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1.
Methylation state and DNase I sensitivity of chromatin containing Moloney murine leukemia virus DNA in exogenously infected mouse cells 总被引:6,自引:3,他引:3
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The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before infection were largely methylated. The configuration chromatin containing M-MuLV proviruses was also investigated by digesting A9 nuclei with DNase I, followed by restriction analysis of the remaining DNA. Endogenous MuLV-related DNA was in chromatin relatively resistant to DNase I digestion, whereas the majority of M-MuLV-specific proviruses were in domains of intermediate DNase I sensitivity. Two proviral copies hypersensitive to DNase I digestion were identified. Analogy to the DNase I sensitivity of expressed and nonexpressed globin genes suggested that the proviral copies containing DNase I-hypersensitive sites were transcribed. 相似文献
2.
Kinetics of human immunodeficiency virus type 1 (HIV) DNA integration in acutely infected cells as determined using a novel assay for detection of integrated HIV DNA 总被引:1,自引:0,他引:1
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We have developed a novel linker-primer PCR assay for the detection and quantification of integrated human immunodeficiency virus type 1 (HIV) DNA. This assay reproducibly allowed the detection of 10 copies of integrated HIV DNA, in a background of 2 x 10(5) cell equivalents of human chromosomal DNA, without amplifying extrachromosomal HIV DNA. We have used this assay and a near-synchronous one-step T-cell infection model to investigate the kinetics of viral DNA accumulation following HIV infection. We report here that integrated HIV DNA started accumulating 1 h after the first appearance of extrachromosomal viral DNA and accounted for approximately 10% of the total HIV DNA synthesized in the first round of viral replication. These results highlight the efficient nature of integrase-mediated HIV integration in infected T cells. 相似文献
3.
A growing body of evidence indicates that proviral DNA load quantitation is an important parameter in establishing the dynamics of HIV infection. Proviral DNA load can be determined during the follow-up of infected individuals to evaluate reservoir status in addition to viral replication. Hence, the study of viral reservoirs, represented by HIV-1 latently infected cells, including resting memory CD4+ T cells, monocytes and macrophages, by which HIV-1 can be reactivated, opens new perspectives in the assessment and the comprehension of HIV-1 infection. However, the identification of viral reservoirs, that can store both wild and drug resistance viruses, is one of the most important steps in developing treatment strategies because it is now clear that viral reservoirs not only prevent sterilizing immunity but also represent a major obstacle to curing the infection with the potent antiretroviral drugs currently in use. Even if only careful evaluation of virological and immunological markers is necessary to fully characterize the course of HIV-1 infection and to provide a more complete laboratory-based assessment of disease progression, the availability of a new standardized assay such as DNA proviral load will be important to assess the true extent of virological suppression in treated patients and to verify the efficacy of new immune-based therapies aimed at purging HIV-1 DNA reservoirs. Several studies demonstrate, in fact, that HIV-1 cellular DNA load may be an indicator of spread of infection whereas the plasma RNA load is indicates active infection. This article will review the importance of monitoring HIV-1 proviral load DNA during the follow-up of HIV-1 infected subjects, suggesting that additional information complementing HIV RNA load could provide crucial information to monitor viral replication and the effectiveness of HAART therapy. 相似文献
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5.
Xiying Qu Pengfei Wang Donglin Ding Lin Li Haibo Wang Li Ma Xin Zhou Shaohui Liu Shiguan Lin Xiaohui Wang Gongmin Zhang Sijie Liu Lin Liu Jianhua Wang Feng Zhang Daru Lu Huanzhang Zhu 《Nucleic acids research》2013,41(16):7771-7782
HIV-infected individuals currently cannot be completely cured because existing antiviral therapy regimens do not address HIV provirus DNA, flanked by long terminal repeats (LTRs), already integrated into host genome. Here, we present a possible alternative therapeutic approach to specifically and directly mediate deletion of the integrated full-length HIV provirus from infected and latently infected human T cell genomes by using specially designed zinc-finger nucleases (ZFNs) to target a sequence within the LTR that is well conserved across all clades. We designed and screened one pair of ZFN to target the highly conserved HIV-1 5′-LTR and 3′-LTR DNA sequences, named ZFN-LTR. We found that ZFN-LTR can specifically target and cleave the full-length HIV-1 proviral DNA in several infected and latently infected cell types and also HIV-1 infected human primary cells in vitro. We observed that the frequency of excision was 45.9% in infected human cell lines after treatment with ZFN-LTR, without significant host-cell genotoxicity. Taken together, our data demonstrate that a single ZFN-LTR pair can specifically and effectively cleave integrated full-length HIV-1 proviral DNA and mediate antiretroviral activity in infected and latently infected cells, suggesting that this strategy could offer a novel approach to eradicate the HIV-1 virus from the infected host in the future. 相似文献
6.
