An RNA-centric dissection of host complexes controlling flavivirus infection.
MECHANISM OF TETHERIN INHIBITION OF ALPHAVIRUS RELEASE.
Journal of virology
Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.
View details for DOI 10.1038/s41564-019-0518-2
View details for PubMedID 31384002
STAG2 deficiency induces interferon responses via cGAS-STING pathway and restricts virus infection.
2018; 9 (1): 1485
Tetherin is an interferon-inducible, antiviral host factor that broadly restricts enveloped virus release by tethering budded viral particles to the plasma membrane. In response, many viruses have evolved tetherin antagonists. The human tetherin gene can express two isoforms, long and short, due to alternative translation initiation sites in the N-terminal cytoplasmic tail. The long isoform (L-tetherin) contains 12 extra amino acids in its N-terminus, including a dual tyrosine motif (YDYCRV) that is an internalization signal for clathrin-mediated endocytosis and a determinant of NF-?B activation. Tetherin restricts alphaviruses, which are highly organized enveloped RNA viruses that bud from the plasma membrane. L-tetherin is more efficient than S-tetherin in inhibiting alphavirus release in 293 cells. Here, we demonstrated that alphaviruses do not encode an antagonist for either tetherin isoform. Instead, the isoform specificity reflected a requirement for tetherin endocytosis. The YxY motif in L-tetherin was necessary for alphavirus restriction in 293 cells, but was not required for rhabdovirus restriction. L-tetherin's inhibition of alphavirus release correlated with its internalization but did not involve NF-?B activation. In contrast, in U-2 OS cells the YxY motif and the L-tetherin N-terminal domain were not required for either robust tetherin internalization or alphavirus inhibition. Tetherin forms that were negative for restriction accumulated at the cell surface of infected cells, while the levels of tetherin forms that restrict were decreased. Together, our results suggest that tetherin-mediated virus clearance plays an important role in the restriction of alphavirus release, and that cell type-specific cofactors may promote tetherin endocytosis.IMPORTANCE The mechanisms of tetherin's antiviral activities and viral tetherin antagonism have been studied in detail for a number of different viruses. Although viral countermeasures against tetherin can differ significantly, overall tetherin's antiviral activity correlates with physical tethering of virus particles to prevent their release. While tetherin can mediate virus endocytic uptake and clearance, this has not been observed to be required for restriction. Here we show that efficient tetherin inhibition of alphavirus release requires efficient tetherin endocytosis. Our data suggest that this endocytic uptake can be mediated by tetherin itself or by a tetherin cofactor that promotes uptake of an endocytosis-deficient variant of tetherin.
View details for DOI 10.1128/JVI.02165-18
View details for PubMedID 30674629
SETD3 is an actin histidine methyltransferase that prevents primary dystocia.
Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types. Here, using a genome-wide CRISPR-Cas9 screening approach to identify host dependency factors and novel innate immune regulators of rotavirus (RV) infection, we demonstrate that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids. Mechanistically, STAG2 deficiency results in spontaneous genomic DNA damage and robust interferon (IFN) expression via the cGAS-STING cytosolic DNA-sensing pathway. The resultant activation of JAK-STAT signaling and IFN-stimulated gene (ISG) expression broadly protects against virus infections, including RVs. Our work highlights a previously undocumented role of the cohesin complex in regulating IFN homeostasis and identifies new therapeutic avenues for manipulating the innate immunity.
View details for PubMedID 29662124
A CRISPR toolbox to study virus-host interactions
NATURE REVIEWS MICROBIOLOGY
2017; 15 (6): 351-364
For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.
View details for PubMedID 30626964
Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2017; 114 (18): E3642-E3651
Viruses depend on their hosts to complete their replication cycles; they exploit cellular receptors for entry and hijack cellular functions to replicate their genome, assemble progeny virions and spread. Recently, genome-scale CRISPR-Cas screens have been used to identify host factors that are required for virus replication, including the replication of clinically relevant viruses such as Zika virus, West Nile virus, dengue virus and hepatitis C virus. In this Review, we discuss the technical aspects of genome-scale knockout screens using CRISPR-Cas technology, and we compare these screens with alternative genetic screening technologies. The relative ease of use and reproducibility of CRISPR-Cas make it a powerful tool for probing virus-host interactions and for identifying new antiviral targets.
View details for DOI 10.1038/nrmicro.2017.29
View details for Web of Science ID 000401062000010
View details for PubMedID 28420884
A Small-Molecule Oligosaccharyltransferase Inhibitor with Pan-flaviviral Activity.
2017; 21 (11): 3032?39
Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways.
