Research Strategy

1. Research Strategy

2. Research Strategy • Organize the Research Strategy in the specified order. • Start each section with the appropriate section heading—Significance, Innovation, Approach.

3. Significance • Explain the importance of the problem or critical barrier to progress in the field that the proposed project addresses. • Explain how the proposed project will improve scientific knowledge or technical capability in one or more broad fields. • Describe how the concepts, methods, and technologies that drive this field will be changed if the proposed aims are achieved.

4. Significance • This is the first part of the Research Strategy and should be approximately ½ page to 1 page in length. • Do NOT review the literature but DO provide sufficient background so that the reader will understand the research to be performed AND why it is important or SIGNIFICANT.

5. Significance • DO include references for the background information that you provide. These can be numbered as you would for a journal article because your proposal will have a Reference list at the end. • Do spell out a term the first time you use it and include the standard abbreviation in parentheses. Use the abbreviation from that point.

6. Good example of Significance • Human papillomaviruses (HPV) cause a range of benign and malignant tumors. A subset infecting the anogenital region comprise the most prevalent sexually transmitted disease in the world today. Moreover, persistent infection with such viruses can cause cervical or anal cancer. A vaccine against the two most prevalent HPVs, HPV16 and 18, is currently being used in the US and UK. However, the vaccine is prophylactic and so of no use to women (approximately half a million worldwide) already infected. New therapeutic treatments are required to be developed to alleviate the burden of disease in these people. The virus capsid, composed of proteins L1 and L2, is absolutely required for virus transmission and spread. Expression of capsid proteins is restricted to cells in the uppermost epithelial layer. Thus this region may be vulnerable to topically applied therapies. The goal of these studies is to understand the regulation of virus capsid protein expression and is an essential first step in the development of novel antiviral therapies.

7. Significance • We will address an important and little trodden area of investigation in the mechanisms of herpes simplex virus type 1 (HSV-1) latency, namely the contribution of host factors and host pathways in the establishment, maintenance and reactivation of HSV-1 latency. To date, studies have concentrated on the viral products, especially the latency associated transcripts or LATS and their role in latency. Although there have been studies demonstrating the importance of cellular factors, such as the neurotrophin, NGF in maintaining latency, there has not been a systematic effort to focus on cellular mechanisms that control the reactivation of latent HSV-1 in sensory neurons. The current proposal represents such an effort wherein we will test the hypothesis that a combination of neurotrophin withdrawal and cytokine secretion triggers reactivation. These studies will directly test the possibility that the withdrawal of specific neurotrophins and the induction of specific cytokines results in reactivation and will further investigate which intracellular signaling pathways may be involved. Because this is a poorly explored area to date, the information forthcoming will undoubtedly move the field forward and provide an essential foundation on which to base further studies.

8. Significance • Host innate immunity represents the first line of defense against invading pathogens and shapes the course and outcome of pathogen infection. Highly conserved sensors that detect invading pathogens and downstream signaling pathways are critical for host innate immune responses. Upon infection, pattern recognition receptors (PRRs), such as toll-like receptors and cytosolic NOD-like or RIG-I-like receptors (NLR and RLR), sense pathogen-associated molecular patterns. Recent studies have shown that RIG-I and MDA-5 are major cytosolic sensors for the detection of infecting virus. Upon association with RNA, RIG-I and MDA-5 heterodimerize with the mitochondrion antiviral signaling (MAVS) adaptor, which activates two closely related kinase complexes, IKKα/β/γ and TBK-1/IKKε. Through phosphorylation, these kinases promote the activation of NFκB and interferon regulatory factors (IRFs), which are transcription factors that, in turn, up-regulate the expression of antiviral pro-inflammatory cytokines and interferons, thereby constituting potent antiviral innate immunity. Viruses have evolved diverse strategies to evade host antiviral cytokine production. A focus of my research is to understand how herpesviruses, which establish a lifelong persistent infection in immuno-competent hosts, contend with innate immune responses. Interestingly, studies using human immunodeficiency virus, KSHV, and γHV68 suggest that viruses may exploit innate immune signaling for their own infection. We recently reported that γHV68, a model murine herpesvirus closely related to human KSHV and EBV, activated IKKβ kinase and further, activated IKKβ phosphorylated the essential viral transcription factor, RTA, to promote viral transcriptional activation and lytic replication. Moreover, we also showed that MAVS and IKKβ are necessary for γHV68 to induce the degradation of RelA, a major subunit of the transcriptionally active NFκB dimer, thereby preventing NFκB activation and host cytokine production . Thus, the Specific Aims of this proposal address a novel mechanism whereby γHV68 infection activates the RIG-I-MAVS-IKKβ pathway to enable viral transcriptional activation and disable host cytokine production. This work will establish an unprecedented example of virus triggered innate immune signaling to promote viral infection and will re-shape our view of “antiviral innate immunity”