Epstein-Barr virus-positive and -negative B-cell lines can be infected with human immunodeficiency virus types 1 and 2. 总被引:7,自引:5,他引:2
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Human immunodeficiency virus type 1 (HIV-1) can infect CD4+ lymphocytes, monocytes-macrophages, and various other cell lines, including B-cell lines. To study the parameters of B-cell infections, we examined the susceptibility of 24 B-lymphoid cell lines to both HIV-1 and HIV-2 infections. These cell lines included a series of Epstein-Barr virus (EBV) genome-negative Burkitt's lymphoma cell lines and their EBV-converted counterparts. To infect these cells we used two HIV-1 isolates and one HIV-2 isolate. Infections were monitored with a cytoplasmic RNA dot-blot and a syncytium assay. HIV infection was also studied by a novel method based on electrophoresis of DNA liberated from cells that were lysed in situ in the well of an agarose gel. All human B-cell lines could be infected with HIV-1, regardless of the presence of EBV genomes; thus, EBV infection had no major effect on HIV susceptibility of B-cell lines. Integrated proviral HIV genomes could be detected by Southern blot analysis of DNA extracted from long-term, non-HIV-producing B-cell lines. This study suggests that B-lymphoid cells may serve as reservoirs for latent or persistent HIV infections in vivo, even in the absence of EBV infection. 相似文献
7.
PCR-in situ hybridization detection of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes of patients with HTLV-1-associated neurologic disease. 总被引:1,自引:0,他引:1
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M C Levin R J Fox T Lehky M Walter C H Fox N Flerlage R Bamford S Jacobson 《Journal of virology》1996,70(2):924-933
PCR-in situ hybridization (PCR-ISH) was developed and utilized to determine the distribution of human T-cell lymphotropic virus type 1 (HTLV-1) tax proviral DNA in peripheral blood lymphocytes (PBL) from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). PCR-ISH of HTLV-1 tax DNA in PBL from patients with HAM/TSP revealed that 1 in 5,000 to 1 in 10,000 PBL contained virus. PCR-ISH was sensitive, because a positive signal was consistently demonstrated from the HTLV-1-infected cell lines HUT-102 (which contains four to six copies of HTLV-1 proviral DNA per cell) and MT-1 (which contains one to three copies of HTLV-1 proviral DNA per cell). Also, intracellular amplification by PCR-ISH significantly increased sensitivity compared with conventional ISH and was shown to be specific for HTLV-1 tax DNA. These results are in contrast to solution-phase PCR amplification in which greater than 1% of cells were estimated to be infected. The discordance between these results is discussed and may indicate that more than one copy of HTLV-1 tax proviral DNA is present in an individual PBL. 相似文献
8.
A bovine leukemia virus (BLV)-producing cell line, fetal lamb kidney cells infected with BLV (FLK) contains one or a few copies of BLV proviral DNA in its genome. These cells contain 0.002% of viral RNA which sediments, in a sucrose gradient, at about 35S and between 18S and 28S.In cattle affected by enzootic bovine leukosis, tumor cells and circulating lymphocytes also contain one or a few copies of BLV proviral DNA integrated in their genome. However, in all cases tested (except one), no viral RNA was detected in these cells in conditions where one or two copies of viral genomic RNA per cell would have been easily detected. 相似文献
9.
