immobile by treatment with reserpine, SNC80 dose-dependently reversed akinesia (EC(50) 7.49 mg/kg). These effects were dose-dependently inhibited (IC(50) 1.05 mg/kg) by a selective delta opioid antagonist (naltrindole) and by SCH23390 (1 mg/kg), but not by haloperidol (1 mg/kg). SNC80 also reversed parkinsonian symptoms in the MPTP-treated marmoset. At 10 mg/kg (ip), scores measuring bradykinesia and posture were significantly reduced and motor activity increased to levels comparable with pre-MPTP-treatment scores. Any treatment that serves to increase delta opioid receptor activation may be a useful therapeutic strategy for the treatment of Parkinson's disease, either in the early stages or as an adjunct to dopamine replacement therapy. Furthermore, enhanced enkephalin expression observed in Parkinson's disease may serve to potentiate dopamine acting preferentially at D1 receptors.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1994

Lack of peroxisome proliferation in marmoset liver following treatment with ciprofibrate for 3 years.

The effect of treatment of marmosets with ciprofibrate for 3 years on activities of hepatic enzymes, hepatic histomorphology, and ultrastructure were investigated. Male and female marmosets were dosed with ciprofibrate (2, 10, and 20 mg/kg) by oral gavage once daily for 3 years. No effect on liver weight (adjusted for body weight) or liver morphology was observed. The activities of catalase, glutathione peroxidase, alpha-glycerophosphate dehydrogenase, benzphetamine N-demethylase, and ethoxyresorufin O-deethylase were unaffected by treatment with ciprofibrate. Activity of glutathione transferase was increased in the low dosage group but unaffected in the mid and high dosage groups. Modest increases in activities of peroxisomal beta-oxidation (2.5-fold, maximal), carnitine acetyl transferase (1.7-fold, maximal), and carnitine palmitoyl transferase (2-fold, maximal) were observed. Cytochemical staining and quantitative image analysis failed to indicate any effect on peroxisomal number, size, or volume density. Similarly, there was no increase in lipofuscin deposition. This study provides data on the effects of a potent peroxisome proliferator on primate liver following a dosing period much greater than that used in previously published studies and is further evidence that the marmoset is relatively insensitive to the well-documented effects that ciprofibrate and other peroxisome proliferators have on rat liver.

Animals↗
PubMed1999

Intergenic splicing between a HERV-H endogenous retrovirus and two adjacent human genes.

We previously reported that a long terminal repeat (LTR) of a human endogenous retrovirus of the HERV-H family promotes expression of a cellular fusion transcript in teratocarcinoma cell lines. This transcript was termed PLA2L due to two regions of similarity to the secreted form of phospholipase A2. In this study, evidence is presented indicating that this transcript appears to be the result of intergenic splicing between the HERV-H element and two independent downstream genes. The 5' gene has been named HHLA1 (HERV-H LTR-associating 1) and is of unknown function but shows sequence conservation in other mammals. The 3' gene is now known to encode human otoconin-90 (OC90) which, in mice, is a major protein expressed in the fetal inner ear. Evidence for intergenic splicing of these two genes includes: (1) the isolation of LTR-driven HHLA1 transcripts, unspliced to otoconin-90 exons, with variable sites of polyadenylation; (2) the cloning of both the putative human intergenic genomic region and the novel 5' terminus of the mouse otoconin-90 gene; (3) the identification of homologous potential signal sequences in the 5' region of mouse otoconin-90 and in the middle of the PLA2L transcript; and (4) the lack of detectable chromosomal rearrangements involving this region in teratocarcinoma cells. The PLA2L transcript therefore represents a rare example of intergenic splicing of two closely linked genes. We hypothesize that human HHLA1 and OC90 are normally expressed independently from different promoters but are expressed from the LTR promoter and spliced together in teratocarcinoma cells. It is tempting to speculate that the high activity of the LTR promoter in this cell type may induce transcriptional fusion between these two genes.

5' Untranslated Regions↗
PubMed1998

Epstein-Barr virus-infected marmoset cells transfected with c-myc do not form lymphomas in mice with severe combined immunodeficiency.

