Control of Herpes Simplex Virus latency by cellular DNA repair proteins

Funding Type: 
SEED Grant
Grant Number: 
RS1-00449
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Herpes Simplex Virus types1 and 2 are human viruses which cause cold sores and genital herpes, respectively. Current estimates suggest that 80% of the US population are infected with HSV-1 and 20% with HSV-2. Both viruses establish latency in neurons of the infected individual, and periodically reactivate in response to stress or sunlight. During latency, the virus is asymptomatic but during reactivation, sores reappear at the site of the initial infection. Current therapies do not prevent reactivation , they only limit the spread of virus once it has already reactivated. We have recently discovered a link between cellular DNA repair proteins and HSV replication, and we have found that this the link is different between lytic and latent infection. During lytic infection, the cellular DNA repair proteins are activated by the virus, whereas during latent infection they are not. Our hypothesis is that DNA repair proteins control the switch between lytic and latent infection and this hypothesis forms the basis of this proposal. If correct, this finding could be exploited therapeutically to force the virus into a permanent state of latency. The natural site of HSV latency is neurons and we have found that the interactions we have observed between DNA repair proteins and HSV are specific to human cells. This finding limits the usefulness of rodent cells for our experiments but we feel that neuronally differentiated human embryonic stem (hES) cells may provide us with a unique opportunity to test our hypothesis in a biologically relevant setting. We propose to establish a model of HSV latency in hES cells which have been differentiated to a neuronal lineage. Modeling latency of a human virus in human neuronal cells will in itself be a significant advance in a field which has been traditionally limited to rodent and rabbit models, which do not recapitulate all aspects of the human disease. We will then use this model to investigate whether inhibiting certain DNA repair proteins can prevent HSV reactivation. Establishing a link between viral latency and DNA repair proteins may have implications beyond the field of HSV research; this example may turn out to be a paradigm for viral latency in general. If correct, our hypothesis will not only greatly expand our understanding of viral latency but may have important therapeutic applications, potentially leading to novel therapies designed to prevent rather than treat HSV reactivation.
Statement of Benefit to California: 
The proposed research will benefit California in two ways. Firstly, it will contribute to our knowledge base of infectious diseases and give new insights into the complex interactions between virus and host cell. Secondly, it may lead to novel therapies to target HSV infections. This will be particularly relevant since the CDC reports that STDs are the most common communicable disease in California and HSV-2 positive individuals have recently been shown to be more likely to acquire and transmit HIV. This proposal represents a novel application of human stem cell technology and could provide a paradigm for studying a range of viruses in the most relevant human cell type.
Progress Report: 
  • Human embryonic stem cells (hEScs) are derived from embryos early in development before their fate has been determined, and can differentiate into all of the cell types of the body. By exploiting the potential of hESCs to differentiate into multiple lineages medicine stands to benefit enormously. To do so requires a comprehensive understanding of the optimal conditions to grow and differentiate these cells without inducing tumors. What is clear is that the physical, three dimensional (3D) microenvironment in which the hESCs reside, regulates directly or indirectly their tissue-specific differentiation. hESc make physical contact with both an insoluble extracellular scaffold termed the extracellular matrix (ECM) and to other cells through specific proteins (receptors) on their own surface. These interactions are responsible for the structural integrity of the body and are at the center of structural transformations that characterize embryogenesis. While this structural role has long been appreciated it has only recently been shown that these points of contact can be the focus of transmission of mechanical forces originating within and outside the cell and that these forces can be converted into the more familiar signals that influence cell fate decisions. A major goal of our work is to define how these cell derived and externally transmitted forces might regulate hESC behavior. Our hypothesis is that these mechanical forces alter hESC fate by regulating the activity of enzymes called RhoGTPases, that are strongly implicated in ESC behavior. During this past year we have optimized the preparation and culture of hESc on a two dimensional (2D) synthetic matrices of defined composition and stiffness that recapitulate the range of mechanical environments hESC experience during embryogenesis as well as those unnaturally stiff mechanical environments that are currently used for their propagation. For these studies we used inert acrylamide gels cross linked with an ECM molecule (laminin) important in early embryonic development. On 2D surfaces feedback loops originating from cell derived contractile forces sense the mechanical “give” of the surface, and dynamically change their organizational and signaling state both at the single cell level and the multicellular level. Using these 2D gels we have defined the range of mechanical environments in which hESc exhibit mechanosensitivity. We have also shown that this causes changes in their external and internal organizational states across a range of scales from the subcellular (becoming stiffer on stiffer substrates), to the cellular (spreading more on stiffer substrates) to the multicellular (compacting under enhanced cell-cell adhesion on softer substrates). Surprisingly, we have found that the standard (very stiff) substrates that are used routinely for maintenance of a non-differentiated state (pluripotent self renewal) are mechanically suboptimal: hESc have higher rates of cell death and lower rates of growth than on surfaces orders of magnitude softer (operationally termed mid range). At the softest end, although we have found that hESc largely maintain pluripotential self renewal they show signs of either low level differentiation or a move towards a state poised to differentiate. This suggests that precise control of the mechanical environment is an important parameter in the establishment of safe and effective propagation of hESc for regenerative medicine and might be exploited for directed differentiation. To complement these studies we are also developing approaches to imparting external mechanical forces to hESc growing in a 3D context. We have both established novel and robust protocols for the efficient encapsulation of hESc in 3D deformable hyaluronic acid (HA) hydrogels and shown that they support pluripotent self renewal and constructed a bioreactor that will impart oscillatory and static compressive loads to hESCs in these gels. We anticipate that these studies will further illustrate the role(s) by which mechanical forces influence hESC fate and provide additional insight into the underlying molecular mechanisms.

© 2013 California Institute for Regenerative Medicine