SV40 virions and viral RNA metabolism are associated with cellular substructures.

Ben-Ze’ev, Avri; Abulafia, Rivka; Aloni, Yosef

doi:10.1002/j.1460-2075.1982.tb00017.x

Cited by 38 publications

(16 citation statements)

References 31 publications

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“…The E7 protein, like most nuclear matrix proteins, was removed from the complete matrix by 2 M NaCl extraction and was not retained with the core filaments. Previous studies have localized the viral oncoproteins simian virus 40 large tumor antigen and adenovirus Ela (which have many properties in common with E7) to a nuclear matrix structure prepared with high salt (52,53). In the present work, HPV-16 E7 is localized on a nuclear matrix prepared with moderate salt but removed by higher salt concentrations.…”

Section: Discussionmentioning

confidence: 58%

Human papillomavirus 16 E7 protein is associated with the nuclear matrix.

Nickerson

Penman

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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The cellular localization of the human papillomavirus (HPV)-16 E7 gene product in the cell lines CaSki and SiHa has been determined by both biochemical and immunocytochemical methods. These measurements show E7 to be localized in the cell nucleus, specifically with the nonchromatin nuclear structure or nuclear matrix. This localization of E7 required an unambiguous fractionation of the nuclear constituents. This was achieved by using a gentle sequential fractionation procedure to prepare the scaffold consisting ofthe nuclear matrix and intermediate filaments (NM-IF). Chromatin was cleaved with nuclease and the resulting nucleosomes eluted with 0.25 M ammonium sulfate. Immunotaining of cells after this extraction procedure with monoclonal antibodies (mAbs) to E7 revealed a fine grained, punctate nuclear fluorescence in CaSki and SiHa, which was absent in normal cervical keratinocytes and the HPV-negative cell line C33.1. Western blots of cell fractions with these mAbs showed that E7 was localized in the NM-IF fraction in SiHa and CaSki but was not detected in HPV-negative cells. A second protein ofslightly higher mobility is identified by these antisera in HPV-16-containing cells. The data suggest that the previous Inability to directly visualize E7 by immunocytology is due to the masking ofepitopes by cellular components and not to low levels of protein.The papillomaviruses are small DNA viruses that infect mucocutaneous surfaces where they induce benign epithelial proliferations or warts. Those viruses that infect the human anogenital mucosae are of particular interest because of persuasive evidence of an association between infection with these viruses and anogenital cancer (1, 2). The DNA sequences of human papillomavirus (HPV)-16 and to a lesser extent are found in at least 80% of cervical carcinomas and in high frequency in high-grade cervical intraepithelial lesions (3-6). HPV DNA has also been detected in cell lines derived from cervical carcinomas with SiHa and CaSki containing HPV-16 DNA and HeLa and C 41 containing HPV-18 DNA (7). Further evidence for an etiological role for these viruses in cervical carcinogenesis comes from their ability to immortalize genital keratinocytes in vitro (8)(9)(10).Transcriptional analysis of infected carcinoma tissue and cell lines has revealed that transcription of the viral DNA is confined to the early region of the viral genome irrespective of whether this DNA is episomal or integrated (11,12). The most abundant mRNA species encodes the E7 protein (11), which is the major transforming protein for . The E7 oncoprotein has been identified in both carcinoma biopsies and in the cell lines CaSki and SiHa by immunoprecipitation and was shown to be a 20-kDa phosphoprotein (11), apparently with a cytoplasmic locale (16).However, the functional properties of E7, which include immortalization (in association with E6) of keratinocytes (17-19), cooperation with ras in rodent cell transformation (14), and binding to the 105-kDa retinoblastoma gene product (20) suggest a n...

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Section: Discussionmentioning

confidence: 58%

Human papillomavirus 16 E7 protein is associated with the nuclear matrix.

