Nurul H. Sarkar

Purification and properties of murine mammary tumor virus DNA polymerase

Abstract Murine mammary tumor virus (MuMTV) DNA polymerase was purified by affinity chromatography on polycytidylate-agarose followed by phosphocellulose ion-exchange chromatography. The resulting enzyme preparation contained two major polypeptides, of 85,000 and 50,000 daltons as determined by SDS-polyacrylamide gel electrophoresis, and appeared, except for low levels of RNase H, free of DNase and RNase activity. The molecular weight of the purified native enzyme as determined by velocity sedimentation, was approx. 108,000. MuMTV DNA polymerase appears to be a zinc metalloenzyme and requires at least one reduced sulfhydryl group for the expression of catalytic activity. Apparent K m values determined for synthetic template-primers and deoxynucleoside triphosphates were 1 μg/ml and 10–12 μ M , respectively. Examination of the optimal biochemical conditions for DNA synthesis on a variety of template-primers revealed that Mg 2+ was the preferred divalent cation for DNA synthesis. Mn 2+ could partially substitute for Mg 2+ , although it inhibited DNA synthesis when added in the presence of Mg 2+ . MuMTV DNA polymerase exhibited a preference for (dC) n ·(dG) 12−18 among all the synthetic template-primers tested. Activated DNA was preferred as a heteropolymeric template for DNA synthesis when compared with viral 70 S RNA with either endogenous primers or with (dT) 10 as a primer. Rates of heat inactivation of the MuMTV DNA polymerase were found to vary depending upon the template-primer used to measure that inactivation.

Replication of mouse mammary tumor virus in tissue culture: I. Establishment of a mouse mammary tumor cell line, virus characterization, and quantitation of virus production

Abstract The establishment of an epithelial cell line (MuMT-73) derived from spontaneous mammary tumors of BALB/cfC3H mice as a source for the continuous production of mouse mammary tumor virus (MuMTV or B particles) is described. Morphological, immunological, and biochemical techniques were used to characterize the virus. It was found that, under conditions of normal cellular growth, the cells produced only type-B particles; no concomitant synthesis of Type-C particles was detected. A unique feature of this cell line is that the cells do not produce detectable amounts of intracytoplasmic type-A particles, but many B particles were found to assemble at the cell surface with gradual development of the nucleocapsid. The kinetics of virus release into tissue culture medium after transfer of cells was studied quantitatively by particle counting and viral reverse transcriptase (RDDP) measurements. When the medium was changed every 24 hr, the cells continuously produced virus at a slow rate for up to 6 to 8 days and thereafter at an increased rate for up to 12 days. After this period, virus production gradually declined but did not decrease to the basal level. During the period of high level virus production, about 30–40% of the cells were found to produce MuMTV antigens. By estimating the total number of virus particles in the culture medium and by counting the number of antigen-positive cells, it was found that over a 20-hr period during optimum virus production a cell would release approximately 800 virus particles. Treatment of the cells with either hydrocortisone or dexamethasone resulted in an increased production of type-B particles. An additional increase in virus production was obtained when the cells were treated with insulin together with either hydrocortisone or dexamethasone.

Separation of epidermal cells by density gradient centrifugation on a continuous colloidal silica (Percoll) gradient

Abstract The epidermis, which is composed mainly of keratinocytes, undergoes continual proliferation and differentiation. Beginning as a basal cell, the keratinocyte matures and ascends through the spinous cell, granular cell, and horny cell layers. To study keratinocyte differentiation in vitro , it is necessary to separate the keratinocytes at their successive stages of maturation. A method for separation of epidermal keratinocytes is reported here. The method utilizes centrifugation on a continuous colloidal silica (Percoll) density gradient, which separates the keratinocytes according to their inherent density. The resulting arrangement of the keratinocytes in the centrifuge tube generally corresponds to their arrangement in situ , with basal cells at the bottom and the more differentiated cells above. There is a continuous decrease in the nuclear:cytoplasm ratio of keratinocytes as the cells mature. Using this technique, it was shown that the decrease in the nuclear:cytoplasm ratio corresponds to decreasing density of keratinocytes as they mature. Using hematoxylin and eosin staining and electron microscopic studies, it was shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. The method is simple and reproducible, and should greatly facilitate biochemical analyses of the various maturation steps of epidermal keratinocytes.

Polypeptides of the mouse mammary tumor virus: II. Identification of two major glycoproteins with the viral structure

