共查询到20条相似文献,搜索用时 62 毫秒
1.
Ultrastructural Studies of H-1 Parvovirus Replication VI. Simultaneous Autoradiographic and Immunochemical Intranuclear Localization of Viral DNA Synthesis and Protein Accumulation
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The localization of H-1 viral replicative-form double-stranded DNA and progeny single-stranded DNA replication in parasynchronously infected, simian virus 40-transformed newborn human kidney cells was studied with high-resolution electron microscope autoradiography (80-nm silver grains). We analyzed wild-type H-1 and ts1 H-1 (a conditional mutant defective in progeny single-stranded DNA synthesis). The proportion of the total DNA synthesis that was viral was estimated to be >90% by comparing the amount of [(3)H]thymidine uptake in cultures infected with wild-type H-1 versus ts14 (an H-1 mutant defective in DNA replication). Simultaneous staining with cytochrome c-conjugated anti-H-1 immunoglobulin G was performed to ensure that cells incorporating [(3)H]thymidine (2- to 60-min pulses) were H-1 infected. The sites of H-1 replicative-form (in ts1-infected cells) and progeny (in wild-type-infected cells) DNA synthesis were identical. Immunospecifically labeled nuclei at the earliest stages of infection exhibited dense clusters of silver grains over material extruded from nucleolar fibrillar centers. These foci became larger with increasing cellular damage, forming a limited number of H-1 DNA synthetic centers in the euchromatin. Each island-like focus was surrounded by tufts of heterochromatin containing high concentrations of unassembled H-1 capsid proteins. In late phases of infection, the heterochromatin became completely marginated, and the nucleoplasm contained only euchromatin that exhibited randomly distributed sites of H-1 DNA replication. This indicates that H-1 DNA synthesis begins at localized euchromatic or nucleolar sites and then spreads outward. Immunostained heterochromatin and nucleolar chromatin never incorporated [(3)H]thymidine. Our results suggest that H-1 proteins and cellular cofactors associated with the fibrillar component of the nucleolus and the euchromatin may play a role in the regulation of H-1 DNA synthesis. 相似文献
2.
Infectious process of the parvovirus H-1: correlation of protein content, particle density, and viral infectivity. 总被引:13,自引:12,他引:1
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
P R Paradiso 《Journal of virology》1981,39(3):800-807
The infectious particles of the parvovirus H-1 were characterized with respect to protein content, density in CsCl, and specific infectivity. Heavy-full and light-full particles were purified from infected simian virus 40-transformed newborn human kidney (NB) cells and from simian virus 40-transformed hamster kidney (THK) cells. Analysis of the protein content of these particles demonstrated that the ratio of viral protein VP2' to VP2 was the same in heavy-full and light-full particles derived from the same cell line, but differed significantly between the two hosts. However, the infectivity of the particles from each cell line was the same for all four viral species.. Also, in vitro conversion of VP2' to VP2 did not enhance the particle infectivity of either heavy-full or light-full virus. When the fate of input virus was studied with 125I-labeled H-1, the conversion of VP2' to VP2 occurred in a time-dependent manner up to 24 h postinfection. Simultaneous with the proteolytic cleavage, there was a shift in the density of the heavy-full virus to the light-full density. However, protein analysis of the 125I-labeled light-full virus at various times postinfection indicated that they were not enriched in VP2 when compared with heavy-full virus or the total virus population. Thus, the cleavage of VP2' to VP2 is not responsible for the shift in density from heavy-full to light-full virus, and although these events might be required for infection they appear not to be interdependent. 相似文献
3.