Nickerson

Penman

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…To localize the subnuclear distribution of p53, nuclei were treated with DNase and salt to remove -95% of the DNA and histones. This fractionation yields a solubilized chromatin fraction and leaves behind the skeletal framework of the nucleus, the nuclear matrix (Berezney and Coffey, 1974;Ben-Ze'ev et al, 1982;Capco et al, 1982). We compared the relative amount of p53 in the solubilized chromatin fraction and in the nuclear matrix fraction in SV40-transformed and methylcholanthrene-transformed fibroblasts by the immunofluorescence and immunoprecipitation procedures as used above.…”

Section: Subnuclear Localization Ofp53 In Transformed Fibroblastsmentioning

confidence: 99%

P53 transformation-related protein accumulates in the nucleus of transformed fibroblasts in association with the chromatin and is found in the cytoplasm of non-transformed fibroblasts.

Rotter¹,

Abutbul²,

Ben-Ze’ev³

1983

The EMBO Journal

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The subcellular localization of the p53 molecule was studied in transformed and non‐transformed fibroblasts. A newly established transformed cell line obtained by treating primary embryonic mouse cells in vitro with the chemical carcinogen methylcholanthrene was compared with the embryonic parent fibroblasts. The transformed cells lost the spindle shape characteristic of the parent fibroblasts, acquired an accelerated growth rate, developed into tumors when injected into syngeneic mice and expressed high levels of p53 synthesis estimated by immunoprecipitation of [35S]methionine‐labeled cell extracts. The cellular localization of the p53 molecule was studied by immunofluorescent staining of fixed cells with monoclonal antibodies and by immunoprecipitation of [35S]‐methionine‐labeled p53 from various subcellular fractions. p53 was mainly found in the nucleus of the transformed fibroblast, while in the parent non‐transformed primary embryonic cells, p53 was detected in the cytoplasm in a Triton X‐100 soluble fraction, and associated with the cytoskeleton. The modulated distribution of p53 was also confirmed by analyzing a wide range of independently established transformed and non‐transformed fibroblastic cell lines growing in vitro. The switch from the cytoplasmic localization of p53 in the non‐transformed fibroblasts to a chromatin‐associated accumulation in the transformed cells suggests a possible mechanism by which this protein may function in the transformed fibroblasts.

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“…Although the validity of comparisons between X. laevis oocytes and somatic cells can be questioned, it should be noted that Graessman et al (13) previously showed that when large, but not In infected cells the vast majority of both VP-1 and the L-T antigen are located in the nucleus, as determined both by indirect immunofluorescent antibody staining (12) and by immunoprecipitation of detergent extracts of the cytoplasmic and nuclear portions of radioactively labeled cells (1,28). It was thus rather surprising that only a relatively small proportion of the VP-1 and VP-3 capsid proteins were found in the oocyte nucleus.…”

mentioning

confidence: 99%

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Michaeli¹,

Prives²

1985

Mol. Cell. Biol.

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Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after inJection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.The permissive host cell for simian virus 40 (SV40) is the kidney cell of the African green monkey. Primary cultures and established cell lines from this tissue respond to viral infection in a temporally regulated manner. The early phase of infection is characterized by transcription of RNA from the early strand (E-strand) of the viral genome, and these RNAs are processed and translated to the large T (L-T) and small t (S-T) tumor antigens. The transition to the late phase is coincident with the onset of viral DNA replication. During the late phase, L-T and S-T antigens continue to be synthesized, but the majority of the viral RNA is transcribed from the opposite late strand (L-strand) of the viral genome. This RNA is processed and translated to the major (VP-1) and minor (VP-2 and VP-3) capsid proteins (for a review see reference 39). Although very small amounts of late RNA have been detected early in lytic (3, 23) or abortive (7) infection, the great increase in this RNA synthesized late in lytic infection is most likely due to more than one factor. First, the number of templates for transcription, arising from the L-T antigen-mediated induction of viral DNA synthesis, increases vastly late in infection. The high ratio of L-to E-strand-specific RNA, then, is most likely due to t...

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SV40 virions and viral RNA metabolism are associated with cellular substructures.

Cited by 38 publications

References 31 publications

Human papillomavirus 16 E7 protein is associated with the nuclear matrix.

Human papillomavirus 16 E7 protein is associated with the nuclear matrix.

P53 transformation-related protein accumulates in the nucleus of transformed fibroblasts in association with the chromatin and is found in the cytoplasm of non-transformed fibroblasts.

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

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