Retroviral DNA synthesis and integration

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 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

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

Andrake, M.D., Merkel, G., Ramcharan, J., Skalka, A.M.

The retroviral DNA integration reaction

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.

Principles of a solution
assay to measure
integrase joining activity
by fluorescence

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, 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 a full-length retroviral integrase, using data from small angle X-ray scattering, crystallography, and biochemical studies

Andrake MD, Bojja R, Merkel G, Henderson A, Kummerling M, Yarychkivska O, Skalka AM.

Model of the structure of
a retroviral integrase

We have shown previously that the retroviral integrases, composed of three linked domains, are dynamic proteins and, indeed, structural flexibility is likely to be essential for IN to perform the distinct steps required for integration. Moreover, we have demonstrated that IN proteins bind together to form multimers when performing the various steps of integration. 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 are using light scattering experiments to characterize the multimers that form in solution for wildtype ASV IN, and C-terminal domain (CTD) substitutions that render this protein deficient in dimerization. Past studies have shown a prominent role for the CTD in multimerization. The ability of these multimerization deficient derivatives to bind and process the viral DNA substrates and to catalyze joining is being investigated.

In collaboration with our FCCC colleague, Dr. Zimei Bu, we have used small angle X-ray scattering (SAXS) to determine the size and shape of both monomeric and dimeric native ASV IN proteins. The SAXS-determined volume and length of the monomeric IN derivative are consistent with the light scattering data, and the deduced shape establishes constraints for the relative arrangement of the three domains. These experimentally defined, low resolution structures were then combined with atomic resolution data of individual IN domains, and flexible fitting methods were employed to develop a structural model for full length ASV IN in solution. The SAXS determined length and volume of the wildtype and another dimeric ASV IN derivative also correspond to the light scattering data for a dimer in solution, and provide some initial insights into IN multimerization. Predictions of these models are now being tested using a variety of genetic, biophysical and biochemical methods. Top

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. It is hypothesized that errors in placement, removal, or reading of epigenetic marks can cause human disease (e.g., cancer) through inappropriate silencing of specific genes, including tumor suppressor genes. In addition, silencing of viral genomes by epigenetic mechanisms can contribute to pathogenesis by promoting a latent viral state, as in the case of HIV/AIDS. Furthermore, epigenetic silencing may present a barrier to gene therapy in some settings. 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. Certain drugs, such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi), relieve epigenetic silencing by inhibiting enzymes that modify chromatin. However, current epigenetic therapies are limited to these two targets. Our current studies are based on the hypothesis that siRNA-based knockdown of key silencing regulators will phenocopy the effects of such drugs and provide an avenue for analysis and discovery of new epigenetic therapies.

In the context of our studies on epigenetic silencing of integrated retroviral DNA, we began by asking which cellular factors played a functional role in this process. We devised an siRNA functional screen to identify such cellular factors, beginning with human (HeLa) host cells. The system utilizes retroviral DNA harboring a silent GFP reporter gene to serve as beacon to monitor the effects of siRNA knockdown of individual epigenetic regulators. We speculated that cellular factors may engage and initiate the formation repressive chromatin on viral DNA, and have obtained evidence that a host cell antiviral response mediates initiation of epigenetic silencing in this system. However, our recent findings have indicated that once silencing is initiated, this system provides a valid model for uncovering general, cellular mechanisms for the maintenance of epigenetic silencing. Because the retroviral reporter DNA can be deposited in a variety of cell types, it can serve as an adaptable platform to probe the functional roles of epigenetic factors in different types of normal or diseased cells.

Our first study using this system has been completed. HeLa cell populations harboring widely distributed, epigenetically silenced, retrovirally encoded GFP genes were interrogated with an siRNA library targeting 200 candidate epigenetic regulatory factors, and the reactivation of silent GFP was measured. From these candidates, 15 validated hits were identified, which represent a unique network of factors that are required for silencing in our experimental system.

These initial studies have uncovered a specific set of essential epigenetic silencing factors and evidence for functional crosstalk among proteins with diverse roles. The results provide an important, first view of an epigenetic network in human cells. They also offer insights as to how factors may cooperate to maintain gene silencing during normal development, or in disease, and have implications for identifying silencing factors that may serve as targets for epigenetic therapies. Efforts are currently underway to broaden our screen to uncover additional regulatory factors and new epigenetic marks in HeLa and other human cell types, and to investigate the functions of these factors and signals.

Current Projects:

1. Functional identification of epigenetic silencing factors in diverse cellular backgrounds using siRNA screening, including epigenetic and genome-wide siRNA libraries (Poleshko, Shalginskikh, Katz)
2. Role of specific epigenetic regulators in tumor suppressor gene silencing (Poleshko, Peretz, Katz)
3. 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, Katz)
4. Detection and analysis of novel roles for factors that mediate epigenetic silencing (Dandekar, Savage, Lipsman, Manion, Katz)
5. Role of integration sites in retroviral silencing (Dandekar, Katz)