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At least 829 records · Page 46Linked to original sources
PubMed1989

Replication of baboon endogenous virus in human cells. Kinetics of DNA synthesis and integration.

For the baboon endogenous virus to infect human cells a specific region on chromosome 6 is required for viral DNA replication and integration. In studying the kinetics of baboon endogenous virus DNA replication we show that linear DNA was synthesized as the predominant species after infection and that unintegrated DNA persisted after many cell passages. Examination of integrated DNA revealed the failure of the virus to integrate at early passages. With continuous replication, however, virus integration was observed, but at multiple sites in the host cell.

Animals↗
PubMed1990

History, precedent, and progress in the development of mammalian cell culture systems for preparing vaccines: safety considerations revisited.

The use of cell substrates to propagate viruses or recombinant plasmids for vaccine productions has been the subject of long evolutionary conflict, primarily from the standpoint of product safety, and especially from the viewpoint of cancer induction. The present concern is for safety of vaccines made using transformed or neoplastic mammalian cells that may contain endogenous contaminating viruses or integrated gene sequences from oncogenic viruses. There is also concern for use of plasmid vectors employing promoter elements from oncogenic viruses. The principal concern for safety lies with retention of residual DNA in the vaccine, especially since induction of cancer is a single-cell phenomenon, and a single functional unit of foreign DNA integrated into the host cell genome might serve to induce cell transformation as a single event or part of a series of multifactorial events. Current proposed standards for vaccines would permit contamination with up to 100 pg of heterologous DNA per dose. This is equivalent to about 10(8) "functional lengths" of DNA. Total safety would seem to require complete absence of DNA from the product. While preparation of biologicals used to treat serious disease might demand the use of mammalian cells, this is not the situation with vaccines that are given prophylactically to persons who might be given equally efficacious vaccines produced in bacterial cells or in yeast that have attributes for greater safety. Careful assessment of safety and risk vs. benefit of continuous mammalian cell-produced vaccines should be made by technically expert scientists in the relevant disciplines and a consensus needs to be evolved in the scientific community at large.

Animals↗
PubMed2006

[Sequence analysis of integration sites of reticuloendotheliosis virus LTR in fowlpox vaccine virus genomes].

By use of genomic DNA prepared from 5 fowlpox virus (FPV) vaccines made in China (from Shandong, Beijing, Liaoning, Zhejing and Shanghai respectively) as the templates, reticuloendotheliosis virus (REV) LTR was amplified in PCR with a pair of primers synthesized according to the sequences flanking the integrated REV-5'LTR in FPV genomes published in US and Australia. Sequence analysis indicated that the REV-5'LTR integration sites in genomes of all 5 Chinese PFV vaccine products were identical to American and Australian FPV vaccines with integrated REV-5' LTR. Among the 5 FPV vaccine products made in China, 3 productes Vac-B-Ch, Vac-D-Ch and Vac-E-Ch had the integrated REV-5' LTR sequences of 223bp with 100% homology to that in American vaccine Vac-3-Am and Australian vaccine Vac-M3-Au. The integrated 505bp REV-LTR sequences in another Chinese products Vac-A-Ch and Vac-C-Ch had 99.6% homology to the integrated REV-LTR of American vaccine Vac-1-Am and Australian vaccine Vac-S-Au. However, REV-5'LTR integrated in all 5 FPV vaccines made in China gave only 75.4%-91.5% homology to LTR of a Chinese field strain HA9901 of REV. Based on the above results, it is reasonable to speculate that the virus stocks for the 5 Chinese FPV vaccine products were rather originally imported with their integrated REV LTR than recombinated with LTR of REV local field strains in China.

China↗
PubMed2003

Intermediate filament integrity is required for Junin virus replication.