Epstein-Barr virus (EBV) has been associated with several malignant processes in man, most notably Burkitt lymphoma in previously healthy individuals and lesions resembling large cell non-Hodgkin lymphomas in organ transplant recipients. Mice with the severe combined immunodeficiency phenotype (SCID mice) are exquisitely susceptible to the development of EBV-associated lymphoproliferative lesions following the intraperitoneal (ip) inoculation of EBV-infected human lymphocytes. Recently, we reported that EBV-infected marmoset lymphocytes do not form lymphomas in SCID mice following ip injection, while human lymphocytes infected with the same EBV strains do. On the assumption that the EBV-infected marmoset cells were lacking a factor necessary for tumor formation, we transfected a plasmid containing c-myc into EBV-infected marmoset cells (B95-8, FF41, and W91 cells). Despite expression of the c-myc protein as determined by immunoblot and flow cytometry when probed with a monoclonal antibody, no increase over baseline lesion development was seen in SCID mice inoculated with 5 x 10(6) c-myc-expressing marmoset lymphoblastoid cells. Thus, cells that express c-myc and harbor EBV are not sufficient to form lymphomas in certain immunocompromised hosts.

Animals↗
PubMed1996

Assessment of functional impairment following permanent middle cerebral artery occlusion in a non-human primate species.

The purpose of the present study was to examine and quantify the functional consequence of a focal cerebral ischaemic lesion in a primate species, the marmoset. Following craniotomy and retraction of the frontal and temporal lobes, the middle cerebral artery was permanently occluded by means of electrocoagulation. Three and eight weeks after surgery, various behavioural tests were used to give a quantifiable measure to the neurological deficits produced. These tests required the monkeys to reach into tubes for foodbits, retrieve rewards from the steps of two designs of 'staircases', respond to one of two simultaneously presented rewarded tubes, remove adhesive labels attached to their feet, and respond to sensory stimuli. Unilateral motor impairment of the contralateral forelimb and neglect of contralateral tactile stimuli were seen in all subjects, and spatial neglect was also present in some monkeys. Subsequent histological analysis revealed unilateral cortical damage in all subjects with varying amounts of injury to the caudate and the putamen in some animals. These results demonstrate the potential for the use of this species in future investigations to examine the effect of neuroprotective treatment on functional outcome after a focal ischaemic insult.

Animals↗
PubMed1998

Subchronic toxicity of Di(2-ethylhexyl)phthalate in common marmosets: lack of hepatic peroxisome proliferation, testicular atrophy, or pancreatic acinar cell hyperplasia.

To evaluate the toxicological effect, di(2-ethylhexyl)phthalate (DEHP) was administered orally at 100, 500, and 2500 mg/kg to four male and four female marmosets in each group for 13 weeks. Its potentials of hepatic peroxisome proliferation, testicular atrophy, and pancreatic acinar cell hyperplasia were evaluated more closely. Clofibrate, which potently causes peroxisome proliferation in rodents, was administered in like manner at 250 mg/kg as a reference drug. DEHP induced significant suppression of weight gain in males at 2500 mg/kg. However, the increase in liver mass and hypertrophy of hepatocytes were not detected in organ weight measurements or histopathological examination. The number of peroxisomes, volume density, peroxisome morphology, and peroxisomal enzyme activities were not different from those in the control group, though the males treated with 500 and 2500 mg/kg DEHP showed 1.3- and 1.4-fold increases in mean peroxisome volume, respectively. In contrast, clofibrate induced 2.2 (in male)- and 1.9-fold (in female) increases in hepatic cyanide-insensitive acyl CoA oxidation system activity, 1. 2 (in male)- and 1.7-fold (in female) increases in hepatic carnitine-dependent acetyltransferase activity, and 1.8 (in male)- and 3.0-fold (in female) increases of carnitine-dependent palmitoyltransferase activity. Cytochrome P-450 contents tended to increase in all males and females administered 500 and 2500 mg/kg of DEHP and clofibrate associated with the increase in hepatic microsomal protein content, suggesting a relationship with the treatment. The atrophic change in the testis or proliferative change in the pancreatic acinar cells seen in rodents were not seen histopathologically; also, no changes were observed in testes weight, testicular zinc level, blood levels of testosterone and estradiol, pancreas weight, and blood levels of cholecystokinin. Finally, no changes considered to be due to the administration of DEHP were noted in blood chemical examination or pathological examination of other organs.

Animals↗
PubMed1993

Epstein-Barr virus glycoprotein gp85 associates with the BKRF2 gene product and is incompletely processed as a recombinant protein.

The Epstein-Barr virus (EBV) glycoprotein gp85 is the EBV gH homologue and is thought to be involved in penetration of virus through the B-cell membrane. However, although the glycoprotein is functionally important, it is found in very low abundance in infected cells and in the virion. To facilitate analysis of the structure and function of gp85, recombinant vaccinia viruses were constructed to express the glycoprotein. Recombinant gp85 was recognized by polyclonal antibody made to a peptide derived from the gp85 sequence, but not by monoclonal antibodies that reacted with the native molecule. Unlike native gp85, the recombinant protein contained no sugars that were resistant to endoglycosidase H and it was not transported to the cell surface. The native protein was found to be associated with two additional glycoproteins with apparent M(r) of 25,000 and 42,000. Antibody made to a peptide derived from a sequence in the BKRF2 open reading frame immunoprecipitated glycoproteins with the mobilities of gp85 and its associated 25,000-Da molecule. These data suggest that the BKRF2 gene product, like that encoded by its positional homologues gL of herpes simplex virus and the UL115 gene product of human cytomegalovirus, associates with gp85 and may be required for glycoprotein processing.

