Hengli Tang - US grants

animal tissue; virus; microscopy; AIDS; immunology; human tissue; sexually transmitted diseases; communicable diseases; lymphatic system; biomedical resource;

DESCRIPTION (provided by applicant): The broad, long-term goal of our program is to advance the knowledge of virus-host cell interactions by characterizing cellular cofactors essential for hepatitis C virus (HCV) infection. HCV infects 170 million people worldwide and is major cause of hepatocellular carcinoma. Understanding how this virus interacts with the host is of critical importance to the development of diagnostics, antiviral drugs, and a prophylactic vaccine. Our previous studies demonstrated that HCV infection of cultured hepatoma cells is critically dependent on a cellular protein, cyclophilin A (CyPA), and that ablating the function of this protein can not only prevent new infections but also suppress an existing infection. In addition, CyPA is a principal mediator of HCV resistance to cyclosporine A (CsA) and its derivatives, which inhibit HCV replication with an undefined mechanism and are currently being evaluated in clinical trials as candidate anti-HCV drugs. The experiments proposed here will investigate the molecular mechanisms that determine the essential cofactor function of CyPA, the action of CsA, and the related CsA resistance. The following experiments will be performed: (1) CsA and small interfering RNA directed at CyPA mRNA will be used to identify the specific function of HCV replicase that requires CyPA as a cofactor. A detailed understanding of why and where the virus needs this cellular chaperone to survive will offer new perspectives on the replication strategy of HCV. (2) Biochemical assay and mutagenesis will be used to characterize the critical interaction between CyPA and the viral replicase, which represents a novel structural interface of virus-host cell interaction that may be disrupted for therapeutic purposes. (3) Macromolecular interaction and reverse genetics will be employed to dissect the mode of action for CsA and molecular basis of CsA resistance with the ultimate goal of predicting and circumventing that resistance. The results of the proposed studies will not only provide significant insights into how highly successful parasites such as human viruses hijack host cell machineries to replicate efficiently but also reveal new therapeutic targets for antiviral intervention. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) infects 3% of the world's population and cause fibrosis, liver cirrhosis and hepatocellular carcinoma (HCC). No prophylactic vaccine exists, and both the current treatment and the drugs in the development pipeline face serious drug-resistance issues. The proposed research will illustrate the molecular basis of drug resistance for a new drug candidate and may also identify a novel aspect of virus-host cell interaction than can serve as target for a new class of drugs.

DESCRIPTION (provided by applicant): The broad, long-term goal of our program is to advance knowledge of virus-host cell interactions by characterizing cellular cofactors essential for hepatitis C virus (HCV) infection. HCV infects 170 million people worldwide and is major cause of hepatocellular carcinoma. Understanding how this virus interacts with the host cell (e.g., what makes a cell susceptible to HCV infection) is of critical importance to the development of diagnostics, antiviral drugs, and a prophylactic vaccine. Our previous studies of pluripotent stem cells and directed hepatic differentiation demonstrated a correlation between hepatic specification during the differentiation process and permissiveness to HCV infection. Characterization of the cellular transition from nonpermissive to permissive revealed candidate genes that are involved in regulating viral susceptibility. One of these cellular factors interacts with HCV glycoprotein E1 and plays an essential role in the HCV life cycle. The experiments proposed here will determine the molecular determinants of HCV permissiveness during hepatic development and identify new cellular cofactors and potential drug targets for HCV therapy. The following experiments will be performed: (1) Suppression of putative proviral factors and overexpression of putative antiviral factors in stem cells. These will be followed by hepatic differentiation and HCV infection of the differentiated human hepatocyte-like cells (DHHs). (2) Mechanistic studies of a novel proviral factor whose role has been validated by our preliminary studies. Specific steps of the HCV life cycle will be examined, and the interaction between viral and host protein will be dissected. (3) In vivo experiments designed to assess the potential of genetically modified DHHs to repopulate mouse liver and confer HCV resistance to the chimeric liver. The results of the proposed studies will not only provide significant insights into the properties of the host cells that govern HCV susceptibility but also reveal new therapeutic targets for antiviral intervention.