The small nonstructural protein (NS2) of the parvovirus minute virus of mice is required for efficient DNA replication and infectious virus production in a cell-type-specific manner. 总被引:27,自引:26,他引:1
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Seven mutations which affect only the small nonstructural protein NS2 were introduced into the infectious clone of the autonomous parvovirus, minute virus of mice (MVM). The majority of these mutants were severely defective for replication following transfection of normal host murine A9 fibroblasts; however, all were found to replicate more efficiently and produce infectious virus in certain other cell types, including human NB324K. The isolation of viral stocks from NB324K cells permitted a more detailed analysis of the mutant defect on A9 cells. NS2 mutant NS2-2018 was shown to be approximately 10-fold deficient for viral monomer replicative-form DNA production within a single-burst cycle in infected A9 cells and produced a reduced amount of progeny single strand. Mutant NS2-2018 generated wild-type levels of monomer replicative-form DNA on NB324K cells but made reduced levels of progeny single strand and small plaques on these cells. The accumulation of NS1 is reduced late in NS2-2018 infection of A9 cells, but NS1 accumulates to wild-type levels late in NB324K cell infections. NS1 nuclear localization is not dependent on NS2 in A9 or NB324K cells. These results indicate that NS2 participates in MVM DNA replication and is required for efficient viral growth. The requirement for NS2 during MVM replication is also host cell specific. This requirement is significantly more pronounced in the normal host murine A9 cells than in certain other cell types, including NB324K. 相似文献
4.
Mapping of the viral mRNA encoding a super-T antigen of 115,000 daltons expressed in simian virus 40-transformed rat cell lines 总被引:16,自引:12,他引:4
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Simian virus 40-transformed V11 F1 clone 1 subclone 7 rat cells produced a considerable amount of an elongated form of large-T antigen with an Mr of 115,000 (115K super-T antigen), but these cells did not produce detectable traces of normal-sized large-T antigen (86,000 daltons) (P. May, M. Kress, M. Lange, and E. May, Cold Spring Harbor Symp. Quant. Biol. 44:189-200, 1980). First, a comparison of the tryptic peptide fingerprints of 115K super-T and large-T antigens suggested that 115K super-T antigen is simian virus 40 coded and contains a duplication of amino acid sequences of large-T antigen. Second, from S1 mapping analysis of 115K super-T mRNA, performed with various restriction fragments of simian virus 40 DNA, it was concluded that super-T mRNA is a form of large-T mRNA containing a tandem duplication of the sequence extending from approximately 0.46 to 0.35 map unit. The duplicated sequence corresponded to that region of the simian virus 40 genome in which 12 of 13 tsA mutation sites are clustered (C. J. Lai and D. Nathans, Virology 66:70-81, 1975). 相似文献
5.
Defective interfering particles of the parvovirus H-1 were produced by serial propagation at high multiplicities of infection. Such particles interfere with the synthesis of capsid proteins and infectious virus of standard H-1. The interference is sensitive to UV irradiation, dependent on the multiplicity of the challenge virus, and is active in heterotypic infections against parvovirus H-3 or LuIII. Defective interfering particle genomes have alterations characterized by integral numbers (1 to 10 or more) of a 60-base-pair addition in the neighborhood of the origin of replicative-form DNA replication and deletions that are located primarily within two regions, 32 to 44 or 80 to 90 on the genome map. Some of the implications of these findings are discussed. 相似文献
6.
Cziepluch C Lampel S Grewenig A Grund C Lichter P Rommelaere J 《Journal of virology》2000,74(10):4807-4815
We have identified a nuclear structure that is induced after infection with the autonomous parvovirus H-1. Using fluorescence microscopy, we observed that the major nonstructural protein (NS1) of H-1 virus which is essential for viral DNA amplification colocalized with virus-specific DNA sequences and sites of ongoing viral DNA replication in distinct nuclear bodies which we designated H-1 parvovirus-associated replication bodies (H-1 PAR-bodies). In addition, two cellular proteins were shown to accumulate in H1 PAR-bodies: (i) the proliferating cell nuclear antigen (PCNA) which is essential for chromosomal and parvoviral replication and (ii) the NS1-interacting small glutamine-rich TPR-containing protein (SGT), suggesting a role for the latter in parvoviral replication and/or gene expression. Since many DNA viruses target preexisting nuclear structures, known as PML-bodies, for viral replication and gene expression, we have determined the localization of H-1 PAR- and PML-bodies by double-fluorescence labeling and confocal microscopy and found them to be spatially unrelated. Furthermore, H-1 PAR-bodies did not colocalize with other prominent nuclear structures such as nucleoli, coiled bodies, and speckled domains. Electron microscopy analysis revealed that NS1, as detected by indirect immunogold labeling, was localized in ring-shaped electron-dense nuclear structures corresponding in size and frequency to H-1 PAR-bodies. These structures were also clearly visible without immunogold labeling and could be detected only in infected cells. Our results suggest that H-1 virus does not target known nuclear bodies for DNA replication but rather induces the formation of a novel structure in the nucleus of infected cells. 相似文献
7.