The role of the cytoskeletal framework in Junin virus (JUNV) replication has already been demonstrated with compounds interfering with the microfilament (MF) and microtubule (MT) networks. In this work, we evaluated the role of intermediate filaments (IF) during JUNV infection. We tested the effect of acrylamide, a compound that selectively disrupts IF, in culture of three different cell types: Vero cells, murine astrocytes and human foreskin fibroblasts. Perturbation of intermediate filaments had an inhibitory effect on JUNV production within a range of acrylamide concentration of 0.5-3mM in a dose-dependent manner, without cell viability modification. Recovery experiments showed that viral production was partially increased when medium containing acrylamide was replaced by normal maintenance medium (MM). The adsorption and internalization steps were not affected by IF disruption. The expression of JUNV proteins was highly reduced in the presence of 2mM acrylamide while immunofluorescence staining of IF showed network disruption with the formation of cytoplasmic aggregates containing vimentin or glial fibrillary acidic protein (GFAP). We conclude that the IF network may play a role in the early step of JUNV multiplication, subsequent to virus entry and that its integrity is a necessary condition for the normal replication of JUNV in neural and fibroblast cells as well as in the Vero cell line.

Acrylamide↗
PubMed2003

Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA.

In latent adeno-associated virus (AAV) infection, the viral genome is integrated preferentially into the human chromosome 19 q arm at a specific region designated AAVS1, which has an open chromatin conformation as indicated by the presence of a DNase I-hypersensitive site (DHS-S1). We examined whether an insulator, which defines the domain of gene expression by directionally blocking the action of enhancers and by preventing the spread of heterochomatin, is present near the DHS-S1 in the middle of a 2.6-kbp AAVS1-related DNA fragment used in this study. The fragment, cloned into an Epstein-Barr virus (EBV)-based eukaryotic episomal plasmid, was introduced into HEK293 cells. The DHS-S1 on the plasmid replicating in the nuclei was hypersensitive to DNase I digestion, and thus, the EBV plasmid system was used in an enhancer-blocking assay with the 2.6-kbp DNA and two shortened DNAs, of 1.6 kbp and 336 bp, containing DHS-S1. The three DNA fragments, when inserted in the proper direction between the cytomegalovirus immediate-early enhancer and minimal promoter, repressed the expression of a reporter gene. Thus, the enhancer-blocking activity was located within the 336-bp DNA containing the entire region (300 bp) of DHS-S1. To investigate the prevention of repression caused by heterochromatin, a transgene-expressing cassette flanked by the two 336-bp DNAs placed in the enhancer-blocking direction was introduced into HEK293 and HeLa cells. All the cell clones examined with the cassette integrated into cell DNA continued to express the transgene, which indicates that the pair of 336-bp DNA apparently prevented the spread of heterochromatin. The results show that an insulator lies between nucleotides 17 and 354 near the DHS-S1 in AAVS1. In a gel shift test, the 336-bp DNA did not bind an in vitro-prepared CCCTC-binding factor that binds to the chicken beta-globin insulator, suggesting that the AAVS1 insulator requires an as yet unidentified binding protein. The newly identified AAVS1 insulator is likely to contribute to the maintenance of an open chromatin conformation that affects the life cycle of AAV.

Animals↗
PubMed1992

Modulation of the cellular phenotype by integrated adeno-associated virus.

The adeno-associated virus (AAV) rep gene encodes a series of overlapping, multifunctional, nonstructural proteins (Rep proteins) which regulate the viral life cycle and which are also capable of trans-regulating nonviral gene expressions (reviewed in Berns, 1990, Microbiol. Rev. 54, 316-329). To investigate the expression of the AAV rep gene in a cellular chromosomal context, SV40-transformed Chinese hamster embryo (OD4) cells were infected with an AAV/neo hybrid virus and progeny resistant to the antibiotic G418 were selected and amplified. Chromosomal integration and RNA transcription of the AAV and neo DNA inserts were confirmed by Southern and Northern blotting procedures. One of the G418R cell lines stably expressed a protein which reacted specifically with AAV anti-Rep antiserum in Western immunoblots. The stable integration of AAV rep DNA, which did not interfere with cell proliferation under normal growth conditions, was associated with two changes in cellular phenotype: eight of nine lines were markedly more sensitive to UV light (254 nm) than were the parental OD4 cells; and seven of the nine lines had lost the capacity to promote SV40 origin DNA amplification in vitro, in contrast to the parental OD4 cells. OD4 cells transformed to G418R by AAV/neo DNA constructs with a deleted rep gene, or by a neo DNA construct lacking AAV DNA, did not display these phenotypic changes. It is suggested that stable integration of the AAV rep gene interferes with cellular processes connected with DNA repair and gene amplification.

Animals↗
PubMed1994

Stably integrated mouse mammary tumor virus long terminal repeat DNA requires the octamer motifs for basal promoter activity.

