Proteins of herpesvirus type 2 III. Isolation and immunologic characterization of a large molecular weight viral protein

Strnad, Bruce C.; Aurelian, Laure

doi:10.1016/0042-6822(78)90144-7

Cited by 26 publications

(29 citation statements)

References 29 publications

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“…The Bgl II/Hpa I fragment CD is sufficient in size to code for [10][11][12][13][14][15] proteins. Seven polypeptides of unknown functions, phosphonoacetic acid resistance, and host shut-off genes, unlikely candidates for transformation, have been mapped in this region (20).…”

Section: Resultsmentioning

confidence: 99%

“…The assay was performed in triplicate with optimal concentrations of antigen (20 jig of protein) and IgG (1.4 dilution) (16). Reaction was considered positive if more than 10% of the complement was fixed (13,16).…”

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confidence: 99%

“…Antisera to ICP1O or total viral antigens (Ra-2) were prepared in rabbits by subeutaneous inoculation in the presence of complete Freund's adjuvant (13 ICP10 is an "immediate early" viral protein (14) purified to radiochemical homogeneity by NaDodSO4/acrylamide gel electrophoresis and shown to be immunologically identical (13) to AG-4, the virus-specific antigen that reflects the progression of human squamous cervical cancer (15). Prior to use in these studies, Ra-2 serum was repeatedly adsorbed with uninfected HEp-2 cells (15), and all antisera were fractionated on linear 10-40% (wt/vol) sucrose gradients as described (13). The IgG fractions were used in all assays.…”

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confidence: 99%

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Tumorigenic transformation induced by a specific fragment of DNA from herpes simplex virus type 2.

Jariwalla¹,

Aurelian²,

Ts’o³

1980

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

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Inactivated herpes simplex virus type 2 (HSV-2) (1, 2) and low concentrations of native, noninactivated viral DNA (3, 4) induce the neoplastic transformation of mammalian cells in culture. Transformation appears to be mediated by fragment(s) of the HSV-2 genome: (i) mechanically sheared HSV-2 DNA (Mr 9 X 106) induces neoplastic transformation at a frequency identical to that observed with native DNA (4), (ii) hamster cells transformed by UV-irradiated HSV-2 retain a fraction (8-32%) of the viral DNA sequences (5), and (iii) virus transforming activity is more resistant than its infectivity to radiation damage (1, 2). However, the oncogenic fragment in the HSV-2 genome has not yet been identified.Provided that oncogenic activity does not depend on distant noncontiguous genes and that suitable restriction endonucleases can yield appropriate fragments of HSV-2 DNA that do not express lytic functions, it seems reasonable to propose that neoplastic transformation should be demonstrable with a restriction endonuclease fragment. The data described in this report support the validity of this proposal.MATERIALS AND METHODS Cells and Virus. Syrian hamster embryo (SHE) cells were grown in ERM medium with 10% fetal bovine serum (3). Vero and HEp-2 cells were grown in medium 199 with 10% fetal bovine serum. HSV-2 strain S-1, a human cervical tumor isolate (6), was plaque-purified and grown in HEp-2 cells at a low (0.1 plaque-forming unit/cell) multiplicity of infection. HSV-2 strain 333 was obtained from R. Duff.DNA Purification. Viral DNA was purified from Vero cells infected with HSV-2 strains S-1 or 333 by buoyant density centrifugation in NaI gradients containing ethidium bromide as described (3, 7). To isolate fragments, we crushed gel slices in buffer (10 mM Tris-HCl/1 mM EDTA/0.1% NaDodSO4, pH 8.0) and incubated them overnight at 45°C. Agarose was removed by centrifugation at 10,000 rpm for 20 min, and the supernatant was extracted with phenol/chloroform/isoamyl alcohol and dialyzed against Hepes-buffered saline, pH 7.05 (9). DNA concentration was determined by UV absorbance at 260 nm. Recovery was approximately 75%.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Tumorigenic transformation induced by a specific fragment of DNA from herpes simplex virus type 2.

Jariwalla¹,

Aurelian²,

Ts’o³

1980

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

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“…§1734 solely to indicate this fact. radiochemical homogeneity (8). Monoclonal antibody (mAb) 48S was the gift of M. Zweig (National Cancer Institute, Frederick, MD).…”

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confidence: 99%

“…Immunoprecipitation, electrophoretic separation of radiolabeled proteins on bisacrylamide cross-linked gels, and autoradiography were performed as described (8,9). Using this procedure, we originally estimated the molecular mass of ICP 10 at 160 kDa ±10%.…”

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confidence: 99%

Multistep transformation by defined fragments of herpes simplex virus type 2 DNA: oncogenic region and its gene product.