Retransformation of a simian virus 40 revertant cell line, which is resistant to viral and DNA infections, by microinjection of viral DNA. 总被引:5,自引:0,他引:5
We have isolated morphological transformants of cultured cells as dense foci on a monolayer of normal cells appproximately 4 weeks after microinjection of purified simian virus 40 DNA (200 to 400 molecules per cell) directly into the nucleus. Both Rat 1 (an established contact-inhibited rat embryo fibroblast line) and F1' 1--4 (a 5-fluorodeoxyuridine-selected flat revertant from the simian virus 40-transformed 14B cell line) were transformed with an efficiency of 5 to 10% of the cells injected. F1' 1--4 is not susceptible to retransformation by either viral or DNA infection (by calcium phosphate-facilitated cellular uptake), and as a result it had previously been thought to possess a host mutation preventing expression of the simian virus 40 genome. 相似文献
8.
The Cold Spring Harbor Symposium on The Ribosome was held on 31 May - 5 June in Cold Spring Harbor, NY. USA. 相似文献
9.
NS2 is required for efficient translation of viral mRNA in minute virus of mice-infected murine cells. 总被引:11,自引:11,他引:0
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Detailed analysis of five NS2 mutants of the autonomous parvovirus minute virus of mice (MVMp) has revealed the following. At low multiplicities of infection, NS2 mutants killed NB324K cells as well as wild-type (wt) MVM did and grew to high titers, while in contrast they grew poorly and did not readily kill murine A9 cells. Following CaPO4 transfection of murine fibroblasts, NS2 mutant infectious clones generated approximately 10-fold less monomer replicative-form DNA than wt and no detectable progeny single-stranded DNA. On nonmurine semipermissive NB324K cells, however, these mutant plasmid clones generated near wt levels of all replicative DNA forms. After infection of highly synchronized murine fibroblasts by NS2 mutant virus at inputs equivalent to those of the wt, mutant monomer replicative-form DNA was decreased 5- to 10-fold compared with that of the wt, and progeny single-stranded DNA accumulation was decreased to an even greater extent. Both total and cytoplasmic NS2 mutant RNA was decreased, but the amount of total viral mRNA generated, relative to accumulated viral DNA in the same experiments, was similar to that seen in wt infection. The accumulation of virus-generated proteins was also decreased in NS2 mutant infection; however, the magnitude of this decrease, compared with that of wt infections, was significantly greater than the concomitant decrease in mutant-generated levels of accumulated cytoplasmic RNA, and this effect was most dramatic for VP2. There was no such disparity between the relative accumulation of mutant-generated RNA and protein in cells permissive for the growth of these mutants. These results suggest that translation of MVM viral RNA is specifically reduced in NS2 mutant infection of restrictive cells. Because the affected viral proteins are required for the efficient production of viral replicative DNA forms, these results reveal a fundamental, although perhaps not the only, role for NS2 in parvovirus infection. 相似文献
10.
Slack FJ 《Genome biology》2006,7(9):328-2
A report of the meeting 'Regulatory RNAs', the 71st Cold Spring Harbor Symposium on Quantitative Biology, Cold Spring Harbor, USA, 31 May-5 July 2006. 相似文献
11.
A report on the 11th Cold Spring Harbor Laboratory/Wellcome Trust conference on Genome Informatics, Cold Spring Harbor Laboratories,
New York, USA, November 2-5, 2011. 相似文献
12.
