Ann Skalka, PhD
Senior Advisor to the President
Office Phone: 215-728-2490
Overview of Research Interests
The work in our laboratory is focused on obtaining a detailed understanding of the mechanism by which retroviral DNA is integrated into its host cell chromatin, and discovering the epigenetic factors and processes that affect its subsequent expression. Retroviruses are of special interest, not only because they are agents of disease, including cancer, but also because they are important as vehicles for the insertion of desired genes into target cells for scientific investigation and gene therapy. We exploit a broad range of investigational methods in our studies from biochemical and biophysical analyses of protein function, to in vivo studies of viral growth and cell biology. This comprehensive approach provides unique insights, and excellent opportunities for cross-discipline training and collaboration. Our continuing overarching goals are to uncover new information of fundamental importance to both virus and cell biology, and to identify new targets for therapies to treat disease.
We have made exciting progress in our investigation of the molecular structure of retroviral integrase, the enzyme that mediates insertion of viral DNA into the DNA of its host cell. With our Fox Chase collaborator, Z. Bu, Ph.D., small angle X-ray scattering (SAXS) and molecular modeling have been used to obtain the first available information concerning the shapes and structures of integrase monomers and dimers in solution. This structure can now be used to identify points of contact between the protein subunits, as well as between the protein and its DNA substrates. Such information will not only add to our understanding of this complex reaction, but may also suggest new ways to interfere with this activity, which is critical for viral replication.
We also described a system in which we detected high-frequency epigenetic gene silencing of freshly integrated retroviral DNA. We believe that such silencing may reflect a cellular anti-viral response. We published the results from our proof-of-principle studies using silent “reporter cells” to establish the validity of this system for identification of cellular factors that govern this epigenetic silencing – a phenomenon that is important to our understanding of the antiviral response as well as, the roles of epigenetic factors in normal cell development and cancer. With the help of the Fox Chase Cancer Center Translational Facility, we have used this reporter cell population to develop a robust and sensitive siRNA-based high throughput screen to identify specific host factors that participate in the maintenance of epigenetic silencing. Our results to date provide compelling evidence for roles of specific host factors and indicate that they are involved in cooperative and reinforcing interactions that mediate epigenetic silencing.Top
Studies on The Structure and Function of Retroviral IntegraseMark Andrake, Matthew Miller & George Merkel
Development of a new and rapid, moderate throughput assay for joining activity and demonstration of its utility in analysis of the activities of ASV and HIV-1 integrase proteins
HIV integrase (IN), the viral protein that inserts viral DNA into the host genome, is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of IN, and to measure the potency of prospective inhibitory compounds, we developed a rapid, moderate throughput solution assay for IN-catalyzed joining of viral and target DNAs. This joining assay is based on the detection of a fluorescent tag, can be scaled up for the analysis of numerous samples, and is substantially more sensitive than the standard radioactive gel assays.
We validated this new assay and demonstrated its utility in comparison of the metal dependent activities of IN proteins from HIV-1
and a related retrovirus, Avian Sarcoma Virus (ASV). The results confirm that the ASV enzyme is considerably more active than that of HIV, but with both enzymes the initial rates of joining, and product yields, are higher in the presence of Mn++ than Mg++. Although the pH optima for these enzymes are similar with Mn++, they differ significantly in the presence of Mg++, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by our observation that a compound that can inhibit HIV-1 IN in the presence of either metal is only effective against ASV in the presence of Mn++. In collaboration with Drs. T Burke and X.Z. Zhao at NCI, Frederick, MD, we are pursuing studies that exploit the differences between the active sites of these two enzymes to understand the mechanistic details of retroviral integration, with the aim of designing better inhibitors of this key step in the virus life cycle.Top
Determination of the solution structure of full-length retroviral integrase monomers and dimers, using data from small angle X-ray scattering, crystallography, and biochemical studies analyses.
The retroviral IN, composed of three linked domains, is a dynamic protein and, indeed, structural flexibility is likely to be essential for performing the distinct steps required for integration. We and others have demonstrated that IN proteins bind together to form dimers and higher order multimers, and such interactions are required for the various required activities of this protein. Increased knowledge of the interactions and spatial organization of IN domains in functional IN-DNA complexes will contribute substantially to our understanding of its mechanism of action.
