Department of Molecular Genetics, Microbiology and Immunology

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Michael Leibowitz, MD

Michael Leibowitz, M.D.
Professor
Office: 732-235-4795
Lab: 732-235-5453
leibowit@umdnj.edu

Office: RWJMS 704
Lab: RWJMS 705

Publications

Click Here for PubMed Link to Publications

Research Interests

A Prion-like Element Alters Viral Gene Expression in Yeast. A unique mechanism which regulates viral gene expression is being studied using the cytoplasmically-inherited killer virus of yeast. Killer virus is a double-stranded (ds) RNA virus which confers upon infected yeast cells the two phenotypes of toxin production and secretion (K+) and resistance to the homologous toxin (R+). The replication competent L-A dsRNA genomic segment of the virus is a 4.6 kbp. dsRNA encoding the major capsid protein and a capsid-RNA polymerase fusion protein generated by a -1 translational frame-shift mechanism. The M satellite dsRNA, which depends on L-A dsRNA helper function for its replication, encodes the preprotoxin precursor of the toxin and resistance substance, that are produced in wild-type yeast cells which harbor M dsRNA. The prion-like [KIL-d] cytoplasmic genetic element places the viral M dsRNA segment under epigenetic control by regulatory elements of the host cell. In haploid cells harboring [KIL-d] and M dsRNA, mitotically stable defective expression of toxin and/or resistance is observed. Upon mating the defective phenotypes are "healed," so diploids are wild-type in phenotype. [KIL-d] is cytoplasmically inherited and transmissible by cytoduction (cytoplasmic exchange), but does not map on M or L-A dsRNA, mitochondrial DNA, the 2-micron DNA plasmid, or the [PSI] or [URE3-1] prions. However, [KIL-d] does show prion-like properties on reversion studies. The defective killer virus phenotypes of [KIL-d] haploids harboring M dsRNA revert to the wild-type phenotype with a relatively high frequency of 10-5. These wild-type revertants "back revert" to yield variegated defective phenotypes with a frequency of 10-3. This very high rate of back reversion to variegated phenotypes is not seen after the loss or mutagenesis of any known plasmid, but is similar to the variegated reappearance of the [PSI] prion after it is lost. Thus, [KIL-d] has prion-like genetic properties, although a host gene encoding the putative prion protein has not yet been identified; identification of this protein is the goal of future research.

Prions have been strongly linked to the etiology of spongiform encephalopathies in various mammalian species; however, in the absence of complete elucidation of the pathogenic mechanisms by which prions cause neurological disease, a role for cryptic viruses in these diseases has been proposed as an alternative to the prion hypothesis. The interaction of the prion-like [KIL-d] element with the dsRNA genome of killer virus of yeast suggests a hypothesis unifying the prion and virus models, in which prions might function by modifying phenotypic expression of a cryptic virus. In the yeast system, the epigenetic effects conferred by [KIL-d] on M dsRNA expression are specific for viral gene expression and are not observed on cDNA clones derived from the viral genome. Future work is planned to elucidate the nature of the [KIL-d] prion-like element and the molecular basis of its specific epigenetic effect on the M dsRNA viral genomic segment.

RNA-Targeted Therapeutic Agents and Drug Delivery. In collaboration with Drs. P.J. Sinko, S. Stein and A.B. Rabson, we have developed analogs of the RNA binding domain of the Tat regulatory protein of HIV-1, which inhibit Tat-dependent gene expression from the HIV-1 long terminal repeat (LTR); this inhibition is seen both in chimeric constructs and in HIV-infected cultured cells. We are also investigating the use of Tat analogs as cell permeation peptides, which can be appended to polymeric conjugates bearing drugs or antisense molecules to promote conjugate entry into cells. We are have found that polymeric conjugates can be targeted to specific cell types, such as macrophages, by appending to each conjugate multiple ligands for surface receptors on the targeted cells. In the case of macrophages, we found that multiple copies of the chemoattractant peptide formyl-methionyl-leucyl-phenylalanyl greatly enhanced specific delivery without similar enhancement of macrophage activation, indicating the potential of this technology to deliver drugs to macrophages without activation-mediated toxicity. We are currently investigating the use of specific targeting and cell permeating agents on polymeric drug carriers to develop new drug and antisense delivery methods applicable to treatment of AIDS and cancer. We have also developed various polymeric systems for drug delivery, whose pharmacokinetic properties and applications are being studied.

Inhibitors of Splicing Activity of Ribozyme Intron. Previous work from this laboratory has shown that the anti-pneumocystis drug pentamidine acts as a potent non-competitive inhibitor of the group I self-splicing intron ribozymes expressed in vitro from the nuclear rRNA genes of that organism. However, since pneumocystis cannot readily be grown in culture, we have continued these studies in other organisms. In the case of Saccharomyces cerevisiae, we have found that pentamidine inhibits the splicing of some group I and group II intron ribozymes present in the mitochondrial genome. We have also found that pentamidine is a potent and specific inhibitor of mitochondrial protein synthesis in that organism. It was previously known that in the opportunistic fungal pathogen Candida albicans, some strains harbor the group I ribozyme intron Ca.LSU in their nuclear large rRNA precursor, while others do not. We found that in intron-containing strains, 1 µM pentamidine inhibits both the splicing of intron Ca.LSU and the maturation of pre-rRNA in one minute, while no effect is seen in inronless strains, suggesting that the group I intron might be a sensitive and specific site of pentamidine action in this organism. Group I introns could represent attractive therapeutic targets, since they are present in a number of pathogenic microorganisms but not in mammalian cells.

Further studies of Ca.LSU revealed that this ribozyme could fold into a catalytically active form during incubation in the absence of divalent cations, but in the presence of divalent cations, such as magnesium, manganese or spermidine, aberrant folding occurs. This divalent cation-induced aberrant folding reduces self-splicing activity of Ca.LSU and seems to represent a “kinetic trap” in the multi-step process of RNA folding. Our study showed that pentamidine alters the folding of Ca.LSU ribozyme, making some peripheral regions less sensitive to ribonuclease T1 cleavage and some other regions more sensitive to T1 cleavage. Helix P7, constituting the active site of the ribozyme, was among the sensitized regions, indicating that alteration of the folding of the P7 helix in the presence of pentamidine that may be account for its inhibition of the ribozyme activity. Inhibition of Ca.LSU self-splicing by the presence of pentamidine is antagonized by the presence of divalent cations and spermidine, suggesting that the positive charges of pentamidine play an important role in its interaction with the ribozyme RNA. We are currently using fluorescence methods to study the interaction of ribozyme Ca.LSU with pentamidine, as an approach to study how drugs interact with a therapeutic target on RNA. This work is being done in collaboration with Drs. Y. Zhang (Wuhan University), D. Pilch and G. Brewer.