Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Summary Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV 16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF-or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidinbiotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV 16 DNA and haptenized HSV2 DNA.     

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF- or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidin-biotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV16 DNA and haptenized HSV2 DNA.     

Identification of transcribed regions of Epstein-Barr virus DNA in Burkitt lymphoma-derived cells.

RNA was extracted from the Burkitt lymphoma-derived cell line Raji and from Burkitt lymphoma tumor biopsies, isotope labeled in vitro by iodination with 125I, and hybridized to electrophoretically separated restriction endonuclease fragments of Epstein-Barr virus DNA on nitrocellulose membranes. The results indicated that only certain parts of the Epstein-Barr virus genome are represented as polyribosomal RNA in Raji cells, with a pronounced dominance of RNA sequences complementary to a 2.0 x 10(6)-dalton segment of Epstein-Barr virus DNA located close to the left end of the viral genome. A map of virus-specific polyribosomal RNA sequences was constructed, which indicated that a minimum of three regions of the Epstein-Barr virus genome are expressed in Raji cells. Total-cell RNA preparations from five Burkitt lymphoma biopsies contained RNA sequences homologous to the same regions of Epstein-Barr virus DNA as polyribosomal RNA from Raji cells, albeit at different relative proportions.     

Resolution and characterization of intracytoplasmic forms of reverse transcriptase from Rauscher leukemia virus-producing cells.

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Similar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency integration.     

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.     

Molecular evidence for a type C retrovirus etiology of the lymphoproliferative disease of turkeys.

Recently, we isolated from the blood of lymphoproliferative disease (LPD)-affected turkeys a type C retrovirus distinct from the avian leukosis-sarcoma virus complex and the reticuloendotheliosis virus group. We present molecular evidence for the implication of this virus in the LPD of turkeys. Using complementary DNA of LPD viral RNA, we found that the LPD viral genome is specifically and efficiently transcribed (2,500 copies per cell) in LPD tumor cells. Moreover, the LPD tumor cells contained newly inserted LPD viral information (5 to 10 copies per haploid genome), which was not present before the infection. From the absence of LPD virus-specific sequences in the normal cell genome of turkeys, it was concluded that the LPD virus is not an endogenous virus of turkeys. DNA-DNA annealing experiments revealed that the degree of sequence homology between LPD viral complementary DNA and cellular DNA of turkeys was not higher than that between LPD viral complementary DNA and cellular DNA of other species, thus indicating that the virus does not originate from turkeys.     

Production and characteristics of seven rat embryo cell lines transformed by adenovirus type 5 and its DNA

Seven cell lines transformed by adenovirus type 5 and its DNA were obtained. It was shown that different cell lines contain the fragments of viral DNA which differ in length and number of copies per DNA of diploid cells. They contain from the left end 6% of the viral DNA to complete or almost complete viral genome. All studied cell lines were sensitive to reinfection with adenovirus type 5. They produced no virus being cocultivated with cell sensitive to the virus. No cell line was able to induce tumors even in immunosuppressed newborn rats. All cell lines formed colonies in soft agar. The level of virus-specific antigens was higher in cells that contained a large part of the viral genome. The methods used did not allow to correlate the biological properties of the transformed cells with the length and the number of copies of the integrated part of the viral genome.     

应用两种基因组快速扩增方法进行病毒芯片杂交鉴定

为了摸索均衡的病毒基因组扩增方法,建立高通量的病毒检测基因芯片技术平台,本研究以甲病毒属的辛德比斯病毒作为检测模型,分别以随机PCR扩增法和MDA( Multiple Displacement Amplification)扩增法扩增病毒基因组,并以两种扩增产物作为模板,扩增辛德比斯病毒的特异基因片段以验证基因组扩增的均衡性;然后将两种基因组扩增产物标记荧光染料后与基因芯片进行杂交;结果表明从两种基因组扩增产物中正确扩增出了辛德比斯的特定基因片段,作为探针可与基因芯片上的靶标基因特异性结合;基因组扩增产物与基因芯片进行杂交,可成功检测到甲病毒属的特异性信号,充分说明随机PCR扩增法和MDA扩增法用于扩增病毒基因组均具有良好的均衡性,扩增产物可用于病毒性病原体的基因芯片检测。     