In the mouse mammary tumor virus promoter, a tandem of octamer motifs, recognized by ubiquitous and tissue-restricted Oct transcription factors, is located upstream of the TATA box and next to a binding site for the transcription factor nuclear factor I (NF-I). Their function was investigated with mutant long terminal repeats under different transfection conditions in mouse Ltk- cells and quantitative S1 nuclease mapping of the transcripts. In stable transfectants, which are most representative of the state of proviral DNA with respect to both number of integrated DNA templates and chromatin organization, a long terminal repeat mutant of both octamer sites showed an average 50-fold reduction of the basal transcription level, while the dexamethasone-stimulated level was unaffected. DNase I in vitro footprinting assays with L-cell nuclear protein extracts showed that the mutant DNA was unable to bind octamer factors but had a normal footprint in the NF-I site. I conclude that mouse mammary tumor virus employs the tandem octamer motifs of the viral promoter, recognized by the ubiquitous transcription factor Oct-1, for its basal transcriptional activity and the NF-I binding site, as previously shown, for glucocorticoid-stimulated transcription. A deletion mutant with only one octamer site showed a marked base-level reduction at high copy number but little reduction at low copies of integrated plasmids. The observed transcription levels may depend both on the relative ratio of transcription factors to DNA templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting.

Animals↗
PubMed2002

Chromosomal integration of Epstein-Barr virus genomes in nasopharyngeal carcinoma cells.

BACKGROUND: Little has been known about whether Epstein-Barr virus (EBV) could persist in nasopharyngeal carcinoma (NPC) cells by chromosomal integration, and no NPC cell line harboring integrated EBV has been reported. In this study, we explored this issue through isolating EBV-infected NPC cell clones generated from an in vitro infection system and examining the configuration of EBV DNA in these cells. METHODS AND RESULTS: EBV genomes were demonstrated in NPC cell clones using polymerase chain reaction and Southern hybridization. Viral nuclear antigens were also detected by use of an anticomplement immunofluorescence assay and an immunoblotting assay. Gardella gel analysis showed that two of the EBV-positive cell clones, H2B4 and H2B17-7, harbored no extrachromosomal form of the viral genome. Restriction analysis of EBV genomic termini indicated that EBV DNA in these two cell clones was not circularized, and the viral genomes were integrated into chromosomes as demonstrated by fluorescence in situ hybridization. CONCLUSIONS: This is the first in vitro model of EBV persistence in NPC cells by genomic integration, which represents a unique state of virus-cell interaction. Using this model, investigation into the association between EBV integration and chromosomal abnormality in tumor cells will help to reveal the underlying biologic significance.

Carcinoma↗
PubMed2001

Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms.

Jumping translocations (JTs) and segmental jumping translocations (SJTs) are unbalanced translocations involving a donor chromosome arm or chromosome segment that has fused to multiple recipient chromosomes. In leukemia, where JTs have been predominantly observed, the donor segment (usually 1q) preferentially fuses to the telomere regions of recipient chromosomes. In this study, spectral karyotyping (SKY) and FISH analysis revealed 188 JTs and SJTs in 10 cell lines derived from carcinomas of the bladder, prostate, breast, cervix, and pancreas. Multiple JTs and SJTs were detected in each cell line and contributed to recurrent unbalanced whole-arm translocations involving chromosome arms 5p, 14q, 15q, 20q, and 21q. Sixty percent (113/188) of JT breakpoints occurred within centromere or pericentromeric regions of the recipient chromosomes, whereas only 12% of the breakpoints were located in the telomere regions. JT breakpoints of both donor and recipient chromosomes coincided with numerous fragile sites as well as viral integration sites for human DNA viruses. The JTs within each tumor cell line promoted clonal progression, leading to the acquisition of extra copies of the donated chromosome segments that often contained oncogenes (MYC, ABL, HER2/NEU, etc.), consequently resulting in tumor-specific genomic imbalances. Published 2001 Wiley-Liss, Inc.

Carcinoma↗
PubMed1994

Immunolocalization of vaccinia virus structural proteins during virion formation.

