Bin Z He, Ph.D. - US grants

The viral response to cellular antiviral defenses is a key step that determines the outcome of viral infection and pathogenesis. Although this has been extensively studied in other viruses, less is known about how herpes simplex viruses (HSV) modulate cellular antiviral defenses. It has been established that virus infection activates the double-stranded RNA dependent protein kinase (PKR), which phosphorylates the alpha subunit of translation initiation factor 2 (eIF-2alpha), leading to the shutoff of protein synthesis and the inhibition of viral replication. In HSV infected cells the gamma134.5 protein is required to block the shutoff of protein synthesis and this function maps to the carboxyl terminal domain. It is believed that the gamma134.5 protein and protein phosphatase 1 (PP1), in association with additional components, form a functional multi-protein complex that dephosphorylates eIF-2alpha and prevents shutoff of protein synthesis. Thus, HSV appears to use a novel strategy to escape host antiviral response that is different from those used by other viruses. The long-term objective of this research is to understand the molecular mechanism of HSV pathogenesis. Current efforts are focused on the role of the gamma134.5 protein. The specific goals are: (1) To define the minimal functional module of the gamma134.5 protein by deletion analysis. (2) To characterize the PP 1 binding domain and define the role of the putative effector domain of the gamma134.5 protein by deletions or site-specific mutations. (3) To examine the molecular nature of interactions between eIF-2alpha, PP1 and the gamma134.5 protein by analysis of the gamma134.5-PP1 complex in vitro and in virus infected cells. These studies should provide insights into HSV infection that could result in the development of novel therapeutic agents. Since the carboxyl terminus of the gamma134.5 protein can be functionally substituted by the corresponding homologous domain of the cellular protein GADD34/MyD116, these studies may also shed light on cell growth control.

0218736
He
The past decade, dubbed the "Decade of the Brain", has witnessed explosive growth in the ability to observe and measure brain activity in human subjects. The technique of functional magnetic resonance imaging (fMRI) has enabled precise anatomical localization of brain structures that are activated during certain psychological processes. Though fMRI has high spatial resolution, temporal resolution is on the order of a second. In contrast, electrophysiological (EEG type) recordings, which can be obtained from the scalp and measure the electrical activity of ensembles of neurons activated synchronously (event-related potentials (ERP) provide millisecond level temporal resolution but limited spatial resolution. Tremendous efforts have been made to localize electrical source generators within the brain that underlie ERP components evoked by specific sensory, motor, or cognitive events. The approach, called electrophysiological neuroimaing, attempts to image the neural source distribution within the brain from electophysiological recordings obtained over the scalp by deconvolving the volume conduction process.

The focus of this proposal is to achieve high spatial and temporal resolution in brain imaging by mathematically combining MRI and EEG data. The method involves placing constraints derived from one imaging modality on possible inverse solutions of the other modality. Once the algorithms are developed, the method will be applied to obtaining finer localization of brain structures involved in detecting and responding to an error in a cognitive task. The multimodal brain imaging technique will be assessed for applicability and performance in studying executive control systems in healthy subjects and disturbance of executive functions in selected groups of patients with schizophrenia.

0201939
He
Locating the source of cardiac arrhythmia currently requires an invasive and complex procedure. Noninvasive localization of sites of origin of arrhythmia would be of enormous value for numerous patients and would allow cardiologists to focus the intervention at the source of the arrhythmia without the need for lengthy intra-cardiac mapping. Currently, invasive mapping procedures in conjunction with programmed stimulation techniques have been steadily increasing in use in combination with newer therapeutic techniques including implantation of automatic intra-cardiac defibrillators and selective radio-frequency ablation of myocardial tissue. The availability of a noninvasive means of localizing sites of origin of activation in three-dimensional (3D) myocardium will greatly increase the ability to help monitor and guide invasive therapeutic and diagnostic techniques such as programmed stimulation and selective catheter-tip ablation.

The goal of this project is to develop and evaluate a novel electrocardiographic localization methodology for accurate and rapid localization of sites of origin of cardiac activation from noninvasive electrocardiogram (ECG) measurements. To accomplish this goal the following aims will be addressed: (1) To develop and evaluate a novel heart-model-based approach to rapidly and accurately localize sites of origin of cardiac activation from noninvasive body surface ECG measurements, with the aid of an anisotropic realistically shaped heart model. (2) To systematically evaluate the capability of the proposed 3D electrocardiographic localization approach in a series of well-controlled computer simulations. (3) To validate the proposed 3D electrocardiographic localization approach in a clinical setting. The working hypothesis for these aims is that, by incorporating a priori information on physiological processes, one will be able to achieve greater stability and accuracy when solving the inverse problem, allowing accurately localizing sites of origin of cardiac activation.