We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers
in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.Top
Incorporation of ancient viral sequences in the genomes of vertebrate speciesIn collaboration with V. Belyi (Simons Center for Systems Biology, Institute for Advanced Study) & A. Levine (Simons Center for Systems Biology, Institute for Advanced Study)
Integration of sequences related to non-retroviral viruses with single stranded RNA genomes
It has long been appreciated that retroviruses can contribute significantly to the genetic makeup of organisms. Genes related other viruses with single stranded RNA genomes were formerly considered to be most unlikely candidates for such contribution. However, recent studies have indicated that sequences of RNA viruses with no DNA stage in their replication can be incorporated into the genomes of insects and plants. To determine if similar integrations could be detected in vertebrate genomes, we compared sequences representing all known viruses that contain single stranded RNA genomes (other than retroviruses), with the genomes of 48 vertebrate species. We discovered that as long ago as 40 million years, almost half of these species acquired sequences related to the genes of certain of these RNA viruses, most likely facilitated by LINE element retrotransposase. Surprisingly, almost all of the nearly 80 integrations identified are related to only two viral families, the Ebola/ Marburgviruses, and Bornaviruses, which are deadly pathogens that cause lethal hemorrhagic fevers and neurological disease, respectively. The conservation and expression of some of these endogenous sequences, and a potential correlation between their presence and a species’ resistance to the diseases caused by the related viruses, suggest that they may afford an important selective advantage in these vertebrate populations. The related viruses could also benefit, as some resistant species may provide natural reservoirs for their persistence and transmission.Top
Integration of sequences related to viruses with single stranded DNA genomes
Vertebrate genomic assemblies were also analyzed for endogenous sequences related to all known viruses that contain single strand DNA genomes. Numerous high-confidence examples were found of sequences related to the Circoviridae and two genera in the family Parvoviridae, the Parvoviruses and Dependoviruses. The integrations were broadly distributed among 31 of the 49 vertebrate species tested. Our analyses indicate that the age of both virus families may exceed 40-50 million years. Shared features of the replication strategies of these viruses suggest mechanisms by which such integrations may have occurred. It is possible that some of the endogenous viral sequences could offer selective advantage to the virus or the host. We note that rep ORF-derived proteins of some members of these families kill tumor cells selectively (3, 11). The genomic "fossils" we have discovered provide a lower estimate of the actual age of these families and a unique glimpse into virus evolution. Numerous recent integrations suggest that such germline transfer has been continuing to present times.Top
Studies on Epigenetic Control of Retroviral Gene SilencingAndrey Poleshko, Yuval Peretz, Natalia Shalginskikh, Caroline Burlingame, Maria Shubina & Richard Katz
Epigenetic Silencing of Cellular and Viral Gene Expression
Epigenetic silencing mediates the heritable transcriptional shutoff of specific genes during development and cellular differentiation. Such processes are executed by the enzymatic placement, or removal, of specific modifications in chromatin. These include DNA methylation, and a variety of posttranslational histone modifications. These modifications, or "marks" are read by repressive complexes, and may also directly influence the accessibility of chromatin by the transcriptional machinery. . Integrated retroviral DNA is subject to epigenetic gene silencing in human cells, resulting in a latent viral state, or loss of vector transgene expression. It is clear that such silencing is maintained by the cellular epigenetic machinery, however, very little in known about how such cellular machinery initiates the silencing process on newly integrated retroviral DNA.
We previously identified an antiviral mechanism that affects the initiation of epigenetic silencing of foreign retroviral DNA in human cells, and is dependent on the large, multifunctional, and ubiquitous scaffolding protein, Daxx. We showed that Daxx binds to the incoming viral DNA-protein complex, and acts as an adapter to recruit epigenetic factors. Our subsequent investigations have uncovered several early and late events in the silencing process. First, we found that siRNA-based knockdown of Daxx prior to infection transiently abrogated silencing. Second, we showed that DNA methylation of viral DNA is detectable within several days post infection, suggesting that this modification is important for initiating silencing. Third, we obtained evidence that Daxx can recruit the repressive DNA methyltransferases (DNMTs), as siRNA-mediated knockdown of Daxx resulted in loss of viral DNA methylation and reactivation of the silent viral DNA. Lastly, we found that Daxx can physically interact with two DNA methylatransferases, DNMT1 and DNMT3A. These findings have important implications for how human cells can respond to repress foreign DNA expression. Silencing of viral genomes by epigenetic mechanisms can contribute to pathogenesis by promoting a latent viral state, as in the case of HIV/AIDS. Because the epigenetic marks that mediate gene silencing are reversible, there is intense interest in devising therapeutic strategies to reactivate silent genes, and to reverse viral latency in a controlled manner.
Current ongoing projects:Top
1. SAXS analysis of IN protein monomers and dimers in solution (Bojja, Henderson, Andrake)
2. Preparation and analysis of crystals of IN proteins and IN-substrate complexes (Bojja, Andrake)
3. Genomic studies ingrated sequences dertived from non-retroviral RNA and DNA viruses (Skalka, in collaboration with Belyi and Levine, Institute for Advanced Study)
4. Functional identification of epigenetic silencing factors in diverse cellular backgrounds using siRNA screening, including epigenetic and genome-wide siRNA libraries (Poleshko, Shalginskikh, Burlingame, Katz)
5. Role of specific epigenetic regulators in tumor suppressor gene silencing (Poleshko, Peretz, Katz)
6. Role of epigenetic processes in self-renewal and differentiation of cancer stem cells (Peretz, Katz)
7. Role of an antiviral response in initiation of retroviral silencing; analysis of potential cell-specific functional repertoires of epigenetic silencing factors; interplay of DNA methylation and histone modification in silencing (Shalginskikh, Schubina, Katz)
8. Detection and analysis of novel roles for factors that mediate epigenetic silencing (Dandekar, , Katz)
9. Analysis of early events in retroviral infection and the roles of host factors (Schubina, Andrake, Katz, Skalka)