Doctor of Philosophy, University of Georgia (2017)
Integrative analysis of functional genomic screening and clinical data identifies a protective role for spironolactone in severe COVID-19.
Cell reports methods
2023; 3 (7): 100503
We demonstrate that integrative analysis of CRISPR screening datasets enables network-based prioritization of prescription drugs modulating viral entry in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by developing a network-based approach called Rapid proXimity Guidance for Repurposing Investigational Drugs (RxGRID). We use our results to guide a propensity-score-matched, retrospective cohort study of 64,349 COVID-19 patients, showing that a top candidate drug, spironolactone, is associated with improved clinical prognosis, measured by intensive care unit (ICU) admission and mechanical ventilation rates. Finally, we show that spironolactone exerts a dose-dependent inhibitory effect on viral entry in human lung epithelial cells. Our RxGRID method presents a computational framework, implemented as an open-source software package, enabling genomics researchers to identify drugs likely to modulate a molecular phenotype of interest based on high-throughput screening data. Our results, derived from this method and supported by experimental and clinical analysis, add additional supporting evidence for a potential protective role of the potassium-sparing diuretic spironolactone in severe COVID-19.
View details for DOI 10.1016/j.crmeth.2023.100503
View details for PubMedID 37529368
Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression.
The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.
View details for DOI 10.1038/s41592-023-01896-x
View details for PubMedID 37291262
View details for PubMedCentralID 3337962
- A flexible system for tissue-specific gene expression in mice using adeno-associated virus NATURE METHODS 2023
The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.
Science (New York, N.Y.)
Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. Here, we used genome-scale CRISPR screens to identify Lysosomal Enzyme Trafficking factor (LYSET) as essential for infection by cathepsin-dependent viruses including SARS-CoV-2. LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery, and mutations in LYSET can explain the phenotype of the associated disorder.
View details for DOI 10.1126/science.abn5648
View details for PubMedID 36074821
Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.
View details for DOI 10.1038/s41588-022-01131-x
View details for PubMedID 35879412
Immunogenicity of Mumps Virus Genotype G Vaccine Candidates in Jeryl Lynn-Immunized Mice
JOURNAL OF VIROLOGY
Mumps virus (MuV) causes a highly contagious human disease characterized by the enlargement of the parotid glands. In severe cases, mumps can lead to neurological complications such as aseptic meningitis and encephalitis. Vaccination with the attenuated Jeryl Lynn (JL) MuV vaccine has dramatically reduced the incidence of MuV infection. Recently, large outbreaks have occurred in vaccinated populations. The vaccine strain JL was generated from genotype A, while most current circulating strains belong to genotype G. In this study, we examined the immunogenicity and longevity of genotype G-based vaccines. We found that our recombinant genotype G-based vaccines provide robust neutralizing titers toward genotype G for up to 1 year in mice. In addition, we demonstrated that a third dose of a genotype G-based vaccine following two doses of JL immunization significantly increases neutralizing titers toward the genotype G strain. Our data suggest that after two doses of JL vaccination, which most people have received, a third dose of a genotype G-based vaccine can generate immunity against a genotype G strain. IMPORTANCE At present, most individuals have received two doses of the measles, mumps, and rubella (MMR) vaccine, which contains genotype A mumps vaccine. One hurdle in developing a new mumps vaccine against circulating genotype G virus is whether the new genotype G vaccine can generate immunity in humans that are immunized against genotype A virus. This work demonstrates that a novel genotype G-based vaccine can be effective in animals which received two doses of genotype A-based vaccine, suggesting that the lead genotype G vaccine may induce anti-G immunity in humans who have received two doses of the current vaccine, providing support for testing this vaccine in humans.