Ihalainen TO Niskanen EA Jylhävä J Turpeinen T Rinne J Timonen J Vihinen-Ranta M 《Cellular microbiology》2007,9(8):1946-1959
Nuclear positioning and dynamic interactions of viral proteins with nuclear substructures play essential roles during infection with DNA viruses. Visualization of the intranuclear interactions and motility of the parvovirus replication protein (NS1) in living cells gives insight into specific parvovirus protein-cellular structure interactions. Confocal analysis of highly synchronized infected Norden Laboratory Feline Kidney cells showed accumulation of nuclear NS1 in discrete interchromosomal foci. NS1 fused with enhanced yellow fluorescence protein (NS1-EYFP) provided a marker in live cells for dynamics of NS1 traced by photobleaching techniques. Fluorescence Recovery after Photobleaching suggested that the NS1 protein is not freely diffusing but undergoes transient interactions with nuclear compartments. Fluorescence Loss in Photobleaching demonstrated for the first time the shuttling of a parvoviral protein between the nucleus and the cytoplasm as assayed with NS1-EYFP. Finally, time-lapse imaging of infected cells revealed that the intranuclear distribution of NS1-EYFP evolves dramatically starting from the formation of NS1 foci and proceeding to a homogenous distribution extending throughout the nucleus. 相似文献
13.
Mutation of lysine 405 to serine in the parvovirus H-1 NS1 abolishes its functions for viral DNA replication, late promoter trans activation, and cytotoxicity. 总被引:12,自引:12,他引:0
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A consensus sequence in parvovirus nonstructural protein NS1 has been predicted to be an ATP-binding domain associated with an ATPase and a DNA helicase activity. To investigate the function of NS1 in viral gene expression, a site-directed mutagenesis converting NS1 lysine 405 to serine in parvovirus H-1 was carried out by the polymerase chain reaction. As shown previously, a parvovirus genome containing a deleted NS1 gene was excised from a bacterial plasmid and replicated when a wild-type NS1 gene was provided in trans but failed to be excised and replicate when the mutant NS1 gene was supplied. Interestingly, the serine 405 mutation totally lost the activity of trans activation on the virus late promoter (P38) in a chloramphenicol acetyltransferase (CAT) assay and it lost evidence for cytotoxicity in two tumor cell lines (HeLa Gey and NB324K). The serine 405 NS1 protein was translocated normally to the nucleus. These results suggest that the NS1 lysine 405 of H-1 in its putative purine nucleotide-binding site is essential for viral DNA replication and that this domain may be involved in the regulation of the P38 promoter by an unknown mechanism. The loss of NS1 cytotoxicity on tumor cells suggests that NS1 expression is the major cause of cell killing by parvoviruses, which may facilitate further study of the mechanism of oncosuppression by parvoviruses. 相似文献
14.
Aleutian mink disease parvovirus infection of mink peritoneal macrophages and human macrophage cell lines. 总被引:9,自引:8,他引:1
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Aleutian mink disease parvovirus (ADV) mRNAs are found in macrophages in lymph nodes and peritoneal exudate cells from ADV-infected mink. Therefore, we developed an in vitro infection system for ADV by using primary cultures of mink macrophages or macrophage cell lines. In peritoneal macrophage cultures from adult mink, virulent ADV-Utah I strain showed nuclear expression of viral antigens with fluorescein isothiocyanate-labeled ADV-infected mink serum, but delineation of specific viral proteins could not be confirmed by immunoblot analysis. Amplification of ADV DNA and production of replicative-form DNA were observed in mink macrophages by Southern blot analysis; however, virus could not be serially propagated. The human macrophage cell line U937 exhibited clear nuclear expression of viral antigens after infection with ADV-Utah I but not with tissue culture-adapted ADV-G. In U937 cells, ADV-Utah I produced mRNA, replicative-form DNA, virion DNA, and structural and nonstructural proteins; however, virus could not be serially passaged nor could [3H]thymidine-labeled virions be observed by density gradient analysis. These findings indicated that ADV-Utah I infection in U937 cells was not fully permissive and that there is another restricted step between gene amplification and/or viral protein expression and production of infectious virions. Treatment with the macrophage activator phorbol 12-myristate 13-acetate after adsorption of virus reduced the frequency of ADV-positive U937 cells but clearly increased that of human macrophage line THP-1 cells. These results suggested that ADV replication may depend on conditions influenced by the differentiation state of macrophages. U937 cells may be useful as an in vitro model system for the analysis of the immune disorder caused by ADV infection of macrophages. 相似文献
15.
