Laura Coates

In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway

During the productive phase of chronic hepadnaviral infections, virion DNA synthesis occurs in the cytoplasm of the infected hepatocyte, but viral RNA is synthesized in the nucleus, apparently from a covalently closed, circular (CCC) viral DNA. J. Tuttleman, C. Pourcel, and J. Summers (1986a, Cell 47, 451-460) have shown that the intracellular levels of CCC DNA can increase during initiation of infection of duck hepatocytes in vitro with duck hepatitis B virus and during long term culture of infected duck hepatocytes in vitro. This amplification of CCC DNA occurs through the reverse transcription pathway. To distinguish between an entirely intracellular process of amplification and amplification due to multiple infections by extracellular virus in the virus producing cultures, suramin was added to the infected cultures to block superinfection. We found that CCC DNA amplification occurred at least as efficiently in the presence of suramin as in its absence. First, there was a net increase in the total amount of CCC DNA in the cultures both in the presence and in the absence of suramin. Second, synthesis of CCC DNA in the presence and absence of suramin was observed by density labeling of this viral DNA by growth of the cultures in medium containing BUdR. Amplification was also demonstrable in the presence of neutralizing duck antibodies. These results support the hypothesis of Tuttleman et al. (1986a) that CCC DNA amplification in chronically infected cultures and, by inference, the mechanism of persistent infection involves primarily intracellular regulatory mechanisms.

Experimental transmission of duck hepatitis B virus

Susceptibility to experimental infection with duck hepatitis B virus (DHBV) was explored, with the objective of defining procedures that were both rapid and reproducible. For the purpose of these experiments, a small flock of DHBV-free breeders was established as a source of susceptible eggs and ducklings, since ca. 10% of the ducks (all ages) from commercial flocks were DHBV infected. Intravenous inoculation of DHBV into 15-day duck embryos from the DHBV-free flock produced a persistent infection, with a high-titer viremia, in at least 80% of the injected animals. The tissue tropism of DHBV in these experimentally infected animals was similar to that associated with natural, congenital infections from viremic ducks to their progeny. Virus antigen was found not only in hepatocytes and bile duct epithelium of liver, but also in cells associated with exocrine and endocrine pancreas, and in proximal convoluted tubular epithelium of kidney. Infection of embryonic liver was rapid, as evidenced by active synthesis of DHBV-DNA by reverse-transcription of RNA by 24 hr postinjection. During this latter analysis, formation of supercoiled viral DNA appeared to precede the reverse-transcription phase of viral DNA synthesis, suggesting that this species may be important in initiation of infection.

Suramin inhibits in vitro infection by duck hepatitis B virus, Rous sarcoma virus, and hepatitis delta virus.

Suramin blocked in vitro infection by duck hepatitis B virus, a hepadnavirus, and Rous sarcoma virus, a retrovirus. Although suramin was able to inhibit the virus-encoded reverse transcriptase activities of these two viruses, this inhibition did not appear to account for the anti-viral effect of the drug. In particular, suramin was unable to block synthesis within cells of full-length viral DNAs when added subsequent to infection. The results are consistent with the hypothesis that suramin acted by blocking virus uptake or uncoating. As further support of this hypothesis, we found that suramin also blocked infection by hepatitis delta virus, an RNA virus that is not known to employ reverse transcriptase during the initiation of infection.

In Vitro infection of woodchuck hepatocytes with woodchuck hepatitis virus and ground squirrel hepatitis virus

Primary cultures of woodchuck hepatocytes were demonstrated to be susceptible to in vitro infection by both woodchuck hepatitis virus and ground squirrel hepatitis virus, as evidenced by the appearance of DNA species characteristic of hepadnavirus replication. Initiation of infection by woodchuck hepatitis virus was blocked by the presence of suramin, polybrene, or dideoxycytidine. Viral CCC DNA, the putative template for viral RNA transcription, was detected at 2 days postinfection. Accumulation of intracellular intermediates in virion DNA synthesis was negligible until 7-10 days postinfection, but these DNA intermediates then increased dramatically in amount over the next few weeks. Results were obtained which suggested that the prolonged accumulation of intermediates in virion DNA synthesis was an intrinsic property of the infection of individual cells, and not the result of a slow spread of virus through the cultures.

Regression of v-src DNA-induced sarcomas is under host genetic control

Previous results have established that subcutaneous inoculation of chickens (line SC) with a v-src(+) subviral DNA fragment induces the formation of progressor sarcomas at the wing web site of inoculation. Because the sarcoma cells are incompetent for production of exogenous progeny virus, this system is a useful model of tumor expansion by sarcoma cell division, in the absence of infection-mediated recruitment of new tumor cells. The present study was undertaken to define conditions that modulate the pattern of growth (regression vs progression) of v-src DNA-induced sarcomas. These conditions were found to include the line of chicken or the presence on the subviral v-src(+) DNA fragment of a viral replication-specific sequence that includes env.