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.
This proposal explores the potential use of hESCs as a model system to study herpes simplex virus (HSV) latency. The hypotheses are that a lack of activation of DNA repair proteins in neuronal cells limits HSV replication and forces HSV into latency and that activation of DNA repair proteins is necessary for HSV reactivation. The PI has data that show: HSV-1 can infect and establish a long-term quiescent infection in neurons derived from hESCs; a DNA damage response is present in infected non-differentiated hESCs, but not in neuronally differentiated hESCs; the DNA damage responses enhances HSV-1 replication in human cells. In Aim I, the PI will establish and validate a model of HSV latency that reactivates. In Aim II, the PI will characterize the interaction between HSV and cellular DNA damage response proteins during establishment, maintenance and reactivation from latency. In Aim III, the PI will test whether manipulation of DNA repair proteins can be used to control latency. SIGNIFICANCE AND INNOVATION: The mechanism(s) of virus latency has been a subject of great interest to the virology community for many years. One of the best studied models of virus latency involves HSV. Understanding virus latency and using hESCs as a model for virus latency may be important in our understanding of many persistent infections, including AIDS. In addition, the experiments planned in the present proposal may lead to therapeutic approaches for HSV infections, which are of public health concern. At present there are no optimal models for HSV latency and none involving human neuronal cells. The use of human cells may be very important since the PI has preliminary data demonstrating that mouse cells, but not human cells, cannot mount a DNA damage response to HSV-1 and are not compromised for viral replication. For these reasons, the potential use of hESCs as a model would be valuable to the scientific community. The idea that hESCs could be used as a model of HSV latency is a very novel one, as is the hypothesized role of DNA repair pathways in HSV-1 latency. From this perspective, the grant is a very creative one. STRENGTHS: The applicants have considerable expertise in studying viral latency, and have formulated a testable model proposing that the expression of DNA repair proteins plays a critical role in establishing latency and allowing reactivation of viral DNA replication. The experiments are complete and very well designed. WEAKNESSES: Reviewer one: This proposal suffers from two significant weaknesses. First, a latency model using neurons derived from human EC cells (hNT cells) already exists. In this model latency is established using a replication defective virus, and reactivation is achieved by superinfection with WT virus. The applicants do not address why the use of a replication defective virus is necessary if, according to their model, neuronal cells do not normally synthesize the DNA repair proteins that are necessary for viral replication. Moreover, they do not address why neurons differentiated from hES cells might be different from neurons differentiated from hNT cells, especially with regard to viral replication. Therefore, I am unconvinced that there is a strong case for switching to hES cells. Second, a major goal of this application is to test the model that induction of DNA repair proteins is necessary for activation of latent HSV replication. Its not apparent why the same experiments proposed for hES derived neurons could not be done in the already existing model using hNT derived neurons. A further technical problem, mentioned in the application, is that HSV infects the feeder cells used in ES culture. Reviewer two: This is a risky proposal. The entire proposal depends on successfully preparing HSV-1 latently infected neuronally differentiated hESCs in the first specific aim. If the PI is unable to establish latently infected neuronally differentiated hESCs, he will be unable to carry out any of the other goals of the proposal. Although preliminary data are not required for the Seed grant, this reviewer’s enthusiasm would have been greatly enhanced with some evidence that the “long-term, quiescent infection” observed in neuronally differentiated hESCs is latent. There are many theories regarding the mechanisms of HSV latency. Might these other theories be involved as well as DNA repair pathways in the mechanisms of HSV latency? Does the PI believe that DNA repair pathways are important in the initiation, maintenance and reactivation of latency? What is the relationship of LAT to DNA repair proteins – i.e., is the expression of LAT in latently infected cells linked to the DNA repair pathway?