Cross-resistance testing of antihepadnaviral compounds using novel recombinant baculoviruses which encode drug-resistant strains of hepatitis B virus.

Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains.

Regulation of heme metabolism in rat hepatocytes and hepatocyte cell lines: delta-aminolevulinic acid synthase and heme oxygenase are regulated by different heme-dependent mechanisms.

Regulation of delta-aminolevulinic acid (ALA) synthase and heme oxygenase was analyzed in primary rat hepatocytes and in two immortalized cell lines, CWSV16 and CWSV17 cells. ALA synthase was induced by 4,6-dioxohepatnoic acid (4,6-DHA), a specific inhibitor of ALA dehydratase, in all three systems; however, the induction in CWSV17 cells was greater than in either of the other two systems. Therefore, CWSV17 cells were used to explore the regulation of both enzymes by heme and 4,6-DHA. Data obtained from detailed concentration curves demonstrated that 4,6-DHA induced the activity of ALA synthase once ALA dehydratase activity became rate-limiting for heme biosynthesis. Heme induced heme oxygenase activity with increases occurring at concentrations of 10 microM or greater. Heme blocked the 4,6-DHA-dependent induction of ALA synthase with an EC50 of 1.25 microM. Heme-dependent decreases of ALA synthase mRNA levels occurred more quickly and at lower concentrations than heme-dependent increases of heme oxygenase mRNA levels. ALA synthase mRNA remained at reduced levels for extended periods of time, while the increases in heme oxygenase mRNA were much more transient. The drastic differences in concentrations and times at which heme-dependent effects were observed strongly suggest that two-different heme-dependent mechanisms control the ALA synthase and heme oxygenase mRNAs. In CWSV17 cells, heme decreased the stability of ALA synthase mRNA from 2.5 to 1.3 h, while 4,6-DHA increased the stability of the mRNA to 5.2 h. These studies demonstrate that regulation of ALA synthase mRNA levels by heme in a mammalian system is mediated by a change in ALA synthase mRNA stability. The results reported here demonstrate the function of the regulatory heme pool on both ALA synthase and heme oxygenase in a mammalian hepatocyte system.

Use of the hepatitis B virus recombinant baculovirus-HepG2 system to study the effects of (-)-beta-2',3'-dideoxy-3'-thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA.

(-)-Beta-2',3'-Dideoxy-3'-thiacytidine (lamivudine [3TC]) is a nucleoside analog which effectively interferes with the replication of hepatitis B virus (HBV) DNA in vitro and in vivo. We have investigated the antiviral properties of 3TC in vitro in HepG2 cells infected with recombinant HBV baculovirus. Different types of information can be obtained with the HBV baculovirus-HepG2 system because (i) experiments can be carried out at various levels of HBV replication including levels significantly higher than those that can be obtained from conventional HBV-expressing cell lines, (ii) cultures can be manipulated and/or treated prior to or during the initiation of HBV expression, and (iii) high levels of HBV replication allow the rapid detection of HBV products including covalently closed circular (CCC) HBV DNA from low numbers of HepG2 cells. The treatment of HBV baculovirus-infected HepG2 cells with 3TC resulted in an inhibition of HBV replication, evidenced by reductions in the levels of both extracellular HBV DNA and intracellular replicative intermediates. The effect of 3TC on HBV replication was both dose and time dependent, and the reductions in extracellular HBV DNA that we observed agreed well with the previously reported efficacy of 3TC in vitro. As expected, levels of HBV transcripts and extracellular hepatitis B surface antigen and e antigen were not affected by 3TC. Importantly, the HBV baculovirus-HepG2 system made it possible to observe for the first time that CCC HBV DNA levels are lower in cells treated with 3TC than in control cells. We also observed that the treatment of HepG2 cells prior to HBV baculovirus infection resulted in a slight increase in the efficacy of 3TC compared to treatments starting 24 h postinfection. The treatment of HepG2 cells with the highest concentration of 3TC tested in this study (2 microM) prior to the initiation of HBV replication markedly inhibited the accumulation of CCC DNA, whereas treatment with the same concentration of 3TC at a time when CCC HBV DNA pools were established within the cells was considerably less effective. In addition, our results suggest that in HepG2 cells, non-protein-associated relaxed circular HBV DNA and particularly CCC HBV DNA are considerably more resistant to 3TC treatment than other forms of HBV DNA, including replicative intermediates and extracellular DNA. We conclude from these studies that the HBV baculovirus-HepG2 system has specific advantages for drug studies and can be used to complement other in vitro model systems currently used for testing antiviral compounds.

Evidence for an internal mechanism of copper toxicity in aquatic animals.

We exposed frog (Rana pipiens) rectus abdominus muscle to 10(-6) M copper and 10(-5) M acetylcholine separately and in combination to test the hypothesis that copper is directly involved in the muscle spasms of fish dying from exposure to incipient lethal concentrations of copper. Copper alone had little effect but mixtures of copper and acetylcholine caused larger contractions than acetylcholine alone. Exposure of muscle to copper plus acetylcholine for 10-15 min resulted in spontaneous, spasmodic contractions.

Fog dispersal by CO(2) laser pulses: an exact solution including prevaporization heating.

The coupled nonlinear equations governing the transmission of a collimated high-energy laser beam through a fog are solved analytically. The incident radiation is assumed to be of sufficient intensity to vaporize the droplets in the path of the beams, thus enabling a greater percentage than without vaporization of the incident energy to be transmitted. Prevaporization heating is included in the treatment, and the vaporization front provides a moving boundary condition for the solution. The initial laser intensity and aerosol density are taken to be arbitrary functions of time and position, respectively; Effects of scattering, molecular absorption, and heat conduction are ignored.

Single-photon counting system for measuring laser parameters.

A single-photon counting system has been developed for measuring gas laser parameters. A linear accelerator provides nanosecond-wide pulses of heavy charged particles which are injected into a specially designed cell. The charged particles excite gas mixtures, and the temporal response of the emitted radiation is observed as a function of wavelength. The resulting data is analyzed to obtain pertinent parameters such as excited-state lifetimes, reaction rate constants, and quenching rate constants. At the present time nuclear pumper laser candidate gases are being examined.