Proteolytic processing of vaccinia virus core proteins is an essential step in the formation of mature virions and occurs during the process of virion morphogenesis. In order to investigate how the vaccinia virus (VV) structural proteins become integrated into virus particles during normal maturation, immunological reagents were generated against the three major VV core proteins 4a, 4b, and 25K and their precursor molecules P4a, P4b, and P25K. These sera were used in conjunction with immunofluorescent and immunogold labeling of VV-infected tissue culture cells. The immunofluorescent results indicated that all three core precursors and their cleavage products were localized to virosomes. As the infection progressed, punctate staining with these sera became spread throughout the cytoplasm which suggested that individual virion particles were being recognized. Immunoelectron microscopy showed that the core proteins were localized to the center of both immature and mature virus particles. This result was in contrast to the situation observed using antisera directed against L65, a protein previously implicated in the assembly of the viral membrane. Immunogold staining of L65 showed that it was initially located along the inner side of the immature virion membrane and remained with the membrane even as the viroplasm began to condense toward the center of the virus particle. In order to determine whether the core protein localization observed was the result of precursors, products, or both, a synthetic peptide strategy was used to generate an antiserum that recognized only P4a in immunoprecipitation reactions. Immunogold labeling with this reagent indicated that P4a was found in the viroplasm of immature particles and in low levels in the mature virion. Intracellular localization of core and L65 proteins during virion morphogenesis is discussed.

Amino Acid Sequence↗
PubMed1993

SSV1-encoded site-specific recombination system in Sulfolobus shibatae.

We present evidence for the existence of a conservative site-specific recombination system in Archaea by demonstrating integrative recombination of Sulfolobus shibatae virus SSV1 DNA with the host chromosome, catalysed by the SSV1-encoded integrase in vitro. The putative int gene of SSV1 was expressed in Escherichia coli yielding a protein of about 39 kDa. This protein alone efficiently recombined linear DNA substrates containing chromosomal (attA) and viral (attP) attachment sites; recombination with either negatively or positively supercoiled SSV1 DNA was less efficient. Intermolecular attA x attA and attP x attP recombination was also promoted by the SSV integrase. The invariant 44 bp "common attachment core" present in all att sites contained sufficient information to allow recombination, whilst the flanking sequences effected the efficiency. These features clearly distinguish the SSV1--encoded site--specific recombination system from others and make it suitable for the study of regulatory mechanisms of SSV1 genome--host chromosome interaction and investigations of the evolution of the recombination machinery.

Bacteriophages↗
PubMed1992

Genetic alterations by human papillomaviruses in oncogenesis.

The integration sites in the cellular genome of human papillomavirus are located in chromosomal regions always associated with oncogenes or other known tumor phenotypes. Two regions, 8q24 and 12q13, are common to several cases of cervical carcinoma and can have integrated more than one type of papillomavirus DNA. These two chromosomal regions contain several genes implicated in oncogenesis. These observations strongly imply that viral integration sites of DNA tumor viruses can be used as the access point to chromosomal regions where genes implicated in the tumor phenotype are located, a situation similar to that of non-transforming retroviruses.

Chromosomes, Human, Pair 12↗
PubMed2005

The Oryctes virus: its detection, identification, and implementation in biological control of the coconut palm rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae).

In view of the increasing and devastating damage by rhinoceros beetle (Oryctes rhinoceros) to coconut palms in the middle of last century, many efforts were made to find an efficient natural control factor against this pest, which could not be controlled by pesticides. The basic procedures of these monitoring programmes are outlined together with the final detection of a virus disease in oil palm estates in Malaysia in 1963. In extensive laboratory studies, the virus was isolated and identified as the first non-occluded, rod-shaped insect virus, morphologically resembling the baculoviruses. Infection experiments clarified the pathology, histopathology, and virulence of the virus and demonstrated that the virus was extremely virulent to larvae after peroral application. These findings encouraged the first pilot release of virus in 1967 in coconut plantations of Western Samoa where breeding sites were contaminated with virus. Surprisingly, the virus became established in the Samoan rhinoceros beetle populations and spread autonomously throughout the Western Samoan islands. As a consequence, there was a drastic decline of the beetle populations followed by a conspicuous recovery of the badly damaged coconut stands. This unexpected phenomenon could only be explained after it was shown that the adult beetle itself is a very active virus vector and thus was responsible for the efficient autodissemination of the virus. The functioning of the beetle as a 'flying virus factory' is due to the unique cytopathic process developing in the midgut after peroral virus infection. Pathological details of this process are presented. Because of the long-term persistence of the virus in the populations, rhinoceros beetle control is maintained. Incorporation of virus into integrated control measures and successful virus releases in many other countries are recorded.