采用基因微阵列技术对常见呼吸道病毒进行检测的初步研究

建立一种高通量的基因微阵列检测技术,对常见呼吸道病毒感染进行监控.根据公开发表的8个病毒科38种常见呼吸道病毒的序列,计算其保守区域,设计病毒的特异性检测探针,制备呼吸道病毒检测基因微阵列.利用随机引物PCR方法标记样品中的病毒靶序列,标记产物与基因微阵列上的探针杂交,清洗、扫描后进行结果分析.采用流感病毒、麻疹病毒、腮腺炎病毒和风疹病毒作为报告病毒,并对80例上呼吸道感染患者的咽拭子标本进行验证测试.初步结果表明,该呼吸道病毒微阵列基因芯片检测是可行的,在利用基因微阵列技术对病毒监控方面进行了有益的尝试,得到了有经验的信息.     

Host restriction of friend leukemia virus; fate of input virion RNA

Host restriction of oncogenesis by RNA tumor viruses may be studied in vitro by measuring the replication of the lymphatic leukemia component of the Friend Virus Complex (LLV-F) in either NIH-Swiss or Balb/C mouse embryo cells. These cells derive from mice differing at the Fv-1 locus, which controls the replication of all murine RNA leukemia viruses. Studies of early events in the replication of LLV-F were carried out by following the infection of permissive and restrictive mouse embryo cells by ³²P labeled LLV-F. ³²P labeled viral genome RNA rapidly becomes associated with cell nuclei and may be found integrated to the same extent with high molecular weight host DNA of either permissive or restrictive cells. These results suggest that Fv-1 mediated host restriction of LLV-F occurs at a step following integration of viral genome RNA into host DNA.Two other conclusions are suggested by these data. The nucleus appears to be the site of activation and synthesis of DNA of the infecting virus; and the “provirus”, at least transiently, is represented as an RNA-DNA hybrid molecule covalently integrated with host cell DNA.     

用SARS冠状病毒全基因组芯片杂交方法分析SARS-CoV

为从临床样品中检测和分析SARSCoV病毒打基础,并为分析SARSCoV病毒的复制和转录等机理提供一种有效方法。以SARS冠状病毒TOR2株序列作为标准设计和制备一种覆盖SARS冠状病毒全基因组的寡聚核苷酸芯片,探针长度为70nt,每相邻的探针序列重复25nt,共660条。用该芯片分析了细胞培养的SARSCoV病毒总RNA、7个SARSCoV病毒的基因克隆片段。对RNA样品用随机引物进行反转录PCR获得cDNA。对DNA用随机引物扩增和dUTPcy3标记。结果用这种芯片杂交检测SARSCoV病毒RNA可见阳性信号呈全基因组分布,并且有多处连续的阳性信号点;用正常人的白细胞RNA为对照,杂交未出现明显阳性信号。检测7个SARSCoV病毒基因克隆片段,在该片段相应的探针区段出现连续阳性信号点。这种方法可有效地检测和分析样品中SARS冠状病毒全基因组的信息。     

针对多种强致病性病毒的基因芯片检测方法的建立

为了制备灵敏的可检测多种烈性病毒性病原体的基因芯片,本研究设计了针对21种烈性病毒性病原体的基因芯片检测探针,每种5条,长50 bp.并以甲病毒属的基孔肯亚病毒和黄病毒属的黄热病毒细胞培养物为检测模型,摸索了合适的病毒基因处理与扩增方法.将提取的病毒RNA先用DNase Ⅰ处理,以去除掉其中的DNA分子,然后利用病毒属特异性引物进行反转录,以引导病毒基因组的合成,从而尽可能地减少宿主细胞基因成分的干扰.进行随机PCR扩增后将扩增产物与基因芯片进行杂交,分别出现了4条基孔肯亚病毒探针信号和5条黄热病毒的探针信号,说明所设计的检测探针具有较好的特异性,可用于这2种病毒的特异性检测.这种病毒基因样品的处理和扩增方法也为此基因芯片的临床应用奠定了基础.     

