This competitive renewal application continues to focus on the early stages of retroviral replication. The overarching goal of this application is to uncover critical cell-virus interactions that determine the success or failure of retroviral gene expression in human cells, as modulated by epigenetic silencing. It was established decades ago that the prototype avian sarcoma virus (ASV) DNA provirus is subject to epigenetic silencing in mammalian cells, and this system will continue to be studied as a model. The applicants have discovered that epigenetic silencing of ASV can be mediated by the rapid engagement of an antiviral host adaptor protein, Daxx, which recruits the cellular epigenetic silencing machinery. Furthermore, through development and implementation of an innovative, robust, and comprehensive siRNA-based screen, a number of novel and expected human host cell factors that play a role in this response were identified. These findings have opened up the exciting new areas of investigation to be pursued in this competing renewal.
In Aim 1, the molecular mechanisms that govern Daxx function as an antiviral factor will be determined. Results of these studies will provide valuable insight into general intrinsic host defense mechanisms.
In Aim 2 mechanistic details for the function of several novel factors identified by siRNA screening will be studied, with a focus on those likely to engage viral DNA sequences. The hypotheses that specific viral DNA binding factors either participate in nucleation of silencing factors or act as a transcriptional barrier, will be tested.
Aim 3 will exploit new comprehensive and systematic approaches to distinguish the physical and functional epigenetic features of silent proviruses that are established soon after infection (initiation) and after long-term passage (maintenance). Specific hypotheses concerning the roles of antiviral factors, and the interplay of proviral and cellular chromatin, in silencing will be considered. Epigenetic mechanisms have an essential role in the regulation of gene expression during development, in differentiated adult tissues, and in human disease, including cancer. Consequently, results from the proposed research will be highly relevant to virology, as well as the cellular biology of normal and cancer cells.
Cells employ epigenetic silencing mechanisms to repress expression of retroviral genes, and the proposed research will increase our understanding of how such silencing is initiated and maintained in human cells. The expression of cellular genes is also regulated by epigenetic mechanisms during development, and failure or inappropriate silencing can lead to developmental errors and diseases such as cancer. These studies will help to uncover new strategies by which appropriate silencing may be restored, and disease prevented or treated.
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|Poleshko, Andrey; Katz, Richard A (2014) Specifying peripheral heterochromatin during nuclear lamina reassembly. Nucleus 5:32-9|
|Poleshko, Andrey; Kossenkov, Andrew V; Shalginskikh, Natalia et al. (2014) Human factors and pathways essential for mediating epigenetic gene silencing. Epigenetics 9:1280-9|
|Poleshko, Andrey; Mansfield, Katelyn M; Burlingame, Caroline C et al. (2013) The human protein PRR14 tethers heterochromatin to the nuclear lamina during interphase and mitotic exit. Cell Rep 5:292-301|
|Shalginskikh, Natalia; Poleshko, Andrey; Skalka, Anna Marie et al. (2013) Retroviral DNA methylation and epigenetic repression are mediated by the antiviral host protein Daxx. J Virol 87:2137-50|
|Bojja, Ravi S; Andrake, Mark D; Weigand, Steven et al. (2011) Architecture of a full-length retroviral integrase monomer and dimer, revealed by small angle X-ray scattering and chemical cross-linking. J Biol Chem 286:17047-59|
|Katz, Richard A; Merkel, George; Andrake, Mark D et al. (2011) Retroviral integrases promote fraying of viral DNA ends. J Biol Chem 286:25710-8|
|Peletskaya, Elena; Andrake, Mark; Gustchina, Alla et al. (2011) Localization of ASV integrase-DNA contacts by site-directed crosslinking and their structural analysis. PLoS One 6:e27751|
|Belyi, Vladimir A; Levine, Arnold J; Skalka, Anna Marie (2010) Sequences from ancestral single-stranded DNA viruses in vertebrate genomes: the parvoviridae and circoviridae are more than 40 to 50 million years old. J Virol 84:12458-62|
|Poleshko, Andrey; Einarson, Margret B; Shalginskikh, Natalia et al. (2010) Identification of a functional network of human epigenetic silencing factors. J Biol Chem 285:422-33|
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