Animals↗
PubMed2005

Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase.

The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3'-processing [dinucleotide released from each 3' end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the 13C/15N natural abundance. The combination of these two types of parameters in calculations significantly improved the DNA structure determination. The well-known features of A-tracts were clearly identified by RDCs in the first part of the molecule. The binding/cleavage site at the viral DNA end is distinguishable by a loss of regular base stacking and a distorted minor groove that can aid its specific recognition by IN.

Binding Sites↗
PubMed1997

Avian leukemias and lymphomas: interplay between retroviruses and herpesviruses.

Avian leukemias and lymphomas are caused primarily by retroviruses and herpesviruses. The protooncogenes activated by avian retroviral insertions in B & T-cell lymphomas will be summarized, with discussion on a new common insertion site, bravo, associated with RAV-O LTR insertion. Two novel interactions between avian retroviruses and Marek's disease herpesvirus (MDV) will be described: one involves direct interactions between putative viral oncoproteins and the other integrative recombination between these two viruses.

Alpharetrovirus↗
PubMed1986

Stable integration of woodchuck hepatitis virus DNA in transplanted tumors and established tissue culture cells derived from a woodchuck primary hepatocellular carcinoma.

The fate of integrated woodchuck hepatitis viral (WHV) DNA was systematically investigated in DNA samples from primary hepatocellular carcinoma (HCC) of woodchucks, solid tumors transplanted in athymic mice derived from a primary HCC of woodchuck, and an established cell line of tissue culture originating from the transplanted tumor. In four of five woodchuck primary HCCs, WHV DNA integration was demonstrated in addition to various amounts of extrachromosomal replicative intermediate WHV DNA. The integration pattern of the primary HCCs does not indicate a common integration site on the host chromosome. The integration pattern in the established cells is identical to that in the transplanted tumor and similar but slightly different from that of the primary HCC. No extrachromosomal or replicative intermediates of WHV DNA were detected in the transplanted tumors or in the established cells of tissue culture. There are three integration sites on the chromosomes of the established cells. Results of Southern hybridization and restriction maps of cloned fragments suggest that all of these integrated WHV DNA sequences are not a complete genome but a part of the genome. A small portion corresponding to the cohesive region of the genome was not detected in all of these integrated WHV DNA. A positive role of WHV DNA integration on the generation of HCC is strongly suggested by the high incidence of WHV DNA integration in woodchuck primary HCCs and the stable maintenance of a certain mode of WHV DNA integration in the hepatoma-derived cell populations during passages of transplantation or serial growth of tissue culture.

Animals↗
PubMed1988

Integration of bovine leukaemia virus in the ovine genome.

Circulating lymphocytes and tumour cells from 12 sheep experimentally infected with bovine leukaemia virus (BLV), for periods of time varying from 9 to 48 weeks, were analysed for evidence of integrated and unintegrated provirus. Hybridization analysis demonstrated that the provirus was integrated at one or two sites in all cases. Integration was observed at different sites in the animals studied and there was no evidence of unintegrated virus molecules in infected sheep lymphocytes or tumour cells. The data obtained support a monoclonal origin of different tumours in the same sheep.

Animals↗
PubMedDate not supplied

Detection of hepatitis B virus DNA in hepatocellular carcinoma: methylation of integrated viral DNA.

In order to determine whether integrated hepatitis B virus DNA sequences in primary liver tumours are methylated we have analysed tumour DNA digested with either MspI or HpaII restriction endonuclease by Southern hybridization. Our results demonstrate methylation in 11 of 17 tumour DNA samples. Where possible, we have examined the tumour tissues for expression of HBsAg and HBcAg using the indirect immunoperoxidase technique. One tumour was positive for both HBsAg and HBcAg and a second was positive for HBsAg alone. Both of these tumours were in the group in which methylation of integrated HBV DNA sequences could not be detected. We postulate that methylation of integrated HBV DNA sequences may influence HBV gene expression in hepatocellular carcinoma.

Carcinoma, Hepatocellular↗