Animals↗
PubMed1993

An origin of DNA replication (oriP) in highly methylated episomal Epstein-Barr virus DNA localizes to a 4.5-kb unmethylated region.

Epstein-Barr virus (EBV) reveals two types of interactions with human B lymphocytes. In immortalized lymphoblastoid cell lines (LCL), nine virus-coded proteins are expressed: six nuclear (EBNA1-6) and three membrane proteins (LMP1, TP 1,2). In Burkitt's lymphoma (BL) tumors and representative cell lines (type I) only EBNA-1 is expressed. The viral genomes are carried by the cells in an extrachromosal, episomal form. We have earlier described an overall difference in EBV DNA methylation between LCLs of normal B-cell origin and type I BL lines. EBV genomes are hypomethylated in the former, methylated in the latter. The origin of replication (oriP) for the episomal DNA is exempted from this overall methylation. This unmethylated region was analyzed in the BL derived type I cell line Rael. It starts just upstream of the coding exons for the small untranslated RNAs EBER I/II. It extends throughout oriP and includes several control elements between oriP and the first promoter for EBNA transcription (BCR2). The viral DNA was methylated around and downstream of that promoter, which is not used for EBNA-1 expression in Rael.

Animals↗
PubMed1993

Regulation of Epstein-Barr virus BamHI-H divergent promoter by DNA methylation.

The regulatory mechanisms which control latency and reactivation of the Epstein-Barr virus (EBV) are not fully understood. To determine whether DNA methylation is involved, we examined the BamHI-H divergent promoter, which also encompasses the origin for lytic replication (oriLyt) of EBV. The divergent promoter was highly methylated in the stringently latent HH514cl16 cell line and largely unmethylated in the semipermissive FF41-1 cell line. Expression vectors in which the divergent promoter directed transcription of the chloramphenicol acetyltransferase gene were made. Using in vitro methylation and transient transfections, we found an inverse correlation between the number of sites methylated and level of gene expression. Similar patterns of inhibition were observed when the methylated promoter was activated by BZLF1 or BRLF1 and in lymphoid or epithelial cells. The role of two CpG dinucleotides in the BRLF1 binding sites of the divergent promoter was determined by site-directed mutagenesis. The results indicated that site-specific methylation of these CpGs was not solely responsible for inhibition of expression by methylation. DNA methylation also reduced DNA replication mediated by oriLyt. These results suggest that hypermethylation of the divergent promoter and oriLyt may suppress transcription and lytic replication of EBV.

Animals↗
PubMed1994

Modulation of vimentin, the CD40 activation antigen and Burkitt's lymphoma antigen (CD77) by the Epstein-Barr virus nuclear antigen EBNA-4.

The growth transformation of human B cells by Epstein-Barr virus (EBV) is controlled by the coordinate expression of 10 latent viral genes. This transforming capacity is believed to be fundamental to the involvement of the virus in human malignancies of B cell origin. EBV-negative Burkitt's lymphoma (dG75) clones stably expressing one of these EBV-coded antigens, EBNA-4, have been established using an episomal-based plasmid. EBNA-4 expression was found to upregulate the cytoskeletal protein vimentin as well as surface expression of the activation antigen CD40. In addition, the presence of EBNA-4 resulted in downregulation of the Burkitt's lymphoma-associated antigen (BLA/CD77). These studies show for the first time that EBNA-4 modulates the expression of several cellular genes implicated in cell-growth transformation.

Animals↗
PubMed1995

HERV-H endogenous retroviruses: presence in the New World branch but amplification in the Old World primate lineage.

The evolutionary origin and age of the HERV-H family of human endogenous retrovirus-like sequences was investigated in this study. HERV-H elements exist in approximately 900 partially deleted copies and 50-100 more intact forms in humans and Old World monkeys. However, their possible presence in more divergent species is unknown. We have isolated a 1.6-kb genomic DNA segment from the New World monkey marmoset that had been PCR amplified using human HERV-H primers. DNA and protein comparisons and database searches indicate that this marmoset clone is more closely related to human HERV-H elements than to any other sequence, indicating that HERV-H-related sequences do exist in New World monkeys. In contrast to the high copy numbers of deleted elements in Old World primates. Southern blot analysis shows that such elements are present in less than 50 copies in two different species of New World monkey. To estimate evolutionary ages of the common deleted form of the element, a selected DNA segment from the pol region was compared from multiple human HERV-H elements. This comparison suggests that many HERV-H elements of the abundant deleted subfamily integrated approximately 30-35 million years ago. Very similar percentage divergence values between 5' and 3' long terminal repeats of individual elements of the deleted subfamily also suggest that these elements are close in age. These results indicate that HERV-H elements first appeared in the germline prior to the New World/Old World divergence over 40 million years ago. Interestingly, they remained in low numbers in the New World branch while a subfamily underwent a major amplification in Old World primates before the time of divergence of hominoids from Old World monkeys.