View details for DOI 10.1128/jvi.01983-21
View details for Web of Science ID 000784247300004
View details for PubMedID 35389265
View details for PubMedCentralID PMC9044963
GENOME-WIDE, BIDIRECTIONAL CRISPR SCREENS IDENTIFY MUCINS AS CRITICAL MODULATORS OF SARS-COV-2 INFECTION
AMER SOC TROP MED & HYGIENE. 2021: 151
View details for Web of Science ID 000778105602160
Cracking the cell access code for a deadly virus
2020; 588 (7837): 223–24
View details for Web of Science ID 000624296500003
Regulation of Mumps Virus Replication and Transcription by Kinase RPS6KB1
JOURNAL OF VIROLOGY
2020; 94 (12)
Mumps virus (MuV) caused the most viral meningitis before mass immunization. Unfortunately, MuV has reemerged in the United States in the past several years. MuV is a member of the genus Rubulavirus, in the family Paramyxoviridae, and has a nonsegmented negative-strand RNA genome. The viral RNA-dependent RNA polymerase (vRdRp) of MuV consists of the large protein (L) and the phosphoprotein (P), while the nucleocapsid protein (NP) encapsulates the viral RNA genome. These proteins make up the replication and transcription machinery of MuV. The P protein is phosphorylated by host kinases, and its phosphorylation is important for its function. In this study, we performed a large-scale small interfering RNA (siRNA) screen targeting host kinases that regulated MuV replication. The human kinase ribosomal protein S6 kinase beta-1 (RPS6KB1) was shown to play a role in MuV replication and transcription. We have validated the role of RPS6KB1 in regulating MuV using siRNA knockdown, an inhibitor, and RPS6KB1 knockout cells. We found that MuV grows better in cells lacking RPS6KB1, indicating that it downregulates viral growth. Furthermore, we detected an interaction between the MuV P protein and RPS6KB1, suggesting that RPS6KB1 directly regulates MuV replication and transcription.IMPORTANCE Mumps virus is an important human pathogen. In recent years, MuV has reemerged in the United State, with outbreaks occurring in young adults who have been vaccinated. Our work provides insight into a previously unknown mumps virus-host interaction. RPS6KB1 negatively regulates MuV replication, likely through its interaction with the P protein. Understanding virus-host interactions can lead to novel antiviral drugs and enhanced vaccine production.
View details for DOI 10.1128/JVI.00387-20
View details for Web of Science ID 000537852600013
View details for PubMedID 32295907
Hardwiring Tissue-Specific AAV Transduction in Mice Through Engineered AAVR Expression
CELL PRESS. 2020: 253
View details for Web of Science ID 000530089301121
- Cracking the cell access code for the deadly virus VEEV. Nature 2020; 588 (7837): 223–24
Structural and cellular biology of adeno-associated virus attachment and entry.
Advances in virus research
2020; 106: 39–84
Adeno-associated virus (AAV) is a nonenveloped, ssDNA virus in the parvovirus family, which has become one of the leading candidate vectors for human gene therapy. AAV has been studied extensively to identify host cellular factors involved in infection, as well as to identify capsid variants that confer clinically favorable transduction profiles ex vivo and in vivo. Recent advances in technology have allowed for direct genetic approaches to be used to more comprehensively characterize host factors required for AAV infection and allowed for identification of a critical multi-serotype receptor, adeno-associated virus receptor (AAVR). In this chapter, we will discuss the interactions of AAV with its glycan and proteinaceous receptors and describe the host and viral components involved in AAV entry, which requires cellular attachment, endocytosis, trafficking to the trans-Golgi network and nuclear import. AAV serves as a paradigm for entry of nonenveloped viruses. Furthermore, we will discuss the potential of utilizing our increased understanding of virus-host interactions during AAV entry to develop better AAV-based therapeutics, with a focus on host factors and capsid interactions involved in in vivo tropism.
View details for DOI 10.1016/bs.aivir.2020.01.002
View details for PubMedID 32327148
Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses.
The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.
View details for DOI 10.1016/j.cell.2020.12.004
View details for PubMedID 33333024
View details for PubMedCentralID PMC7723770
Capsid engineering overcomes barriers toward Adeno-associated viral (AAV) vector-mediated transduction of endothelial cells.
Human gene therapy
Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but are inefficiently transduced with Adeno-associated viral (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable already few hours post transduction. Notably, AAV-VEC transduced not only proliferating EC, but also quiescent EC although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid-engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.
View details for DOI 10.1089/hum.2019.027
View details for PubMedID 31407607
Genome-Wide CRISPR/Cas9 Screening Identifies GPR108 as a Highly Conserved AAV Entry Factor
CELL PRESS. 2019: 313–14
View details for Web of Science ID 000464381003125
GPR108 Is a Highly Conserved AAV Entry Factor.
Molecular therapy : the journal of the American Society of Gene Therapy
Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.
View details for DOI 10.1016/j.ymthe.2019.11.005
View details for PubMedID 31784416
Enterovirus pathogenesis requires the host methyltransferase SETD3.
Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.
View details for DOI 10.1038/s41564-019-0551-1
View details for PubMedID 31527793