Effect of Helper Virus on the Number of Murine Sarcoma Virus DNA Copies in Infected Mammalian Cells
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Cell lines of four mammalian species were each examined for the number of Moloney murine sarcoma virus (M-MSV) DNA copies in total cellular DNA after M-MSV transformation. Sarcoma-positive, leukemia-negative (S+L-) M-MSV-transformed cells were compared to M-MSV-transformed cells infected with a replicating leukemia virus. Both unfractionated M-MSV complementary DNA and complementary DNA representing the MSV-specific and the MSV-murine leukemia virus-common regions of the M-MSV genome were hybridized to total cellular DNA of various species. DNAs of mouse, cat, dog, and human S+L-cells contained from less than one to a few proviral M-MSV DNA copies per haploid genome. In contrast, helper virus-coinfected, M-MSV-producing cells of each species showed a 3- to 10-fold increase in M-MSV proviral DNA over that found in corresponding S+L- cells. MSV-specific and MSV-murine leukemia virus-common nucleotide sequences were each increased to a similar degree. A corresponding examination of cellular DNA of leukemia virus-infected normal or S+L- mammalian cells was performed to establish the resulting number of leukemia proviral DNA copies. The infection of normal or S+L- mammalian cells with several leukemia-type viruses that did not have nucleotide sequences closely related to the cell before infection resulted in the appearance of one to three corresponding leukemia proviral DNA copies. 相似文献
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11.
Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair
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The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration. 相似文献
12.
Murine mammary tumor virus: characterization of infection of nonmurine cells. 总被引:19,自引:9,他引:10
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Murine mammary tumor virus (MuMTV) was used to productively infect feline and mink cells. MuMTV "proviral" DNA could be detected in the infected cells by molecular hybridization using radioactive MuMTV complementary DNA as a probe. Kinetic analysis of MuMTV proviral DNA synthesis after infection showed that maximum MuMTV DNA synthesis was achieved by 8 h; however, this was followed by a decline in detectable proviral DNA and eventual stabilization at a lower level. MuMTV synthesis in feline cells was greatly stimulated by the synthetic glucocorticoid, dexamehtasone. On the other hand, MuMTV synthesis in mink cells was relatively at a much higher level in absence of dexamethasone and the stimulation with dexamethasone was not as marked as in the case with infected feline cells. Thermal denaturation of hybrids between MuMTV complementary DNA and infected mink cell RNA revealed no difference from homologous hybrids. 相似文献
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14.
Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.
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The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA. 相似文献
15.
Beretta A Clerici M Hasson H Fumagalli L Trabattoni D Lillo F Ferrante P Saniabadi AR Adachi M Lazzarin A 《Journal of biological regulators and homeostatic agents》2000,14(1):27-31
AIDS pathogenesis results from a complex array of immune alterations which include, among others, changes in the pattern of cytokine production. Some monocyte-derived cytokines, like TNFalpha play a major role in HIV pathogenesis. TNFalpha transactivates HIV NF-kB thereby inducing viral replication, potentiates HIV replication in lymphomonocytes TNFalpha is one of the main factors of HIV-induced cachexia and might be involved in HAART-associated lipodystrophy. In addition, monocytes are infectable by HIV in vitro and infected monocytes can be recovered from the blood of HIV infected patients. For these reasons, we tested whether renewal of the pool of circulating monocytes by selective monocyte apheresis may improve the immune reconstitution which follows treatment with highly active anti-retrovirals (HAART). HIV-infected HAART receiving (> 1 year) patients who were either virologically non-responders (HIV-1 RNA >50,000 copies/ml) or immunologically non-responders (CD4 counts < 200) were treated with a novel monocyte apheresis device (G-1 Adacolumn). Plasma HIV viral load, proviral DNA and phenotypic and functional immunological analyses were performed. G-1 apheresis was well tolerated, not accompanied by adverse responses, and followed by clinical improvement. TNFalpha production was suppressed and CD4 T cell counts increased. In one G-1 patient with elevated HIV-1 proviral DNA a significant reduction (from 1,500 to 40 copies/10(5) cells) was observed. Neither immunologic nor virologic parameters were modified in the control patients who received HAART alone. Thus, purging of circulating monocytes by G-1 apheresis has a dramatic suppressive effect on TNFalpha production and is followed by both clinical and immunovirological improvement. G-1 apheresis should be considered in patients in whom HAART is only partially effective. 相似文献
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Gallego S Mangano A Gastaldello R Sen L Medeot S 《Memórias do Instituto Oswaldo Cruz》2004,99(4):377-380
This study aimed at implementing a Nested-polymerase chain reaction (Nested-PCR) for the molecular diagnosis of human T-cell lymphotropic virus type I/II (HTLV-I and HTLV-II) infections in peripheral blood mononuclear cells of infected subjects in Argentina. The sensitivity and specificity of the assay for the detection of regional strains were assessed by comparing them with the molecular assay of reference PCR-hybridization. The Nested-PCR detected 1 MT-2 cell (> or = 8 proviral copies)/1x10(6) non-infected cells showing high sensitivity for provirus detection. While both molecular assays showed high specificity (100%) for HTLV-I and HTLV-II detection, the sensitivity values differed: 100% for Nested-PCR and 67% for PCR-hybridization assay. Moreover, this technique showed less sensitivity for the detection of DNA sequences of HTLV-II (33%) than for the detection of DNA sequences of HTLV-I (75%). The high sensitivity and specificity of the Nested-PCR for regional strains and its low costs indicate that this assay could replace the PCR-hybridization assay for the molecular diagnosis of HTLV-I/II infections. It will be interesting to assess the usefulness of this assay as a tool for the molecular diagnosis of HTLV-I/II infections in other developing countries. Other studies that include a greater number of samples should be conducted. 相似文献
18.