Abstract In order to localize the major structural polypeptides of the murine mammary tumor virus (MuMTV), three subviral fractions were prepared, examined by electron microscopy, and analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate. Treatment of intact virus with 0.05 N HCl at 4° for 20 min resulted in the removal of the characteristic surface projections of the virus. Viral membrane fragments with attached projections (rosettes), consisting of stalks and knobs, were prepared by treating MuMTV with Tween-80 and ether. The buoyant density (in sucrose gradients) of the particles after removal of the projections was 1.181 g/cm 3 , as compared with 1.171 g/cm 3 for the intact virus. PAGE analysis of the viral material released by the action of acid (for 20 min at 4°) revealed that it contained mainly two glycoproteins of 55,000 (gp55) and 68,000 (gp68) molecular weight (MW), gp55 being the major species. The polypeptide composition of the projectionless particles was found to be complex; the major component was a glycoprotein of 34,000 molecular weight (gp34). Although intact virus contained only one glycoprotein (gp55) in the 50,000–60,000-MW range, the projectionless particles were found to contain two minor components within the same MW range, only one of which was a glycoprotein. The nonglycopeptide component with an apparent MW of 51,000 (p51) appears to be a normal minor protein constituent of MuMTV. The viral rosettes were found to contain gp34, gp55, gp68, and an additional glycoprotein of MW 90,000 (gp90). A comparison of the morphology and polypeptide composition of the intact virus and subviral components suggests that the knobs of the viral projections are composed of gp55. The gp34 component is associated with the viral membrane and may be situated within the membrane bilayer from which the projections protrude.

Structural alterations in the long terminal repeat of an acquired mouse mammary tumor virus provirus in a T-cell leukemia of DBA/2 mice

ML, a transplantable T-cell leukemia of DBA/2 mice, expresses the gag and env gene products of the murine mammary tumor virus (MuMTV). Analysis of the genomic DNA of ML cells using the restriction enzyme HindIII and hybridization with MuMTV-specific probes revealed that the ML cells contained two or more newly integrated MuMTV proviruses (ML-MuMTV). Further analysis of these proviruses with a combination of Mspl and Pstl enzymes showed that the long terminal repeat (LTR) (ML-MuMTV LTR) of the ML-MuMTV provirus(es) was structurally different from the LTRs of both exogenous and endogenous MuMTV proviruses of DBA/2 mice. In order to characterize the nature of the structural alterations in the ML-MuMTV LTR, we cloned a 4.0-kb HindIII fragment containing the 3 half of an acquired provirus. Sequence analysis of the ML-MuMTV LTR of this acquired provirus revealed a deletion of a 387-bp segment that maps between the 5 nucleotide 616 and the 3 nucleotide 1003 of the normal MuMTV LTR and duplication of a 102-bp fragment that mapped between 514 and 616. In addition to two point mutations in the direct repeat, the proviral ML-MuMTV LTR has also acquired 9- and 7-bp segments at the 5 and 3 sites of the duplicated 102-bp segment, respectively. Since direct repeats in the U3 regions of a number of LTRs have been found to be associated with enhancer function, we examined the enhancer function of the U3 region sequences of the ML-MuMTV LTR using enhancer-dependent transient expression assay of chloramphenicol acetyltransferase (CAT) gene in NIH 3T3 cells. Our studies have shown that the U3 region sequences of the rearranged ML-MuMTV LTR have the ability to enhance the expression of the CAT gene 12- to 15-fold more than the U3 region sequences from the normal MuMTV LTR. The presence of a direct repeat in the ML-MuMTV LTR and its ability to enhance the transcription of adjacent genes is analogous to the LTRs of certain murine leukemia viruses.

Effects of interferon on the production of murine mammary tumor virus by mammary tumor cells in culture

A cell line established from the mammary tumor of a GR mouse produces murine mammary tumor virus upon stimulation- with dexamethasone in vitro. Treatment of these stimulated GR cells with 50 U/ml of partially purified mouse interferon inhibited the extracellular virus production by about 80% as measured by virus-associated reverse transcriptase activity or by metabolic labeling of the virus with [3H]uridine or 14C-amino acids. There was no inhibition of cellular RNA or protein synthesis under these conditions. The same effects were observed when the same dose of pure mouse interferon was used. Though the interferon-treated cells produced fewer extracellular virus particles, the intracellular levels of viral RNA as measured by hybridization analyses and the intracellular levels of the major viral envelope and core proteins as measured by radioimmunocompetition assays were not lowered in the interferon-treated cells. Electron microscopy of thin sections from the control and interferon-treated cells revealed no gross differences in any subcellular structures including that of the viruses. Scanning electron microscopy showed, however, an increased number of virus particles on the cell surface of the interferon-treated cells. These results suggest that the impairment of extracellular mammary tumor virus production by interferon treatment of GR cells is caused by an interferon-induced block at a very late stage of virus morphogenesis.

The relative hydrophobicity of oncornaviral structural proteins

Abstract The interaction of the major structural polypeptides from the murine mammary tumor virus (MuMTV) and Rauscher murine leukemia virus (R-MuLV) with alkyl-agarose derivatives containing hydrocarbon arms of various lengths was examined. Both R-MuLV and MuMTV polypeptides were selectively adsorbed to octylimino (C 8 )-agarose and decylimino (C 10 )-agarose columns. Elution of proteins was accomplished using buffers containing 8.5 M ethylene glycol. In certain cases, polypeptides could only be removed from the columns by the addition of detergent to ethylene glycol-containing buffers. By examining the chromatographic behavior of polypeptides using (C 8 )- and (C 10 )-agarose columns and the conditions required for the elution of adsorbed proteins, we were able to determine the relative potential for hydrophobic interaction (degree of hydrophobicity) for each viral protein. The most hydrophobic polypeptide of MuMTV was found to be the glycoprotein gp34, while p15(E) and the gag precursor prp67 appeared to be the most hydrophobic of the R-MuLV proteins. The least hydrophobic polypeptides of MuMTV were p23 and p16, while those of R-MuLV were the two major internal proteins, p10 and p12. Four degrees of apparent hydrophobicity, from least hydrophobic to strongly hydrophobic, could be discerned by this procedure. The major core proteins (MuMTV p28, R-MuLV p30) of both viruses were slightly hydrophobic, while their major glycoproteins were moderately hydrophobic. The degree of hydrophobicity of oncornaviral polypeptides, as determined by hydrophobic chromatography, appeared to bear some relation to the different subviral components with which the proteins were associated. Our results indicate that hydrophobic chromatography may provide a new parameter for the characterization of oncornaviral polypeptides. This procedure should also prove applicable to the study of polypeptides from other viral systems.