Parvovirus b19 DNA CpG dinucleotide methylation and epigenetic regulation of viral expression

CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression.The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections.The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19.     

Persistence of herpes simplex virus type-2 genome in a human leukemic cell line

To study the nature of virus-cell interaction in persistently infected cells we have examined production of infectious virus, synthesis of viral DNA and DNA polymerase in a human leukemic cell line K562. It was found that only one of three K562 cell lines was permissive for limited growth of HSV-2 and infectious virus was released in a cyclical fashion. Intranuclear inclusions with electron-dense fibrils and particles resembling viral structures were observed in the virus-infected but not control K562 cells. Viral DNA synthesis could not be detected by centrifugation in CsCl density gradients; but was readily identified by Southern blot hydridization of virus-infected intracellular DNA with purified viral DNA. Viral DNa polymerase was synthesized by infected cells during active infectious virus production. In one of the two K562 cell lines that did not produce infectious virus, a few DNA fragments from infected cells were found to hybridize with purified viral DNA. These results suggest that variable lengths of HSV-2 genome can be harbored and propagated by different human leukemic K562 cells.     

Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.     

Integration of progeny simian virus 40 DNA into the host cell genome

A procedure was developed for the separation of cellular DNA of productively infected monkey kidney cells from free simian virus 40 DNA. The application of this procedure allowed the investigation of progeny viral DNA integration into the host cell DNA by nucleic acid hybridization techniques. The purification consisted of precipitation of the cellular DNA by Hirt's (1967) method, velocity centrifugation in alkaline sucrose gradients, equilibrium centrifugation in ethidium bromide/CsCl solution, and an additional velocity centrifugation in an alkaline sucrose gradient. The efficiency of each step of the procedure was determined by monitoring the amount of contaminating free viral DNA. Purified cellular DNA, isolated from cells late after infection, contained approximately 0/sd006% free viral DNA, but as much as 2% integrated simian virus 40 DNA. This corresponds to more than 20,000 integrated virus genome equivalents per cell, as determined by DNA-DNA reassociation kinetics. Integration of simian virus 40 DNA into the cellular DNA became detectable at 24 hours after infection, and increased with the increase in the rate of viral DNA synthesis.     

Herpes simplex virus DNA in transformed cells: sequence complexity in five hamster cell lines and one derived hamster tumor.

Analyses of the hybridization kinetics of labeled herpes simplex virus 2 (HSV-2) DNA with DNA from five hamster cell lines transformed by UV light-irradiated HSV-2 revealed the following. (i) Viral DNA sequences were detected in all five cell lines tested. (ii) None of the cell lines contained the full complement of HSV-2 DNA. (iii) The amount of viral DNA present in the cells varied in different transformed cell lines and ranged from 8 to 32% of the HSV-2 DNA genome in 1 to 3 copies/cell. (iv) Two parallel passages of the same cell line (333-2-29) differed in the amount of viral DNA they contained. We also compared the viral DNA sequences present in (i) one transformed cell line (333-8-9) propagated serially in culture for 80 passages, (ii) a tumor produced by inoculation of a newborn hamster with the 333-8-9 cells, and (iii) a cell line derived from a hamster tumor as above and propagated in culture for 32 passages. The results show that viral DNA present in the hamster tumor and in the cells derived from the tumor had a lower sequence complexity than that present in the original serially passaged 333-8-9 cell line.     

Unintegrated viral DNA sequences in a hamster tumor induced by bovine papilloma virus

A fibrosarcoma was induced in a hamster by bovine papilloma virus type 2 (BPV2). The content of BPV2 DNA sequences was measured by DNA-DNA and cRNA-DNA hybridizations. The tumor contained approximately 300 BPV2 genome equivalents per cell. Southern blot hybridization indicated that the viral DNA was in free form, the entire genome most likely being present. In situ hybridization with BPV2 cRNA showed that multiple genome copies were present in each cell. Neither virus particles nor virus coat antigens could be detected in the tumor. A cell line was established from the fibrosarcoma, and the cells contained multiple copies of the BPV2 genome. The latter was in free form, and all of the DNA sequences appeared to be present in multiple copies and in all cells. An extensive search failed to reveal the presence of virus or viral antigens.