DESCRIPTION (provided by applicant): Ebola virus is a highly dangerous pathogen causing hemorrhagic fever in humans. Currently, there is no vaccine or effective treatment available for Ebola virus infection. While multiple factors contribute to the pathogenesis of Ebola virus infection, the ability of Ebola virus VP35 to abate the host interferon response is critical in determining the outcome of viral infection. VP35 is an essential component required for the Ebola virus RNA replication and nucleocapsid assembly. In addition, VP35 is implicated as an interferon antagonist. When expressed in mammalian cells, VP35 blocked interferon-beta expression and induction of interferon-stimulated response genes triggered by RNA or virus infection. These observations are consistent with previous findings that in cells infected with Ebola virus, production of interferon or expression of interferon-inducible genes is suppressed, for example, MHC class I, double-stranded RNA-dependent protein kinase, and interferon regulatory factor-1. Recent studies showed that mice lacking an interferon alpha/beta response resemble primates in their susceptibility to rapidly progressive, overwhelming Ebola virus infection. These studies suggest that VP35 is a viral factor that interferes with one or more components of the interferon pathways, therefore inhibiting host interferon response that results in rapid spread and dissemination of viruses. In this proposal, the biological functions of VP35 in interferon response will be investigated using mammalian cell lines and a surrogate infection system. Deletions or site-specific mutations will be used to define the functional modules of VP35. Furthermore, biochemical and genetic approaches will be employed to examine the nature of interactions between VP35 and the interferon pathways. Because of the potential of VP35 as a vaccine and antiviral target, the proposed studies should not only provide insight into the pathogenesis of Ebola virus infection but could also lead to the development of antiviral therapeutics.

DESCRIPTION (provided by applicant): The gamma 134.5 protein, present both in herpes simplex virus 1 and 2 (HSV), plays an essential role in viral virulence. Unlike wild-type virus, HSV mutants that lack the gamma 134.5 gene are incapable of replicating and causing diseases. However, the underlying mechanisms are not incompletely understood. The major goal of the proposed research is to investigate the biological functions of gamma 134.5 during HSV infection. HSV gamma 134.5 contains an amino-terminal domain, a linker region of triplet repeats, and a carboxyl- terminal domain. A critical function of gamma 134.5 is to block the interferon response mediated by double-stranded RNA dependent protein kinase PKR. In addition, gamma 134.5 is involved in virus egress. This activity maps to the amino-terminus of gamma 134.5 that shuttles between the nucleus, nucleolus and cytoplasm. It is thought that coordinated actions of functional elements in the gamma 134.5 protein and cellular factors facilitate virus egress that contributes to viral virulence. To test this hypothesis, mutational analysis will be performed to define functional modules of gamma 134.5. Recombinant viruses will be constructed to study viral replication, spread, and the interferon response in infected cells. Experiments will also be performed to identify targets of gamma 134.5. Furthermore, studies will be carried out to explore the molecular nature by which gamma 134.5 promotes viral infection. Taken together, these studies will provide insights into the mechanisms of HSV pathogenesis. PUBLIC HEALTH RELEVANCE: In summary, herpes simplex viruses are human pathogens that cause diseases such as genital herpes, keratitis, and encephalitis. This research is designed to investigate how a pathogenic factor of herpes simplex viruses facilitates viral infection, which may lead to the development of novel vaccines and antiviral therapeutics.

DESCRIPTION (provided by applicant): Herpes simplex viruses (HSV) establish persistent infections where the viruses modulate host immune systems in a complex way. Although many factors are involved, emerging evidence indicates that the interaction of HSV and dendritic cells is a key step which dictates the outcome of viral infection and pathogenesis. As sentinels, dendritic cells bridges innate and adaptive immunity. It is well documented that upon infection immature dendritic cells take up viral antigens, undergo maturation. In this process, co-stimulatory molecules such as MHC class II, CD40, CD80, and CD86 are up-regulated. Additionally, mature dendritic cells release inflammatory cytokines such as IL-6, IL-12, TNF-?, and type I interferon. These cellular factors coordinate with dendritic cells to control viral infection. Nevertheless, HSV replication compromises dendritic cell functions. Currently, little is known about underlying mechanisms due to the complex nature of HSV life cycles. The objective of this research is to understand the viral mechanisms through which HSV ?134.5 modulate dendritic cell maturation and functions. It is believed that this viral factor perturbs one or more of cellular components of Toll-like related innate signaling pathways in dendritic cells and impairs subsequent T cell activation in HSV infection. As such, multi-faceted approaches will be taken to investigate the roles of viral and cellular elements. Systematic analysis will be carried out to define critical functional domains in the context of viral replication and the induction of co-stimulatory molecules and cytokines. Additionally, studies will be performed to examine components and the nature of HSV and dendritic cell interactions relevant to viral pathogenesis. Together, these studies will not only advance our understanding of the mechanisms of viral infection but also provide an insight into dendritic cell functions in viral infections. PUBLIC HEALTH RELEVANCE: Herpes simplex viruses are human pathogens that cause diseases such as genital herpes, keratitis, blindness, and encephalitis. Herpes simplex virus infection is also a risk factor in HIV transmission. This research is designed to investigate how herpes simplex viruses impair the functions of immune cells which restrict viral infections. The proposed research may facilitate the development of novel vaccines and antiviral therapeutics.

? DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) is a human pathogen which infects the epithelial cells of the mucosal tissues, leading to productive infection. Following primary replication the virus invades the sensory neurons to establish latency. Reactivation from latency occurs periodically, which is a lifelong source of virus responsible for recurrent infections. Therefore, a central issue in HSV biology is how HSV switches between lytic and latent cycles. Although transcriptional regulation of viral gene expression plays a crucial role little is known about other means of control, in particular innate immunity. Emerging evidence suggests that human is predisposed to HSV encephalitis when a mutation occurs in TANK-binding kinase 1(TBK1), which is a key component of Toll-like receptor dependent and independent pathways. This research will explore the hypothesis that TBK1-mediated innate immunity regulates HSV replication, spread, latency or reactivation. Upon activation TBK1 mediates the expression of type I IFN and inflammatory cytokines. Moreover, TBK1 promotes autophagy. In this process, TBK1 interacts with an array of host adaptor proteins, possibly in a signal specific manner. Thus, studies will be carried out to investigate the mechanisms of TBK1 regulation in response to HSV infection. The focus is the molecular nature of TBK1 that governs HSV infection in epithelial and neuronal cells. Furthermore, genetic studies will be performed to evaluate the impact of TBK1 on viral replication, penetrate, latency and reactivation. In parallel, analysis will be integrated to determine the IFN response and autophagy. Collectively, these studies will provide an insight into innate immune regulation of HSV persistency and pathogenesis.

? DESCRIPTION (provided by applicant): Herpes simplex virus 1 (HSV-1) infection of the eye is the leading cause of corneal blindness by an infectious agent in the developed countries. In addition, HSV-1 causes encephalitis and contributes to 35-50% of new cases genital herpes. HSV-1 usually infects the epithelium where it undergoes viral gene expression, DNA replication, and maturation. Following lytic infection the virus goes into latency in the trigeminal ganglia, which represents a lifelong source of virus for recurrent lesions. A licensed vaccine currently does not exist. Although a variety of strategies are undertaken, precise determinants of an effective HSV vaccine is unclear. This partly stems from the complex nature of HSV. There is now increased recognition that Toll-like receptor and cytosolic receptor pathways play a pivotal role in initiating antiviral immunity. Upon activation, these pathways regulate the expression of type I interferon, cytokines, and co-stimulatory molecules. Notably, TANK binding kinase 1 (TBK1), a key factor in innate immune pathways, dictates vaccine responses. Preliminary studies suggest that an HSV mutant lacking the Us3 gene activates dendritic cells which are sentinels of innate and adaptive immunity. We propose to develop engineered HSV vaccines with superior immunity against wild type virus. Accordingly, we will selectively modify the HSV genome to delete the Us3 gene. Furthermore, we will harness TBK1 to potentiate protection. With state of the art technology, we will investigate protective efficacy in relation to viral replication and latency. We will also explore the underlying mechanism(s) by which engineered HSV skews innate immunity in dendritic cells, which translates into protective immunity. The long-term goal of this research is the development of vaccines for prevention and therapy of HSV induced-diseases.