DESCRIPTION (provided by applicant): The broad, long-term goal of our research program is to advance knowledge of virus-host cell interactions by characterizing cellular cofactors essential for viral infections, particularly positive-strand RNA viruses such as hepatitis C virus (HCV) and dengue virus (DENV). These viruses infect millions of people worldwide and pose major threats to human health. Understanding how these viruses interact with the host cell is of critical importance to the development of diagnostics, antiviral drugs, and a prophylactic vaccine. In preliminary studies, we have uncovered a critical role of lipid droplets and one of its associated proteins in DENV and HCV entry into host cells at the membrane fusion step. Although a role of the lipid droplets in viral assembly has been reported, a function of these organelles in viral entry and membrane fusion has not been proposed before. In this study, we will investigate the mechanism of action for lipid droplets and its associated protein, CIDEB, to facilitate the entry of diverse RNA viruses. The following specific aims are proposed: (1) to define the roles of lipid droplets and their recruitment of early endosomes in virus entry. Our working hypothesis of this aim is that LDs and the recruitment of virus-containing early endosomes to LDs are required for completing viral entry. We will use pharmacological and genetic manipulations to reduce LDs in the target cells of DENV and HCV and then determine if infection by these viruses is inhibited at specific steps such as endocytosis, membrane fusion, or capsid release. (2) to determine the function of CIDEB during the infection cycle of positive-strand RNA viruses. We have demonstrated the CIDEB knockdown (KD) with RNAi reduced DENV/HCV infection and hypothesize that CIDEB can serve as a new host target for treating infections by positive-stranded RNA viruses. We will use CIDEB knockout (KO) models, generated using TAL effector nuclease (TALEN) technology, to determine the importance of CIDEB in the life cycles of various RNA viruses. The results of the proposed studies will not only provide significant insights into the virus- and host-related functions of lipid droplets and CIDEB but also may reveal new therapeutic targets for antiviral intervention directed at medically important RNA viruses.

? DESCRIPTION (provided by applicant): The broad, long-term goal of our research program is to advance knowledge of virus-host cell interactions by characterizing cellular cofactors essential for viral infections, particularly positive- strand RNA viruses such as dengue virus (DENV) and hepatitis C virus (HCV). The current proposal focuses on DENV, which infects millions of people worldwide and poses major emerging threat to human health. Understanding how DENV interacts with the host cell is of critical importance to the development of antiviral drugs and a prophylactic vaccine, both of which are currently lacking. Building upon our previous work on in vitro hepatic differentiation and HCV infection which led to the identification of host determinants of HCV susceptibility, we propose to investigate the cellular parameters that contribute to DENV permissiveness in both monocytes and hepatic cells. We have uncovered a discrete cellular transition to DENV permissiveness during directed differentiation of pluripotent stem cells in preliminary experiments. We will scrutinize the gene profiles during this transition period to identify putative host factors required for DENV infection which in turn may be exploited as new drug targets for antiviral therapy. In addition, we will analyze the contribution of downregulated antiviral proteins to the transition to DENV susceptibility and investigate the mechanism of action for any antiviral proteins that are effective at suppressing DENV infection. The results of the proposed studies will not only provide significant insights into how DENV hijacks cellular proteins and machineries to facilitate its own replication in human cells; but also may reveal new therapeutic targets for antiviral intervention directed at important human pathogens.

SUMMARY ? Overview Modeling of infectious diseases that affect the human central nervous system (CNS), such as those associated with Zika virus (ZIKV) and West Nile virus (WNV), has been challenging due to the inaccessibility of the relevant cell types. Reprogramming human somatic cells, such as skin fibroblasts, into induced pluripotent stem cells (iPSCs) provides a genetically tractable and renewable source of human neural cell populations. We can differentiate these iPSCs into many of the cell types critical for the study of neurotropic viruses, but typically this is performed in monolayer cultures to allow for more control and to generate more homogeneous cell populations, but this methodology lacks the self-organizing properties and interactive dynamics among different cell populations observed during organ development. Recently, more complex structures resembling whole developing organs, named organoids, have been generated from human iPSCs via 3D culturing methods. This emerging new technology has the potential to significantly advance our understanding of infectious diseases and for future therapeutic development. The success of this approach, however, critically depends on how well organoids mimic biological structures, recapitulate human physiology and disease pathology, and incorporate components critical to disease and human host responses. We propose to develop a robust platform for organoid development to model brain development that can be adopted by single labs for basic research, and is amenable to translational studies and drug development and testing. Our Research Center is comprised of three Research Projects, a Scientific Core, and an Administrative Core led by experts in virology, stem cell biology, neural development, and bioengineering. We will focus on ZIKV and WNV, two neurotropic flaviviruses, to develop our organoid platform, which can then be used by the scientific community to investigate other infectious diseases that affect the nervous system. Importantly, ZIKV and WNV are thought to impact the CNS at different stages of development, with ZIKV having been recently implicated as being causal for microcephaly in some pregnant women. This affords us the opportunity to develop an organoid platform with proof-of-principle testing with viruses suspected to have cell type- and stage-specific tropism. Project 1 will focus on technology development to generate more mature organoids and the scaling up of robust assays to perform medium-throughput compound testing. Project 2 will focus on ZIKV infections in early stage organoids and Project 3 will evaluate co-culture organoid systems to model WNV infections in later stage organoids. The projects will be supported by a Scientific Core that will provide cells and on-site training to Projects 2 & 3, as well as optimization of differentiation protocols and bioinformatics analyses. Finally, the Administrative Core will provide logistical support to facilitate collaborations among investigators and to coordinate the timely release of results and resources to the scientific community.

