Examination of homologies between retroviral oncogenes and transforming sequences defined by transfection reveals that the human bladder carcinoma (EJ) oncogene is homologous to the Harvey sarcoma virus oncogene (ras). Structural analysis limits the region of homology to a 3.0-kilobase SacI fragment of the EJ oncogene. Both EJ and ras DNA probes detect similar transcripts in transfectants derived from bladder carcinoma cell lines.

We have previously demonstrated that DNA of mouse fibroblasts transformed by 3-methylcholanthrene (3-MC) induced foci of transformed cells when applied to monolayer cultures of NIH3T3 cells, which indicates that at least a part of this phenotype is encoded in DNA sequences. However, our conclusions were confined to the effects of DNAs of 3-MC-transformed mouse fibroblasts on recipient NIH3T3 cells, also of mouse fibroblast origin. To elucidate this phenomenon further, we have prepared DNAs from a series of mouse and non-mouse tumour lines of non-fibroblastic origin and investigated whether tumour transforming genes can act across tissue and species barriers to transform NIH3T3 cells. We find that DNAs obtained from human, rabbit and mouse bladder carcinoma lines, a lung carcinoma line and rat neuroblastoma and mouse glioma lines, are able to induce transformation of NIH3T3 cells on transfection.

No abstract

Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nucleaseresistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.The hepatitis B virus (HBV) core antigen (HBcAg) is a 22-kDa protein with multiple functions, including interactions with the pregenomic RNA (pgRNA) and the polymerase during encapsidation (10,31,41) and with the viral DNA during reverse transcription and DNA elongation (18), polymerization to form the nucleocapsid or core particles (4, 13), importing of HBV DNA to the nucleus (51), and targeting to the endoplasmic reticulum for envelope formation (5). The C-terminal region of HBcAg contains a protamine-like domain, which is rich in arginine and presumably binds to HBV RNA and DNA during pgRNA encapsidation and DNA replication (13,18,35). This arginine-rich domain has been shown to be dispensable for core particle assembly but not for viral replication (2, 4, 13, 30).

(12,13).The transfection of these sarcoma virus DNAs led to the observation of foci of transformed cells whose behavior was indistinguishable in many cases from that of virus-infected cells. Some of these transfections utilized donor cellular DNA in which the transforming genome was present in single copy number per haploid cell DNA complement. We reasoned that nonviral transforming genes, if present in unique copy number, might also be transferable via DNA transfection. Specifically, we attempted to demonstrate the existence of genes in the DNA of chemically transformed cells whose introduction into normal recipients would result in focal transformation of the recipient monolayer. MATERIALS AND METHODSCell lines used here are described in Table 1. DNA transfection procedures were as described (11). DNA was prepared from tumors or cell lines as described (23).Chromatin transfection procedures were as described (24) with the following exceptions: (a) recipients were not pretreated with mixtures of colchicine, Colcemid, and cytochalasin D before transfection, (b) gentamicin was not used in these experiments, (c) instead of counting the chromosome number under a microscope, quantitation of chromosomes was done by spectrophotometric absorbance, and (d) 10% dimethyl sulfoxide posttransfectional treatment was not always included.Southern gel-filter transfer was performed as described (12). The soft agar assay was done by pouring 0.3% soft agar (Difco) containing 3000 cells over a 0.6% agar layer in a 6-cm dish. Colonies were scored 14 days later. Transforming virus rescue assays were done as described (11). RESULTSA series of 15 different cell lines (Table 1) were collected from various sources. These cell lines were all of murine origin and most were transformed in vitro by various carcinogens commonly used for in vivo and in vitro chemical carcinogenesis (25,26). DNA from all these lines was prepared and transfected in a fashion identical to that used in the transfection of retrovirus DNAs. The recipient cells used for monitoring the biological activity of these DNAs were a subline of NIH3T3 cells which fulfills two requirements for these experiments. First, these cells take up DNA in a biologically active form at high efficiency compared with most other mouse cell lines that we have tested. Second, these cells are contact inhibited, and the monolayers they form allow relatively easy visualization of transformed foci.After transfection of NIH3T3 cultures, the cells were reseeded and scored for foci 14-20 days after transfection. The scored foci were examined individually and were counted only if the constituent cells were hyperrefractile, grew in a crisscrossed pattern, and formed a colonial morphology that we feel Abbreviation: MSV, murine sarcoma virus. 5714 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.