Identification of the structural proteins of the murine mammary tumor virus that are serologically related to the antigens of intracytoplasmic type-A particles

Abstract The immunological relationship between intracytoplasmic A particles and B particles was investigated in order to determine whether A particles are the preformed cores of mature B particles. A serum prepared in rabbits against purified A particles (obtained from Leydig cell tumors) reacted, in immunodiffusion tests, to extracts from a variety of mouse tumors. It was found that tumors containing both A and B particles as well as those with only A particles contained A particle-associated antigens. Radioimmunoprecipitation and polyacrylamide gel electrophoretic (PAGE) techniques were used to identify those murine mammary tumor virus (MuMTV) proteins that were serologically related to the antigens of A particle. The MuMTV proteins were radiolabeled with either 14C-labeled amino acids in tissue culture or with 125I using the lactoperoxidase method. The solubilized viral proteins were tested with anti-A particle serum, or as controls, with antisera to MuMTV, MuMTV-gp55, and -p28, as well as normal rabbit and goat sera. Rabbit anti-MuMTV serum precipitated all the major polypeptides of MuMTV (gp55, gp36, p28, p18, and p12) when reacted with solubilized MuMTV proteins. However, rabbit anti-A particle serum precipitated only p28, p18, and p12. PAGE analysis of the MuMTV proteins precipitated by goat anti-MuMTV gp55 or by p28 sera showed only gp55 or p28; normal goat or rabbit serum did not precipitate detectable amounts of viral proteins indicating that the reactions were specific. These results suggest that A particles contain antigens which cross-react with the three major internal proteins of MuMTV and support the hypothesis that intracytoplasmic A particles are the preformed cores of B particles.

Murine mammary tumor virus related antigen in human male mammary carcinoma.

An antigen immunologically related to mouse mammary tumor virus (MuMTV) and the major envelope glycoprotein, gp52 of MuMTV, was identified in tissue sections of human male and female mammary ‐ carcinomas using the peroxidase‐antiperoxidase technique. The specificity of the reaction was established by absorption studies. Positive reactions with the gp52 antiserum were seen in mouse and human mammary carcinomas, but not in normal mammary tissues, mammary tissues with benign diseases and in other primary malignant neoplasms. Almost all (32/36, 89%) male mammary carcinomas were positive for the gp52 related antigen. A lesser proportion of tumors among female patients (14/50, 28%) were positive. The gp52 positive tumors were significantly larger than the gp52 negative tumors in female patients (P < 0.05). Gp52 positive tumors were also larger than gp52 negative tumors in male patients, but the difference was not statistically significant. Gp52 reactivity was also detected in metastatic mammary carcinoma in axillary lymph nodes of male and female patients. The presence of gp52 related antigen was not apparently related to tumor grade or lymphocytic infiltrate in the primary tumor. The data do not permit a firm conclusion regarding nodal status in men; no correlation of gp52 activity and nodal status in women was evident. These results indicate that mammary carcinomas in men as well as in women have an antigen related immunologically to MuMTV gp52. Other than tumor size, the antigen seems to be unrelated to major prognostic factors. The significance of the antigen with respect to etiologic features and prognosis in breast cancer remains to be determined.

An enzyme-linked immunoassay for the detection of antibodies to the mouse mammary tumor virus: Application to human breast cancer

An enzyme-linked immunoassay (ELISA) was developed, using the mouse mammary tumor virus (MMTV) fixed to wells of a microtiter plate, for the determination of antibodies to MMTV. The intensity of the final color change was dependent upon virus or viral antibody concentration. MMTV antibody was readily detectable in sera diluted as much as 1 : 2800. Fixed MMTV bound antibodies to an internal viral protein (p 28) as well as to viral envelope components (gp 52, gp 34), demonstrating that the virus was rendered permeable by our procedure. Applying this assay to human sera, significant differences (P less than 0.005) in IgG binding to MMTV were detected between sera of breast cancer patients, benign breast disease patients and healthy individuals. 26% of breast cancer-derived sera contained MMTV binding antibody; 10% of benign sera or 8% of normal sera were also positive. The reactivity of human IgG with MMTV was blocked by prior incubation of the virus with antisera to gp 34 or, to a lesser extent, with gp 52. The results demonstrate that MMTV antibodies can be quantitated by this simple, rapid and inexpensive procedure.