SUMMARY ? Project 2 The broad, long-term goal of our research program is to advance knowledge of viral replication strategies and virus?host cell interactions that are relevant for therapeutic intervention. The viruses that we study are positive- strand RNA viruses including dengue virus (DENV), hepatitis C virus (HCV), and Zika virus (ZIKV). To complement animal models, there is a clear need for better in vitro models to study infectious diseases that affect largely inaccessible organs such as the human nervous system. In the current HTMID CRC proposal with the overarching goal to develop an organoid-based platform using human induced pluripotent stem cells (iPSCs), Project 2 focuses on ZIKV, which poses a major emerging threat to human health due to a large number of recent outbreaks and its association with microcephaly, a neurodevelopmental birth defect. Understanding how ZIKV interacts with the host to cause disease is of critical importance to the development of effective antiviral drugs and a prophylactic vaccine, both of which are urgently needed. We have recently published work showing that ZIKV infects human neural stem cells and also performed a high-throughput screening to identify small molecule compounds that can inhibit ZIKV infection. Here we propose to utilize a 3D cerebral organoid model to investigate the mechanism of ZIKV pathogenesis in human neural development (Specific Aims 1 & 2). We will also use the organoid model to compare and contrast ZIKV with West Nile virus (WNV), another human neurotropic virus of biomedical importance (Specific Aim 3). Finally, we will validate the utility of the organoid model as a drug testing tool using chemical compounds with confirmed antiviral or anti-apoptotic functions (Specific Aim 4). With the successful completion of the project, we expect to: positively identify the type of cells and their ZIKV infection rate during the different stages of brain development; unravel the mechanisms by which ZIKV infection causes neuronal death and developmental defects; reveal any quantitative differences between ZIKV and WNV in infection efficiencies, cellular tropism, and pathology in the 3D model; and confirm lead compounds for inhibiting ZIKV replication in both 2D culture and 3D tissue models. These results will validate the utility of the 3D brain organoid model for both basic and translational research of ZIKV and also provide direct and immediate impact on the mission to develop effective therapy to treat ZIKV infection and its associated neurological diseases.

The broad, long-term goal of our research program is to advance knowledge of virus?host cell interactions that are relevant for disease prevention. The viruses that we study are positive- strand RNA viruses such as dengue virus (DENV), hepatitis C virus (HCV), and Zika virus (ZIKV). The current proposal focuses on ZIKV, which has re-emerged worldwide and poses a major emerging threat to human health. Understanding how ZIKV interacts with the host cell is of critical importance to the development of antiviral drugs and a prophylactic vaccine, both of which are currently lacking. Building upon our recently published work on the infection and impact of ZIKV on human neural stem cells, we propose to unravel the mechanism by which ZIKV impedes the growth of human cortical neural progenitor cells (hNPCs) and leads to defects in cortex development. Our preliminary data indicate that part of the mechanism for cell cycle arrest is a ZIKV-induced DNA damage response (DDR) which blocks DNA replication and leads to S-phase arrest. Unexpectedly, the ATR/Chk1 DNA checkpoint pathway that normally functions to deal with DNA replication stress in S-phase was not activated, suggesting that ZIKV suppresses ATR/Chk1 activation during DNA replication. The ability to increase DNA replication stress while simultaneously inhibiting ATR responds constitute a potent ?one-two punch? that exacerbates replication defects and ultimately leads to cell cycle arrest. We will carry out experiments to investigate the mechanisms by which ZIKV achieve these feats and identify the viral proteins responses. We will also cross-validate our results with brain organoids and infectious clones of Zika/Dengue chimeric viruses. We expect to clearly understand the mechanisms by which ZIKV perturbs the cell cycle to achieve its pathological effect on hNPC-mediated neural development in vitro. We also expect to reveal the virulent determinant of ZIKV critical for its effect on brain development. These results will not only fundamentally advance our understanding of this important human pathogen but also provide direct and immediate impact on the mission to develop effective therapy to treat ZIKV infection and its associated diseases.