The cytopathic effects induced during parvovirus infection have been widely documented. Parvovirus infection-induced cell death is often directly associated with disease outcomes (e.g., anemia resulting from loss of erythroid progenitors during parvovirus B19 infection). Apoptosis is the major form of cell death induced by parvovirus infection. However, nonapoptotic cell death, namely necrosis, has also been reported during infection of the minute virus of mice, parvovirus H-1 and bovine parvovirus. Recent studies have revealed multiple mechanisms underlying the cell death during parvovirus infection. These mechanisms vary in different parvoviruses, although the large nonstructural protein (NS)1 and the small NS proteins (e.g., the 11 kDa of parvovirus B19), as well as replication of the viral genome, are responsible for causing infection-induced cell death. Cell cycle arrest is also common, and contributes to the cytopathic effects induced during parvovirus infection. While viral NS proteins have been indicated to induce cell cycle arrest, increasing evidence suggests that a cellular DNA damage response triggered by an invading single-stranded parvoviral genome is the major inducer of cell cycle arrest in parvovirus-infected cells. Apparently, in response to infection, cell death and cell cycle arrest of parvovirus-infected cells are beneficial to the viral cell lifecycle (e.g., viral DNA replication and virus egress). In this article, we will discuss recent advances in the understanding of the mechanisms underlying parvovirus infection-induced cell death and cell cycle arrest. 相似文献
16.
Down TA 《Genome biology》2006,7(2):305-2
A report on the Genome Informatics meeting held at Cold Spring Harbor Laboratory, Cold Spring Harbor, USA, 28 October-1 November 2005. 相似文献
17.
A report on the Cold Spring Harbor Laboratory meeting 'Eukaryotic mRNA Processing', Cold Spring Harbor, USA, 20-24 August 2003. 相似文献
18.
Replication process of the parvovirus H-1. X. Isolation of a mutant defective in replicative-form DNA replication. 总被引:10,自引:9,他引:1
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A temperature-sensitive mutant of H-1, ts14, that is partially defective in replicative-form (RF) DNA synthesis has been isolated. ts14 H-1 is characterized by a decrease in plaque-forming ability and production of infectious virus at the restrictive temperature of 39.5 degrees C. RF DNA synthesis of ts14 is reduced to 3 to 7% of that of wild-type H-1 at either the restrictive or the permissive temperature. A complementation analysis of RF synthesis of ts14 and a viable defective H-1 virus, DI-1, or wild-type H-3 indicates that the defective RF DNA synthesis of ts14 is cis-acting. ts14, unlike wild-type H-1, causes a multiplicity-dependent inhibition of DI-1 or H-3, but not LuIII, RF DNA synthesis. Mixed infections of cells with two parvoviruses also exhibited a cross-interference for viral protein synthesis that was multiplicity dependent, ts14 inhibited infectious virus production of H-1 or H-3, but not LuIII. LuIII-or H-3-pseudotype particles were produced by coinfection with H-1. H-3 and H-1 showed similar interactions with ts14, and H-3 DNA was more homologous to H-1 than was LuIII by comparative physical mapping studies. The results suggest that ts14 is a mutant with a defect in a regulatory sequence of its DNA that influence RF DNA replication. 相似文献
19.
W F Bodmer 《BioEssays : news and reviews in molecular, cellular and developmental biology》1987,7(1):41-45
The 1986 Cold Spring Harbor Symposium was on the subject of human genetics; it was the first symposium at Cold Spring Harbor on this topic since 1964. In the opening remarks for the conference, Walter F. Bodmer first summarized the progress in this field since 1964. He then described what is presently known about the functional complexity of the human genome and discussed the case for a definitive characterization and sequencing of the human genome. The following is an abridged and slightly adapted version of this talk; it is reproduced courtesy of the Cold Spring Harbor Laboratory © 1987. 相似文献
20.
Taylor MS 《Genome biology》2001,2(8):reports4018.1-reports40185
A report on the Cold Spring Harbor meeting on Genome Sequencing and Biology, Cold Spring Harbor, NY, USA, 9-13 May 2001. 相似文献