An antiserum against human epidermal keratins was used to detect keratins in frozen sections of various rabbit and human tissues bv indirect immunofluorescence. Strong staining was observed in all stratified squamous epithelia (epidermis, cornea, conjunctiva, tongue, esophagus, vagina, and anus), in epidermal appendages (hair follicle. sebaceous gland, ductal and myoepithelial cells of sweat glands), as well as in Ilassall's corpuscles of the thymus, indicating that all contain abundant keratins. No staining by the antiserum was observed in fibroblasts, muscle of anv tvpe, cartilage, blood vessel, nerve tissue, iris or lens epithelium, or the glomerular or tubular cells of the kidnev. In contrast, the antiserum stained the cells of most epithelia of the intestinal tract, urinary tract (urethra, bladder, ureter, collecting ducts of kidney), female genital tract (cervix, cervical glands, uterus, and oviduct), and respiratory tract (trachea and bronchi). Epithelial cells of the fine ductal system in the pancreas and submaxillary gland also stained well. When primary cultures of epithelial cells derived from bladder, intestine, kidney, and trachea were grown on glass coverslips and stained with anti-keratin, fiber networks similar to those of cultured keratinocytes were observed. These results show that keratins constitute a cytoskeleton in epithelial cells of diverse morphology and embryological origin. The stability of keratin filaments probably confers the structural strength necessary for cells covering a free surface. Keratin staining can be used to obtain information about the origin of cell lines. The stratified squamous epithelium is the most common covering or lining surface of the animal body. It may be of ecto-(lernmal origin (the epidermis) or of endodermal origin (the esophageal epithelium). The dominant cell type of these epithelia (the keratinocvte) contains abundant 80-A filaments composed of keratin proteins. Cells of this type cultivated from different stratified sqltuamolus epithelia are rather similar (1), keratins accounting for about 30% of the cellular protein.Most other epithelia, such as those of the intestinal, respiratory, genital, and urinary tracts, seem quite different. Although the cells of these epithelia frequently contain 80-to 100-A filaments, they are not as abundant as those of the keratinocvte and mav not show their typical lateral aggregation. The cells of epithelia other than stratified squamous are often secretory and have morphology and specialization very different from those of the keratinocvtes.The keratins of human stratum corneum have been purified as a group and used to make a rabbit antiserum (2). This antisertim produces specific precipitin lines when tested against extracts of keratinocytes, and it stains by indirect immunofluorescence a network of fibers wvithin the cytoplasm of keratinocytes (3). Using this serum, wve show here that keratins are present not only in cells of all stratified squjamous epithelia (of hunman and rabbit) tumt also in many other e...

The capsid shell of infectious hepatitis B virus (HBV) is composed of 240 copies of a single protein called HBV core antigen (HBc). An atomic model of a core assembled from truncated HBc was determined previously by X-ray crystallography. In an attempt to obtain atomic structural information of HBV core in a near native, non-crystalline environment, we reconstructed a 3.5Å-resolution structure of a recombinant core assembled from full-length HBc by cryo electron microscopy (cryoEM) and derived an atomic model. The structure shows that the 240 molecules of full-length HBc form a core with two layers. The outer layer, composed of the N-terminal assembly domain, is similar to the crystal structure of the truncated HBc, but has three differences. First, unlike the crystal structure, our cryoEM structure shows no disulfide bond between the Cys61 residues of the two subunits within the dimer building block, indicating such bond is not required for core formation. Second, our cryoEM structure reveals up to four more residues in the linker region (amino acids 140-149). Third, the loops in the cryoEM structures containing this linker region in subunits B and C are oriented differently (~30° and ~90°) from their counterparts in the crystal structure. The inner layer, composed of the C-terminal arginine-rich domain (ARD) and the ARD-bound RNAs, is partially-ordered and connected with the outer layer through linkers positioned around the two-fold axes. Weak densities emanate from the rims of positively charged channels through the icosahedral three-fold and local three-fold axes. We attribute these densities to the exposed portions of some ARDs, thus explaining ARD’s accessibility by proteases and antibodies. Our data supports a role of ARD in mediating communication between inside and outside of the core during HBV maturation and envelopment.