Animals↗
PubMed1995

Transmissible retrovirus in Epstein-Barr virus-producer B95-8 cells.

Epstein-Barr virus (EBV) released from the B95-8 marmoset cell line has served as a prototype for biologic and biochemical studies of EBV. Here we identify and characterize a retrovirus carried by many cultures of B95-8 cells. The experiments were stimulated by the isolation of a cDNA clone from B95-8 cells in which sequences from the EBV large internal repeat were linked to gag sequences similar to those of squirrel monkey retrovirus, human isolate, SMRV-H. However, among 413 amino acids predicted from the nucleotide sequence of the gag region of the B95-8 SMRV isolate there were 48 amino acid changes that distinguished this virus from SMRV-H originally isolated from a human lymphoid cell line by Oda et al. (1988, Virology 167, 468-476). Nucleic acid and antibody probes were developed for the B95-8 isolate of SMRV. Using such probes, we found that SMRV-B95-8 was readily transmissible, independent of EBV, as an infectious virus to human B and T cell lines. SMRV-B95-8 was highly fusogenic in the presence or absence of EBV. The ultrastructural appearance of the B95-8 retrovirus was characteristic of a type D retrovirus. Cells dually infected with EBV and SMRV-B95-8 did not demonstrate increased levels of lytic EB viral replication. SMRV-B95-8 did not by itself cause lymphocyte immortalization or enhance immortalization by EBV. Thus SMRV-B95-8 does not contribute to the major biologic properties of the B95-8 strain of EBV.

Amino Acid Sequence↗
PubMed1996

Epstein-Barr virus single-stranded DNA-binding protein: purification, characterization, and action on DNA synthesis by the viral DNA polymerase.

Epstein-Barr virus (EBV) BALF2 gene product is one of the essential components in the lytic phase of the EBV DNA replication. The BALF2 protein was purified to near homogeneity from the nuclear extract of B95-8 cells with virus productive cycle induced by chemical agents. SDS-polyacrylamide gel electrophoresis showed the presence of a single polypeptide with a molecular weight of 130 K, which was identified as BALF2 protein by Western immunoblot analysis. On Superose 6 HR 10/30 gel filtration the BALF2 protein eluted at a position corresponding to an apparent molecular mass of approximately 128 K, indicating that the BALF2 protein behaves as a monomer in solution. The purified BALF2 protein bound to single-stranded DNA preferentially over double-stranded DNA or single-stranded RNA. Replication of singly primed M13 single-stranded DNA by the EBV DNA polymerase complex in the absence of the BALF2 protein exhibited a highly processive mode of replication and generated full length products in addition to some bands of pausing sites. Although the addition of the BALF2 protein did not affect the replication rate, the average chain length of the replication products was slightly increased with eliminating bands of pausing sites. Similar effects were observed with the reconstituted polymerase complex composed of the BALF5 and BMRF1 Pol subunits. On the other hand, in the absence of the BALF2 protein, the BALF5 Pol catalytic subunit alone extended the primer slightly and paused at specific sites on M13 ssDNA template where stable secondary structure is predicted. However, addition of the BALF2 protein, in contrast to the case of herpes simplex virus ICP8 which does not affect the overall distribution of length of the replication products synthesized by the HSV Pol catalytic subunit (Gottlieb et al., 1990, J. Virol. 64, 5976-5987), stimulated DNA synthesis and yielded a distribution of replication products with long lengths in addition to full length products. Although the BALF2 protein behaved as if it converts a low processive enzyme of the EBV Pol catalytic subunit to a highly processive form like the BMRF1 Pol accessory subunit, challenger DNA experiments revealed that the EBV Pol catalytic subunit is transferred to challenger DNA even in the presence of the BALF2 protein. It is therefore likely that the EBV BALF2 protein functions to melt out the regions of secondary structure on the single-stranded DNA template, thereby reducing and eliminating pausing of the EBV DNA polymerase at specific sites. These properties indicate that the EBV BALF2 protein acts as a single-stranded DNA-binding protein during lytic phase of EBV DNA replication.

Animals↗