Hayashi¹,

Iwasaka²,

Smith³

et al. 1985

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

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("immortalization"). However, the cells remained nontumorigenic, thereby raising obvious questions pertaining to the biological effect of the right-hand side ofBgl II C, which is now represented by the cloned BamHI E fragment (pGH17a, 0.533-0.584 m.u., Fig. 1).The present study was designed to (i) determine whether Bgl II C-induced neoplastic transformation of normal diploid SHE cells is a multistep process and (it) identify the sequences that impart tumorigenic potential. The data indicate that a 4.4-kilobase (kb) subfragment located within the BamHI E fragment of HSV-2 DNA (0.554-0.584 m.u.) and overlapping 16% of the right hand of the Bgi II C fragment converts cells immortalized by fragment EcoRI/HindIII AE to a fully tumorigenic phenotype. The 4.4-kb subfragment encodes a 144-kDa protein that is antigenically and structurally similar to a HSV-2-infected cell protein (ICP 10) that appears to be at least one of the components of the viral ribonucleotide reductase (5). A tumor derivative established with these transformed cells expresses the c-myc oncogene. MATERIALS AND METHODSCells. SHE cells, prepared from 13-day embryo, were obtained from T. Okeda and grown in IBR modified Dulbecco's Eagle reinforced medium with 10% fetal calf serum. HEp-2 cells were grown in medium 199 with 10% calf serum. K562 cells were grown in suspension as described (6).Recombinant Plasmid DNA. Plasmids derived from pBR322 that contain fragments of HSV-2 (333) DNA in the 0.416-0.584 m.u. region (Fig. 1) were obtained from G. S. Hayward (The Johns Hopkins University, Baltimore, MD). The plasmids were propagated in Escherichia coli HB 101 and the DNA was purified from cleared lysates by CsCl/ethidium bromide density-gradient centrifugation.Restriction Enzymes and Gel Electrophoresis. Enzymes were purchased from New England Biolabs and used as recommended by the manufacturer. Cleaved DNAs were electrophoresed on horizontal slab gels of 0.7% agarose in Tris borate buffer (89 mM Tris borate/89 mM boric acid/2 mM EDTA) containing 0.5 Ag of ethidium bromide per ml at 25 V for 15 hr. The DNA was visualized with long-wave UV light. DNA-containing agarose bands were electroeluted and extracted with phenol/chloroform/isoamyl alcohol.Establishment of Immortal/Tumorigenic Lines. HindIfllinearized plasmid DNAs (HindIII cuts once within pBR322) were mixed with 5 ,ug of salmon sperm DNA and precipitated with calcium phosphate, and aliquots (0.5 ml) were applied to subconfluent monolayers of normal SHE cells (passage 4; 2 x 105 cells per 60-mm dish) (3). Cultures were refed fresh medium at 4 hr after transfection and were serially passaged (1:10 or 1:20 split ratio) at confluency. Phenotypic alterations associated with transformation were determined as described (3, 7). Colonies in 0.3% agarose were counted at 3 wk and scored as medium (0.1-0.2 mm) or large (> 0.2 mm) by using a calibrated microscope eyepiece. Cloning efficiency (CE) was expressed as the (number of colonies x 100)/number of seeded cells. Tumorigenic potential was determin...

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Viruses and gynecologic cancers: Herpesvirus protein (ICP 10/AG-4), a cervical tumor antigen that fulfills the criteria for a marker of carcinogenicity

Aurelian

Kessler

Rosenshein

et al. 1981

Cancer

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The studies associating infections by herpes simplex virus type 2 (HSV-2) with carcinoma of the human uterine cervix are reviewed within the context of three possible interpretations. Extensive seroepidemiologic evidence indicates that the virus does not grow preferentially in neoplastic tissue, nor is the association of HSV-2 with cervical carcinoma a reflection of their independent relationship to promiscuity. While a number of infectious agents, including other viruses, are associated with cervical atypia, only HSV-2 is a significant risk factor for CIS. In vitro transformation data supporting the oncogenic potential of the virus are summarized, and evidence is presented that an antigen designated ICP 10/AG-4 is a valid candidate for the role of a virus-encoded protein involved in the maintenance of a transformed phenotype. Antibody to AG-4 is IgM, and it is detected by microquantitative complement fixation. With this assay, it is demonstrated that conversion to anti-AG-4 occurs during primary infection with HSV-2; however, it is transient. In testing 1325 patients, a correlation was observed between antibody to AG-4 and cervical carcinoma. Thus, whereas only 11.7% of controls and 7.7% of patients with cancer at other sites are AG-4 seropositive, as many as 49.6% of patients with dysplasia, 63% of those with CIS, and 72.7% of those with invasive cancer are positive for the antibody. Antibody to AG-4 is related to tumor growth. This is evidence by 1) retrospective analyses demonstrating that the proportion of AG-4 seropositive individuals is directly correlated to the state of the disease and 2) prospective study of 209 patients demonstrating loss of antibody in patients with a successfully removed tumor mass and reappearance of AG-4 antibody in cancer recurrence. The possible use of AG-4 (or its antibody) in the diagnosis and monitoring of cervical carcinoma and its treatment is discussed.

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Proteins of herpesvirus type 2 III. Isolation and immunologic characterization of a large molecular weight viral protein

Cited by 26 publications

References 29 publications

Tumorigenic transformation induced by a specific fragment of DNA from herpes simplex virus type 2.

Tumorigenic transformation induced by a specific fragment of DNA from herpes simplex virus type 2.

Multistep transformation by defined fragments of herpes simplex virus type 2 DNA: oncogenic region and its gene product.

Viruses and gynecologic cancers: Herpesvirus protein (ICP 10/AG-4), a cervical tumor antigen that fulfills the criteria for a marker of carcinogenicity

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