Bachelor of Science, Wuhan University (2010)
Doctor of Philosophy, Princeton University (2016)
Mark Kay, Postdoctoral Faculty Sponsor
While gene transfer using recombinant adeno-associated viral (rAAV) vectors has shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet-derived cells into functional beta cells in animal models, we constructed 2 highly complex barcoded replication competent capsid shuffled libraries and selected for high-transducing variants on primary human islets. We describe the generation of a chimeric AAV capsid (AAV-KP1) that facilitates transduction of primary human islet cells and human embryonic stem cell-derived beta cells with up to 10-fold higher efficiency compared with previously studied best-in-class AAV vectors. Remarkably, this chimeric capsid also enabled transduction of both mouse and human hepatocytes at very high levels in a humanized chimeric mouse model, thus providing a versatile vector that has the potential to be used in both preclinical testing and human clinical trials for liver-based diseases and diabetes.
View details for DOI 10.1172/jci.insight.131610
View details for PubMedID 31723052
Infection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at epithelial surfaces and continues into the peripheral nervous system (PNS). Inflammatory responses are induced at the infected peripheral site prior to invasion of the PNS. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which includes the interferons (IFNs). The fundamental question is how do PNS cell bodies respond to these distant, potentially damaging events experienced by axons. Using compartmented cultures that physically separate neuron axons from cell bodies, we found that pretreating isolated axons with beta interferon (IFN-?) or gamma interferon (IFN-?) significantly diminished the number of herpes simplex virus 1 (HSV-1) and PRV particles moving in axons toward the cell bodies in a receptor-dependent manner. Exposing axons to IFN-? induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFN-? induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated antiviral effects induced by IFN-?, but not those induced by IFN-?. Proteomic analysis of IFN-?- or IFN-?-treated axons identified several differentially regulated proteins. Therefore, unlike treatment with IFN-?, IFN-? induces a noncanonical, local antiviral response in axons. The activation of a local IFN response in axons represents a new paradigm for cytokine control of neuroinvasion.Neurons are highly polarized cells with long axonal processes that connect to distant targets. PNS axons that innervate peripheral tissues are exposed to various situations that follow infection, inflammation, and damage of the tissue. After viral infection in the periphery, axons represent potential front-line barriers to PNS infection and damage. Indeed, most viral infections do not spread to the PNS, yet the mechanisms responsible are not well studied. We devised an experimental system to study how axons respond to inflammatory cytokines that would be produced by infected tissues. We found that axons respond differentially to type I and type II interferons. The response to type I interferon (IFN-?) is a rapid axon-only response. The response to type II interferon (IFN-?) involves long-distance signaling to the PNS cell body. These responses to two interferons erect an efficient and rapid barrier to PNS infection.
View details for DOI 10.1128/mBio.02145-15
View details for Web of Science ID 000373933100060
View details for PubMedID 26838720
Viruses are intracellular parasites that can only replicate and spread in cells of susceptible hosts. Alpha herpesviruses (?-HVs) contain double-stranded DNA genomes of at least 120 kb, encoding for 70 or more genes. The viral genome is contained in an icosahedral capsid that is surrounded by a proteinaceous tegument layer and a lipid envelope. Infection starts in epithelial cells and spreads to the peripheral nervous system. In the natural host, ?-HVs establish a chronic latent infection that can be reactivated and rarely spread to the CNS. In the nonnatural host, viral infection will in most cases spread to the CNS with often fatal outcome. The host response plays a crucial role in the outcome of viral infection. ?-HVs do not encode all the genes required for viral replication and spread. They need a variety of host gene products including RNA polymerase, ribosomes, dynein, and kinesin. As a result, the infected cell is dramatically different from the uninfected cell revealing a complex and dynamic interplay of viral and host components required to complete the virus life cycle. In this review, we describe the pivotal contribution of MS-based proteomics studies over the past 15 years to understand the complicated life cycle and pathogenesis of four ?-HV species from the alphaherpesvirinae subfamily: Herpes simplex virus-1, varicella zoster virus, pseudorabies virus and bovine herpes virus-1. We describe the viral proteome dynamics during host infection and the host proteomic response to counteract such pathogens.