9. Significance • ICP0, an HSV-1 immediate-early protein is an E3 Ub ligase and a potent transcriptional activator of viral gene expression. ICP0 mutants grow inefficiently in cell culture and in vivo and are impaired in their ability to reactivate from latency, indicating that ICP0 plays a pivotal role in the reactivation from latency. During early infection, ICP0 localizes to and disrupts nuclear structures termed nuclear domain (ND) 10, a component of innate immunity. This disruption of ND10 by ICP0 correlates with decreased levels of ND10-associated antigens, such as promyelocytic leukemia (PML) and Sp100, and the degradation of distinct cellular proteins, such as catalytic subunit of DNA-dependent protein kinase. This occurs through the Ubiquitin-proteasome pathway. ICP0-directed proteolysis, via its E3 Ub ligase activity, appears to play a central role in its transactivating activity. However, depletion or inactivation of established targets of ICP0-mediated degradation, such as PML and Sp100, are not sufficient to fully complement the replication of an ICP0 null mutant virus. This observation supports the hypothesis that several cellular targets of ICP0-directed proteolysis are required for efficient viral replication. The identities of these cellular targets are largely unknown. It is plausible that ICP0 directs the degradation of several proteins because other E3 Ub ligases, including the Kaposi sarcoma associated herpesvirus K3 and K5 proteins and the cellular Parkin protein, target multiple proteins for proteolysis. In this proposal we plan to identify new cellular factors whose stability is regulated by ICP0. We propose that degradation of these factors is necessary for efficient HSV-1 replication. This approach will be significant because the genetic screen described in this proposal can be broadly applied to identify proteins and pathways that play important roles in HSV-mediated diseases as well as other infectious and human diseases. Our results can ultimately be used to develop new strategies for preventing or treating HSV-1 infection by modulating or increasing the stability of ICP0’s targets. Furthermore, we anticipate that our research will lead to a greater understanding of how virus-host interactions affect basic cellular processes such as transcription and protein degradation.

10. Significance Adeno associated virus (AAV) is a non pathogenic parvovirus that requires adenovirus or other viruses such as herpes simplex as a helper to replicate. AAV is highly prevalent throughout the human population and is found in a number of different organs. It can replicate to very high DNA copy number and because of the structure of its genome, it can be seen as damaged DNA by the host cell. The effects of the DNA damage response on adenovirus infection have been well characterized, but the effects on the DNA response on AAV infected cells have been much less characterized. Because of the advent of AAV gene therapy vectors, it is important to probe how the cellular DNA damage response affects AAV replication and persistence. These studies will provide important and medically relevant information.

11. Innovation • Explain how the proposal challenges and seeks to shift current research paradigms. • Describe any novel theoretical concepts, approaches or methodologies, instrumentation or intervention(s) to be developed or used, and any advantage over existing methodologies, instrumentation or intervention(s). • Explain any refinements, improvements, or new applications of theoretical concepts, approaches or methodologies, instrumentation or interventions.

12. Innovation • Host immune responses are generally viewed as antiviral. Through investigating the roles of innate immune pathways in γHV68 infection, we made a surprising discovery that γHV68 hijacks the MAVS-IKKβ pathway to promote viral lytic replication and to abolish host cytokine production. These findings challenge the current paradigm because they imply that host innate immune responses can be proviral. As such, γHV68 has evolved to activate the RIG-I-MAVS-IKKβ pathway that facilitates viral lytic replication. This proposal will directly test this hypothesis and will investigate the underlying molecular mechanisms, thereby significantly expanding our understanding of viral exploitation of host innate immune responses

13. Innovation • The finding of autophagy in Varicella-Zoster virus (VZV) infected cells and in zoster vesicles is novel. Further, the central hypothesis of this proposal namely, that VZV glycoprotein E triggers endoplasmic reticulum (ER) stress that leads to the unfolded protein response and that leads to autophagy, has implications for VZV pathogenesis, especially in painful zoster lesions. This avenue of research is highly innovative. The demonstration that what had been described in previous reports as the VZV cellular receptor, namely Insulin Degrading Enzyme (IDE), actually binds to the immature form of gE is a new finding that will direct these studies into a whole new direction from virus entry to studies on autophagy and the ER stress response.

14. Innovation • There are a number of aspects of the regulatory processes that govern chromatin remodeling and gene silencing that we propose to study in this proposal that are novel and innovative. The recruitment of polycomb repressor complexs (PRCs) to silence gene expression during HSV latency is unique among DNA viruses and this may provide the virus with a more easily reversible repression of its lytic genes than if it used constitutive heterochromatin marks to silence its genes. Our discovery that the latency associated LAT intron binds to the PRC2 complex and reduces the amount of H3K27triMe repressive mark on the latent genome highlights a novel regulatory mechanism for heterochromatin remodeling not only among viruses, but also potentially for cells as well. In addition, there are a number of aspects of our experimental approach that are innovative. These include extending our sensitive Taq-man real-time PCR analysis of ChIP assays for PCR2 to a high resolution PCR array that will provide coverage of the entire HSV-1 genome. This technique has greater sensitivity than tiling arrays or ChIP-seq and to our knowledge this is the first time this approach has been used to perform ChIP analyses on latently infected tissue samples.