Herpes simplex virus (HSV) is the most common cause of infectious blindness and viral encephalitis in the Western countries. Primary or recurrent infection can lead to severe disease, yet no licensed vaccine is available. HSV typically initiates infection in the epithelial cells of mucosa and spreads to sensory neurons where the virus establishes latency. Reactivation from latency occurs intermittently, which is a lifelong source for recurrent lesions. Although viral replication in the mucosa or penetration into the nervous system inflicts damages or inflammation, the disease mechanism is less clear. As a large DNA virus, HSV evokes antiviral responses through the innate immune pathways that regulate TANK-binding kinase 1, a key factor required to activate cytokine expression and autophagy in mammalian cells. Remarkably, while the interferon- stimulated gene (STING) drives the cytokine response the tripartite motif protein 23 (TRIM23) serves to mediate autophagy. Despite such regulatory control, HSV is able to compromise host restrictions, which depends on an HSV virulence factor ?134.5. A central hypothesis of this proposal is that HSV differentially reprograms host immunity, where a dynamic interplay between viral and cellular factors may determine HSV spread, virulence and inflammation. Current effort is directed to decipher mechanisms of HSV pathogenesis. Several aspects of HSV infection will be investigated in a multi- faceted approach. Accordingly, recombinant HSV will be generated to determine the nature of HSV interactions with the innate immune factors in epithelial and neuronal cells. This will dissect elements pertinent to viral interference of the nucleic acid sensing complexes and autophagy machineries. Furthermore, genetic studies will explore viral features relevant to ocular replication, spread and neurovirulence. In parallel, gene expression analysis will assesses ocular and neuoinflammation. Collectively, these studies will provide an insight into genetic determinants of HSV virulence, which may inform design of novel antiviral therapeutics or vaccines.

Herpes simplex virus 1 (HSV-1) is a human pathogen which infects the epithelial cells of the mucosal tissues where the virus undergoes entry, replication, assembly and egress, leading to productive infection. Following primary replication, the virus invades the sensory neurons to establish latency. Reactivation from latency occurs periodically, which is a lifelong source of virus responsible for recurrent infections. As a large DNA virus, HSV-1 assembles progeny capsids in the nucleus of infected cells. To exit, HSV-1 traverses the nucleoplasm, crosses the lamina of a dense meshwork of lamin filaments, and buds through the nuclear membranes in a two-step process known as ?envelopment and de-envelopment?. Therefore, the virus drives vesiculation of the inner nuclear membrane and forms enveloped virions in the perinuclear space. Primary virions then fuse with the outer nuclear membrane to release the naked capsids for further maturation. In this process the nuclear egress complex, composed of viral proteins UL31 and UL34, plays an essential role. While UL31/UL34 orchestrates a series of events, the regulatory circuit is unclear in virus-infected cells. Accumulating evidence suggests the hypothesis that additional virus and host factors may work coordinately, which is particularly relevant to temporal or cell-type specific regulation of viral replication. This research will investigate the mechanism of HSV nuclear egress, with a focus on a virulence factor ?134.5. Firstly, genetic analysis will be performed to identify viral determinants that direct host p32/gC1qR and protein kinase C to the sites of nuclear budding. Furthermore, studies will be carried out to examine the nature of host factor recruitment. Secondly, systematic analysis will be integrated to investigate the interplay of ?134.5 and the nuclear egress complex. The objective is to define the pathways that control local dissolution of nuclear lamina and capsid transit in the nucleoplasm upon virus infection. Together, these studies will provide insights into the remodeling of nuclear envelope pertinent to herpesvirus replication and maturation.

Alzheimer's disease (AD) is a neurodegenerative disorder with progressive decline in cognitive functions leading to memory loss and dementia. It affects millions of Americans and causes significant morbidity and mortality. AD is characterized by the accumulation of amyloid-?-containing neuritic plaques and intracellular tau protein tangles in the brain. Growing evidence pinpoints a link between herpes simplex virus 1 (HSV-1) infection and AD. Notably, HSV-1 DNA is detectable in AD amyloid plaques in human brains, and antiviral acyclovir is reported to block the accumulation of the AD- associated proteins beta-amyloid. Multiscale transcriptome analysis of independent Alzheimer's cohorts in the USA suggests that AD pathology traits are closely coupled with neurovirulence factor ?134.5 encoded by HSV-1. However, the way through which HSV-1 is functionally involved remains largely unknown. We recently found that ?134.5 recruits and activates protein kinase C?, a host serine/threonine kinase that upregulates ?-secretase and facilitates AD pathology. As viral ?134.5 also targets Beclin1 in the autophagy pathway, we hypothesize that viral activities mediated by HSV-1 may alter homeostasis of amyloid precursor protein and its metabolites through ?134.5 and facilitates the development of AD. As such, we will study viral regulation of amyloid-? generation a 3D human neural cell culture model of Alzheimer's disease. Recombinant HSV will be constructed to interrogate the expression of ?-secretase. Furthermore, we will investigate amyloid-? clearance. Genetic studies will be carried out to assess viral interference of autophagy machineries. The proposed research will systematically explore pathological features of AD linked to HSV-1 infection. If successful, it will inform design of new therapeutic approach for AD.