View details for DOI 10.1073/pnas.1619266114
View details for Web of Science ID 000400358000009
View details for PubMedID 28416666
View details for PubMedCentralID PMC5422808
Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens
2016; 535 (7610): 159-?
The mosquito-borne flaviviruses include important human pathogens such as dengue, Zika, West Nile, and yellow fever viruses, which pose a serious threat for global health. Recent genetic screens identified endoplasmic reticulum (ER)-membrane multiprotein complexes, including the oligosaccharyltransferase (OST) complex, as critical flavivirus host factors. Here, we show that a chemical modulator of the OST complex termed NGI-1 has promising antiviral activity against flavivirus infections. We demonstrate that NGI-1 blocks viral RNA replication and that antiviral activity does not depend on inhibition of the N-glycosylation function of the OST. Viral mutants adapted to replicate in cells deficient of the OST complex showed resistance to NGI-1 treatment, reinforcing the on-target activity of NGI-1. Lastly, we show that NGI-1 also has strong antiviral activity in primary and disease-relevant cell types. This study provides an example for advancing from the identification of genetic determinants of infection to a host-directed antiviral compound with broad activity against flaviviruses.
View details for PubMedID 29241533
View details for PubMedCentralID PMC5734657
BST2/Tetherin Inhibition of Alphavirus Exit
2015; 7 (4): 2147-2167
The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.
View details for DOI 10.1038/nature18631
View details for Web of Science ID 000379015600044
View details for PubMedID 27383987
View details for PubMedCentralID PMC4964798
Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry
2013; 9 (12)
Alphaviruses such as chikungunya virus (CHIKV) and Semliki Forest virus (SFV) are small enveloped RNA viruses that bud from the plasma membrane. Tetherin/BST2 is an interferon-induced host membrane protein that inhibits the release of many enveloped viruses via direct tethering of budded particles to the cell surface. Alphaviruses have highly organized structures and exclude host membrane proteins from the site of budding, suggesting that their release might be insensitive to tetherin inhibition. Here, we demonstrated that exogenously-expressed tetherin efficiently inhibited the release of SFV and CHIKV particles from host cells without affecting virus entry and infection. Alphavirus release was also inhibited by the endogenous levels of tetherin in HeLa cells. While rubella virus (RuV) and dengue virus (DENV) have structural similarities to alphaviruses, tetherin inhibited the release of RuV but not DENV. We found that two recently identified tetherin isoforms differing in length at the N-terminus exhibited distinct capabilities in restricting alphavirus release. SFV exit was efficiently inhibited by the long isoform but not the short isoform of tetherin, while both isoforms inhibited vesicular stomatitis virus exit. Thus, in spite of the organized structure of the virus particle, tetherin specifically blocks alphavirus release and shows an interesting isoform requirement.
View details for DOI 10.3390/v7042147
View details for Web of Science ID 000353720400030
View details for PubMedID 25912717
Recombineering linear DNA that replicate stably in E-coli
2008; 59 (1): 63-71
The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.
View details for DOI 10.1371/journal.ppat.1003835
View details for Web of Science ID 000330535400057
View details for PubMedID 24367265
STR data for the AmpFlSTR identifiler loci in three ethnic groups (Malay, Chinese, Indian) of the Malaysian population
FORENSIC SCIENCE INTERNATIONAL
2003; 138 (1-3): 134-?
The advent of recombineering technology in Escherichia coli has revolutionized the way recombinant DNA molecules are constructed. We present a novel application of recombineering to linearize DNA by capping their ends with individual telomeres derived from bacteriophage N15, which exists as a linear prophage in E. coli. The N15 telomerase occupancy site was recombined into circular DNA and resolved into individual telomeres by the phage N15 protelomerase enzyme. We demonstrate this technique by assembling linear BACs that replicate stably in their host strain E. coli DH10B. Correct linearization of the BACs was confirmed by restriction mapping using pulsed field gel electrophoresis. The linear BAC DNA can be easily purified using standard plasmid isolation methods and resist degradation from RecBCD nuclease in vitro and in vivo owing to the presence of telomeres. Transfection of a linear 100 kb BAC containing the human beta-globin gene cluster into HT1080 cells produced accurately spliced transcripts, demonstrating that the linear DNA will be useful for subsequent functional studies. This novel recombineering technique may be particularly useful for building large linear constructs for assembling artificial chromosomes with telomeres, and may provide a unique means to clone and study large linear viral genomes that contain hairpin ends.
View details for DOI 10.1016/j.plasmid.2007.09.002
View details for Web of Science ID 000253074300007
View details for PubMedID 17988739