Reduced renal prepro-epidermal growth factor mRNA and decreased EGF excretion in ARF.

Levels of prepro epidermal growth factor (EGF) mRNA in renal cortical tissue and urinary EGF excretion have been determined during cisplatin and ischemia-induced acute renal failure in the rat. Northern analysis of polyadenylated RNAs of kidney cortical tissue showed diminished renal preproEGF mRNA in rats injected with cisplatin (5 mg/kg). The decrease in preproEGF mRNA occurred as early as 12 hours in the kidney and persisted for at least three days after cisplatin injection. The submandibular gland, a major site of EGF synthesis, contained normal levels of preproEGF mRNA. Transplatin, a non-nephrotoxic isomer of cisplatin, did not reduce renal preproEGF mRNA levels. Northern analysis of polyadenylated RNAs of kidney cortical tissue 24 hours after a 50 minute period of renal pedicle clamping also showed reduced preproEGF mRNA levels. By contrast, cisplatin increased renal c-fos mRNA. Urinary EGF excretion was also reduced after cisplatin and ischemia and the decrease in EGF excretion correlated significantly with the degree of renal failure. The data show that nephrotoxic and ischemic renal cell injury reduces preproEGF mRNA and urinary EGF excretion. Reduced preproEGF mRNA and diminished EGF excretion may be important in the functional and regenerative responses to renal injury.

Replicative intermediate of hepatitis B virus in transfected murine fibroblasts.

The NIH 3T3-derived cell line psi AM22b, which carries a defective Moloney murine leukemia virus, was transfected with a plasmid carrying the neo gene and two head-to-tail copies of the hepatitis B virus (HBV) genome positioned with opposing polarities. Both the two HBV dimers and the neo gene were located between two Moloney murine leukemia virus long terminal repeats. Poly(A)+ RNAs isolated from one clone that grew in the presence of G418 contained the two major classes of HBV-specific transcripts (3.5-kilobase pregenome and 2.1-kilobase mRNAs) in approximately equivalent amounts, which was reminiscent of the profiles of viral mRNAs from the livers of infected humans and chimpanzees.

Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions.

Clonal cells derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete hepatitis B surface antigen particles, nucleocapsids, and virions (M. A. Sells, M.-L. Chen, and G. Acs, Proc. Natl. Acad. Sci. USA 84:1005-1009, 1987) which elicit acute hepatitis in chimpanzees (G. Acs, M. A. Sells, R. H. Purcell, P. Price, R. Engle, M. Shapiro, and H. Popper, Proc. Natl. Acad. Sci. USA 84:4641-4644, 1987). We report here the initial characterization of the viral nucleic acids produced in this culture system. Kinetic analyses of nuclear, cytoplasmic, and extracellular HBV DNAs were performed by Southern blotting with radiolabeled HBV strand-specific probes. The results from these analyses indicate that at the stationary cellular growth phase, there is a dramatic increase in the rate at which HBV DNA accumulates. Incomplete double- and single-stranded forms of the HBV genome were detected in the nuclear and cytoplasmic fractions as well as in the extracellular medium. In addition, the nuclear DNA apparently includes multiple complete copies of the HBV genome chromosomally integrated and full-length covalently closed circular HBV DNA. Multiple HBV-specific polyadenylated RNAs with lengths of 3.5, 2.5, and 2.1 kilobases were identified by Northern (RNA) blot analysis. S1 nuclease mapping and primer extension identified a single 3' end and multiple unique initiation sites corresponding to nucleotides just 5' to the pre-S1 region, as well as upstream and within the pre-S2 and precore regions. The nucleic acid profile obtained from these analyses is essentially a facsimile of that obtained by studying liver tissue from HBV-infected individuals.

Murine cells carrying integrated tandem genomes of hepatitis B virus DNA transcribe RNAs from endogenous promoters on both viral strands and express middle and major viral envelope proteins.

Clone 4.10 cells were isolated as a methotrexate-resistant clone arising after cotransfection of mouse 3T3 cells with plasmid DNA containing a head-to-tail dimer of the hepatitis B virus (HBV) genome and DNA coding for methotrexate-resistant dihydrofolate reductase. The majority of methotrexate-resistant clones derived by this procedure have been found to contain multiple copies of the HBV genome, but the intact HBV dimer was rarely preserved. In contrast, 4.10 cells contained at least 40 copies of intact HBV dimer per cell. These cells produced large amounts of 22-nm hepatitis B surface antigen particles that included viral envelope proteins reactive with the pre-S2 region-specific antibody, indicating transcription and translation of the pre-S2 and S regions of the integrated viral genomes. The cells also synthesized viral e antigen, which was released into the culture medium. Characterization of polyadenylated viral RNAs transcribed from the long (minus) strand of the integrated HBV DNA demonstrated the presence of shorter-than-genome-length RNAs containing only X region sequences, shorter-than-genome-length RNAs containing both X and S region sequences, and longer-than-genome-length RNAs containing core, X, and S region sequences. Start sites for transcripts were mapped 5' to and within the pre-S region and 5' to and within the precore region at approximately the same sites as those utilized for HBV transcription during viral replication in infected livers. Polyadenylated RNA transcripts complementary to the short (plus) strand of HBV that initiated and terminated within the intact and integrated head-to-tail tandem viral genomes were also detected.