The core promoter mutants of hepatitis B virus (HBV) emerge as the dominant viral population at the late HBeAg and the anti-HBe stages of HBV infection, with the A1762T/G1764A substitutions as the hotspot mutations. The double core promoter mutations were found by many investigators to moderately enhance viral genome replication and reduce hepatitis B e antigen (HBeAg) expression. A much higher replication capacity was reported for a naturally occurring core promoter mutant implicated in the outbreak of fulminant hepatitis, which was caused by the neighboring C1766T/T1768A mutations instead. To systemically study the biological properties of naturally occurring core promoter mutants, we amplified full-length HBV genomes by PCR from sera of HBeAg ؉ individuals infected with genotype A. All 12 HBV genomes derived from highly viremic sera (5 ؋ 10 9 to 5.7 ؋ 10 9 copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 ؋ 10 7 to 4.6 ؋ 10 7 copies/ml) were core promoter mutants. Of the 11 wild-type genomes and 14 core promoter mutants analyzed by transfection experiments in human hepatoma cell lines, 6 core promoter mutants but none of the wild-type genomes replicated at high levels.

A new procedure for DNA transfection has been developed in a system of chicken embryo fibroblast cells and cloned Rous sarcoma virus DNA by using a polycation reagent as ?t mediator to adsorb DNA to the cell surface and dimethyl sulfoxide as an agent to facilitate the uptake of adsorbed DNA by the cells. In this new, simple, and convenient polycation-dimethyl sulfoxide transfection, which requires no carrier DNA even with small amounts of DNA, the number of transformed cell foci induced by Rous sarcoma virus DNA was proportional to the dose of the transfecting DNA, and chicken embryo fibroblast cells were successfully transformed by v-src-containing subgenomic DNA as well.The transfection technique has been widely used to investigate the biological activities of cellular and viral DNAs directly extracted from various tissues, cells, and virions or those molecularly cloned in bacteria. This technique has contributed greatly to the recent rapid progres § in studies on oncogenes in human cancers (1,21). Of the various methods developed for transfection of tissue-cultured cells (11,14,19,20), the calcium phosphate method has been employed most often because it is more efficient and reproducible than other methods (5). However, the calcium phosphate method has narrow optimal conditions for several factors, such as the pH of the buffer used, the amount of the DNAs, and the size of the calcium phosphate-DNA precipitate. Thus, the efficiency of transfection depends greatly on the conditions used (5,8,10). Furthermore, the mechanism for DNA uptake by the cell from calcium phosphate-DNA precipitate has not been fully elucidated (10). We attempted to develop a simpler and easier procedure for DNA transfection by directly adsorbing DNA to the cell surface instead offorming a calcium phosphate-DNA coprecipitate.Transfection seems to involve two steps, adsorption of DNA to the cell surface and then DNA uptake by cells. Polycations are known to enhance adsorption of retroviruses to cells (17,18), probably by interacting with negative charges of both virions and cell surfaces and thus forming bridges between the two. Therefore, DNAs would likewise be adsorbed to the cell surface in the presence of polycations since DNA molecules have a polyanionic character. In calcium phosphate transfection, brief treatment of cells with 25 to 30% dimethyl sulfoxide (DMSO) appears to increase the transfection efficiency (8, 9, 16). DMSO is considered to enhance uptake of adsorbed DNAs by increasing the permeability of cell membranes. Therefore, we first examined the effects of the combination of the polycation Polybrene, which facilitates retrovirus adsorption (17), and DMSO on DNA transfection. As shown below, the Polybrene-DMSO combination was found to be very effective.In this study, the transfecting agent used was a molecularly cloned Rous sarcoma virus (RSV) DNA derived from plasmid pSRA-2, which was prepared by DeLorbe et al. (3). This plasmid DNA contains the entire genomic sequence of RSV virus in a permuted form. Before transfection, ...

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We have molecularly cloned the provirus of the avian musculoaponeurotic fibrosarcoma virus AS42.

The erbB gene of an avian erythroblastosis virus, AEV-H, was determined to be 1812 nucleotides long and was predicted to code for a protein of 67,638 daltons. Unexpectedly, a sequence of 285 amino acids in the middle of the protein showed a significant homology (38%) with the sequence in the carboxy terminus of p60src. The nucleotide sequence of a mutant of AEV-H, td-130, which induces sarcomas but not erythroblastosis in chicken, was also analyzed. A deletion of 169 nucleotides was identified in the 3' half of the erbB gene, indicating that the gene codes for a truncated protein with the predicted molecular weight of 46,667. These findings suggest that the homologous domain of erbB protein with its N-terminal portion is sufficient for the transformation of fibroblasts and that one-third of the carboxy-terminal domain has a key role for the transformation of erythroid cells.

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Transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), which contains deletions in the gene responsible for transformation (src gene), are unable to transform chicken embryo fibroblasts in vitro. Injection of some of these td mutants into newborn chickens resulted in the formation of sarcomas from which sarcoma virus was unfailingly recovered. The possibility that transforming RSV was present in the td virus preparations was excluded by further purification of the td viruses. Morphology of the foci induced by the newly recovered sarcoma virus was distinct from that of foci induced by the parental Schmidt Ruppin strain of RSV. It is suggested that the new sarcoma virus was generated as a result of the genetic interaction between the genomes of td virus and chicken cells.

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