Productive infection maintains a dynamic steady state of residual viremia in human immunodeficiency virus type 1-infected persons treated with suppressive antiretroviral therapy for five years
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Havlir DV Strain MC Clerici M Ignacio C Trabattoni D Ferrante P Wong JK 《Journal of virology》2003,77(20):11212-11219
To provide insight into the dynamics and source of residual viremia in human immunodeficiency virus (HIV) patients successfully treated with antiretroviral therapy, 14 intensely monitored patients treated with indinavir and efavirenz sustaining HIV RNA at <50 copies/ml for >5 years were studied. Abacavir was added to the regimen of eight patients at year 5. After the first 9 months of therapy, HIV RNA levels had reached a plateau ("residual viremia") that persisted for over 5 years. Levels of residual viremia differed among patients and ranged from 3.2 to 23 HIV RNA copies/ml. Baseline HIV DNA was the only significant pretreatment predictor of residual viremia in regression models including baseline HIV RNA, CD4 count, and patient age. In the four of five patients with detectable viremia who added abacavir to their regimen after 5 years, HIV RNA levels declined rapidly. The estimated half-life of infected cells was 6.7 days. Decrease in activated memory cells and a reduction in gamma interferon production to HIV Gag and p24 antigen in ELISpot assays were observed, consistent with a decrease in HIV replication. Thus, in patients treated with efavirenz plus indinavir, levels of residual viremia were established by 9 months, were predicted by baseline proviral DNA, and remained constant for 5 years. Even after years of highly suppressive therapy, HIV RNA levels declined rapidly after the addition of abacavir, suggesting that productive infection contributes to residual ongoing viremia and can be inhibited with therapy intensification. 相似文献
19.
目的建立快速、敏感、特异的猴免疫缺陷病毒(SIV)TaqMan探针实时荧光定量PCR检测方法,对SIV病毒核酸进行定量检测。方法RT—PCR扩增SIVmac251保守gag基因序列796bp片段,进行TA克隆,构建标准品质粒pMD—SIVgag。通过对SIV定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量PCR方法的灵敏度、特异性和重复性。结果所建立的SIVQPCR检测方法,质粒DNA模板在10’~10。拷贝之间表现较好线性和相关性,标准曲线所得斜率为-3.26,相关系数为0.999。检测灵敏度达到200拷贝,方法重复性测试,检测25份临床样品CV%均小于1%。结论建立的SIVQPCR检测方法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒(SIV)核酸拷贝量。 相似文献
20.
准确测定HIV 1的前病毒载量和病毒载量的技术 ,在感染者预后和艾滋病患者药物治疗效果的评价以及艾滋病的其它研究方面 ,都具有十分重要的应用价值。以定量的HIV 1DNA和RNA为标准外参照 ,利用SYBRGreen荧光染料和GeneAmp5 70 0SequenceDetectionSystem (5 70 0系统 ) ,建立了测定HIV 1的前病毒载量和病毒载量的荧光实时定量PCR技术。以病毒感染细胞和培养上清为材料 ,测定了三种化合物 (AZT ,GL和WT)对细胞内的前病毒载量和培养上清中的病毒载量的抑制活性 ,并与合胞体形成抑制方法测定化合物抗病毒活性的结果进行了比较。根据病毒载量、前病毒载量和合胞体形成计算出的三种化合物的治疗指数均依次变小 ,提出以荧光实时定量PCR技术测定前病毒载量 ,会在评价药物在体内外根除或减少存在于CD4休止或记忆T淋巴细胞中的HIV 1前病毒方面有特别的价值。 相似文献