Establishment of adeno-associated virus latency by the recruitment of KAP1

Viruses have evolved mechanisms that allow them to control and take advantage of the host cells, which they infect. We are studying a parvovirus, named adeno-associated virus (AAV) that adds an additional layer of complexity by exploiting and controlling a helper virus, which it needs to be able to replicate in cells. We have discovered that in the absence of helper viruses AAV associates with a cellular protein to silence its genome and that the virus can undo this by mediating a modification of the cellular protein that abolishes its silencing activity. Here we propose experiments that will allow us to determine the mechanism by which the cellular protein, KAP1, silences the viral genome. We will also investigate if helper viruses play a role in releasing KAP1 from the AAV genome and thereby allow production of AAV proteins. These products in turn will further modify KAP1 in order to avoid that AAV is silenced again. We will perform experiments to discern how AAV introduces changes to KAP1, which lead to its inactivation. The studies we propose will lead to a better understanding of the AAV life cycle and will provide a framework to study pathogenic parvoviruses such as B19, which can cause severe disease in patients with inherited blood disorders such as sickle cell anemia. Improved knowledge of silencing and reactivation of parvoviruses and by extension possibly other DNA viruses may lead to the development of therapies that control infections. In addition, our findings may also inform how production of gene therapy vectors derived from AAV can be improved. Recent clinical trials have demonstrated safety and efficacy of AAV-mediated gene therapy, however more efficient vector production schemes will be required to be able to offer gene therapy to large patient populations.

Adeno-associated virus (AAV) is a human dependovirus that appears to have evolved an optimal working relationship with host and helper virus. With the support of viruses such as adenovirus and herpes virus, AAV replicates successfully, causing a cytopathic effect only in those cells which were already destined to suffer from helper virus infection; in the absence of co-infection the virus remains latent without causing any apparent harm. It was generally thought that latency is established through integration into the human genome, however our preliminary data indicate that latency of AAV might already be established prior to integration through interactions with the transcriptional co-repressor KAP1, which render the virus 'invisible' until conditions for replication are met. In addition, we have found that relief of repression is achieved in part through actions of the AAV Rep proteins, which lead to phosphorylation of KAP1. We propose experiments to further investigate the mechanism of KAP1-mediated repression of AAV, to determine a role for helper factors in the release of KAP1-mediated repression of the AAV genome and unravel the mechanism of Rep-mediated phosphorylation of KAP1. Knowledge gained from this proposal does not only establish a new concept in AAV biology but will also inform virus-host interactions of other DNA viruses, including pathogenic viruses. In addition, AAV has become a frontrunner as a vector for human gene therapy based on results from a number of clinical trials. However, the research community around AAV biology has remained very small and thus limited molecular insights into its life cycle is available. As an example of how an understanding of the biology of AAV can further the application of AAV vectors is demonstrated by our finding that KAP1 knockdown leads to increased recombinant AAV vector replication, an observation that could be exploited to help overcome the challenges of production for wide clinical use.

This proposal focuses on the study of the interactions between the viral Rep protein and KAP1, a cellular protein that functions as a transcriptional co-repressor by recruiting histone modifying enzymes and heterochromatin-inducing factors. We have discovered that KAP1 associates with the AAV genome and helps establish viral latency. We propose to investigate how latency is established and how the virus has evolved mechanisms to escape from KAP1-mediated repression. Our work introduces a paradigm shift in the view of the life cycle of AAV and potentially other - pathogenic- parvoviruses such as B19. The molecular mechanisms by which B19 produces latency and reactivation are currently unknown. Given the strong similarities between the AAV Rep and the B19 NS1 protein, it is highly likely that KAP1 also plays a role in the life cycle of this pathogenic virus. We could envisage that by manipulating these interactions, treatments that curtail B19 replication in patients with inherited hemolytic anemia could be developed.
The proposed research also opens up possibilities for biotechnological developments that lead to the improvement of AAV gene therapy vector production schemes. We are currently investigating if manipulation of this pathway, potentially in combination with others, leads to a significant increase in recombinant AAV production. We already have established a collaboration with a major pharmaceutical company, and inclusion of a biotechnological translation of this project could be further explored with them.
We are communicating our research results through public access journals, teaching of pre-university and undergraduate students, lay audiences and through interactions with industrial partners.
The beneficiaries of this research potentially include: 1) pharmaceutical industry/biotech companies who have an interest in applying our research for the development of new therapies, 2) patients with diseases that could be treated with gene therapy/ patients with inherited hemolytic anemia such as sickle cell disease, 3) pre-university and university students who have an interest in pursuing a career in science, 4) the postdoctoral associate/Ph.D. student who will receive further training and profit from interactions with industrial partners.