View details for DOI 10.1002/pmic.201400604
View details for Web of Science ID 000356344900002
View details for PubMedID 25764121
Infection by alphaherpesviruses invariably results in invasion of the peripheral nervous system (PNS) and establishment of either a latent or productive infection. Infection begins with long-distance retrograde transport of viral capsids and tegument proteins in axons toward the neuronal nuclei. Initial steps of axonal entry, retrograde transport, and replication in neuronal nuclei are poorly understood. To better understand how the mode of infection in the PNS is determined, we utilized a compartmented neuron culturing system where distal axons of PNS neurons are physically separated from cell bodies. We infected isolated axons with fluorescent-protein-tagged pseudorabies virus (PRV) particles and monitored viral entry and transport in axons and replication in cell bodies during low and high multiplicities of infection (MOIs of 0.01 to 100). We found a threshold for efficient retrograde transport in axons between MOIs of 1 and 10 and a threshold for productive infection in the neuronal cell bodies between MOIs of 1 and 0.1. Below an MOI of 0.1, the viral genomes that moved to neuronal nuclei were silenced. These genomes can be reactivated after superinfection by a nonreplicating virus, but not by a replicating virus. We further showed that viral particles at high-MOI infections compete for axonal proteins and that this competition determines the number of viral particles reaching the nuclei. Using mass spectrometry, we identified axonal proteins that are differentially regulated by PRV infection. Our results demonstrate the impact of the multiplicity of infection and the axonal milieu on the establishment of neuronal infection initiated from axons.Alphaherpesvirus genomes may remain silent in peripheral nervous system (PNS) neurons for the lives of their hosts. These genomes occasionally reactivate to produce infectious virus that can reinfect peripheral tissues and spread to other hosts. Here, we use a neuronal culture system to investigate the outcome of axonal infection using different numbers of viral particles and coinfection assays. We found that the dynamics of viral entry, transport, and replication change dramatically depending on the number of virus particles that infect axons. We demonstrate that viral genomes are silenced when the infecting particle number is low and that these genomes can be reactivated by superinfection with UV-inactivated virus, but not with replicating virus. We further show that viral invasion rapidly changes the profiles of axonal proteins and that some of these axonal proteins are rate limiting for efficient infection. Our study provides new insights into the establishment of silent versus productive alphaherpesvirus infections in the PNS.
View details for DOI 10.1128/mBio.00276-15
View details for Web of Science ID 000355312400045
View details for PubMedID 25805728
Ag receptor engagement triggers lymphocyte activation and proliferation by activating several transcription factors including NF-?B. Caspase recruitment domain (CARD) containing membrane-associated guanylate kinase (MAGUK) protein 1 (CARMA1) is an essential adaptor protein that links Ag receptors to NF-?B activation. Here, we identify stress-induced-phosphoprotein 1 homology and U-box containing protein 1 (STUB1) as a CARMA1-associated protein. STUB1 constitutively interacted with CARMA1, and the interaction was intensified by TCR stimulation. Downregulation of STUB1 expression by RNAi markedly diminished TCR-induced canonical NF-?B activation and IL-2 production. Furthermore, overexpression of STUB1 enhanced the ubiquitination of CARMA1, whereas knockdown of STUB1 abolished the endogenous ubiquitination of CARMA1 induced by TCR stimulation. Subsequently, the ubiquitination of CARMA1 catalyzed by STUB1 was identified as Lys-27 linked, which is important for CARMA1-mediated NF-?B activation. These data provide the first evidence that ubiquitination of CARMA1 by STUB1 promotes TCR-induced NF-?B signaling.
View details for DOI 10.1002/eji.201242554
View details for Web of Science ID 000317859200020
View details for PubMedID 23322406
T-cell receptor (TCR)-induced T-cell activation is a critical event in adaptive immune responses. The engagement of TCR complex by antigen along with the activation of the costimulatory receptors trigger a cascade of intracellular signaling, in which caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) is a crucial scaffold protein. Upon stimulation, CARMA1 recruits downstream molecules including B-cell CLL/lymphoma 10 (Bcl10), mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1), and TRAF6 to assemble a specific TCR-induced signalosome that triggers NF-?B and JNK activation. In this report, we identified protein kinase C? (PKC?) as a CARMA1-associated protein by a biochemical affinity purification approach. PKC? interacted with CARMA1 in TCR stimulation-dependent manner in Jurkat T cells. Overexpression of PKC? inhibited CARMA1-mediated NF-?B activation, whereas knockdown of PKC? potentiated TCR-triggered NF-?B activation and IL-2 secretion in Jurkat T cells. Reconstitution experiments with PKC? kinase-dead mutant indicated that the kinase activity of PKC? was dispensable for its ability to inhibit TCR-triggered NF-?B activation. Furthermore, we found that PKC? inhibited the interaction between MALT1 and TRAF6, but not the association of CARMA1 with PKC?, Bcl10, or MALT1. These observations suggest that PKC? is a negative regulator in T cell activation through inhibiting the assembly of CARMA1 signalosome.
View details for DOI 10.1074/jbc.M111.335463
View details for Web of Science ID 000306414500029
View details for PubMedID 22528498