All Publications

  • CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication. Viruses Shivaprasad, S., Qiao, W., Weng, K. F., Umashankar, P., Carette, J. E., Sarnow, P. 2024; 16 (5)


    The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.

    View details for DOI 10.3390/v16050725

    View details for PubMedID 38793607

  • MYADM binds human parechovirus 1 and is essential for viral entry. Nature communications Qiao, W., Richards, C. M., Kim, Y., Zengel, J. R., Ding, S., Greenberg, H. B., Carette, J. E. 2024; 15 (1): 3469


    Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.

    View details for DOI 10.1038/s41467-024-47825-0

    View details for PubMedID 38658526

    View details for PubMedCentralID 6893760

  • The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation. Proceedings of the National Academy of Sciences of the United States of America Wąchalska, M., Riepe, C., Ślusarz, M. J., Graul, M., Borowski, L. S., Qiao, W., Foltyńska, M., Carette, J. E., Bieńkowska-Szewczyk, K., Szczesny, R. J., Kopito, R. R., Lipińska, A. D. 2024; 121 (11): e2309841121


    The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.

    View details for DOI 10.1073/pnas.2309841121

    View details for PubMedID 38442151

  • LYSET/TMEM251- a novel key component of the mannose-6-phosphate pathway. Autophagy Qiao, W., Richards, C. M., Jabs, S. 2023


    ​​Degradation of macromolecules delivered to lysosomes by processes such as autophagy or endocytosis is crucial for cellular function. Lysosomes require more than 60 soluble hydrolases in order to catabolize such macromolecules. These soluble hydrolases are tagged with mannose-6-phosphate (M6P) moieties in sequential reactions by the Golgi-resident GlcNAc-1-phosphotransferase complex and NAGPA/UCE/uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase), which allows their delivery to endosomal/lysosomal compartments through trafficking mediated by cation-dependent and -independent mannose-6-phosphate receptors (MPRs). We and others recently identified TMEM251 as a novel regulator of the M6P pathway via independent genome-wide genetic screening strategies. We renamed TMEM251 to LYSET (lysosomal enzyme trafficking factor) to establish nomenclature reflective to this gene's function. LYSET is a Golgi-localized transmembrane protein important for the retention of the GlcNAc-1-phosphotransferase complex in the Golgi-apparatus. The current understanding of LYSET's importance regarding human biology is 3-fold: 1) highly pathogenic viruses that depend on lysosomal hydrolase activity require LYSET for infection. 2) The presence of LYSET is critical for cancer cell proliferation in nutrient-deprived environments in which extracellular proteins must be catabolized. 3) Inherited pathogenic alleles of LYSET can cause a severe inherited disease which resembles GlcNAc-1-phosphotransferase deficiency (i.e., mucolipidosis type II).

    View details for DOI 10.1080/15548627.2023.2167376

    View details for PubMedID 36633450

  • The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection. Science (New York, N.Y.) Richards, C. M., Jabs, S., Qiao, W., Varanese, L. D., Schweizer, M., Mosen, P. R., Riley, N. M., Klüssendorf, M., Zengel, J. R., Flynn, R. A., Rustagi, A., Widen, J. C., Peters, C. E., Ooi, Y. S., Xie, X., Shi, P. Y., Bartenschlager, R., Puschnik, A. S., Bogyo, M., Bertozzi, C. R., Blish, C. A., Winter, D., Nagamine, C. M., Braulke, T., Carette, J. E. 2022: eabn5648


    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

  • TMEM41B and VMP1 modulate cellular lipid and energy metabolism for facilitating dengue virus infection. PLoS pathogens Yousefi, M., Lee, W. S., Yan, B., Cui, L., Yong, C. L., Yap, X., Tay, K. S., Qiao, W., Tan, D., Nurazmi, N. I., Linster, M., Smith, G. J., Lee, Y. H., Carette, J. E., Ooi, E. E., Chan, K. R., Ooi, Y. S. 2022; 18 (8): e1010763


    Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses.

    View details for DOI 10.1371/journal.ppat.1010763

    View details for PubMedID 35939522

  • Flock house virus as a vehicle for aphid Virus-induced gene silencing and a model for aphid biocontrol approaches JOURNAL OF PEST SCIENCE Jiang, J., Erickson, A., Qiao, W., Matsumura, E. E., Falk, B. W. 2022
  • A memory of eS25 loss drives resistance phenotypes. Nucleic acids research Johnson, A. G., Flynn, R. A., Lapointe, C. P., Ooi, Y. S., Zhao, M. L., Richards, C. M., Qiao, W. n., Yamada, S. B., Couthouis, J. n., Gitler, A. D., Carette, J. E., Puglisi, J. D. 2020


    In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation and suggests caution in the interpretation of ribosomal protein gene mutation data.

    View details for DOI 10.1093/nar/gkaa444

    View details for PubMedID 32463448

  • Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism. Cell chemical biology Liu, Q. n., Gupta, A. n., Okesli-Armlovich, A. n., Qiao, W. n., Fischer, C. R., Smith, M. n., Carette, J. E., Bassik, M. C., Khosla, C. n. 2020


    Genome-wide analysis of the mode of action of GSK983, a potent antiviral agent, led to the identification of dihydroorotate dehydrogenase as its target along with the discovery that genetic knockdown of pyrimidine salvage sensitized cells to GSK983. Because GSK983 is an ineffective antiviral in the presence of physiological uridine concentrations, we explored combining GSK983 with pyrimidine salvage inhibitors. We synthesized and evaluated analogs of cyclopentenyl uracil (CPU), an inhibitor of uridine salvage. We found that CPU was converted into its triphosphate in cells. When combined with GSK983, CPU resulted in large drops in cellular UTP and CTP pools. Consequently, CPU-GSK983 suppressed dengue virus replication in the presence of physiological concentrations of uridine. In addition, the CPU-GSK983 combination markedly enhanced the effect of RNA-dependent RNA polymerase (RdRp) inhibition on viral infection. Our findings highlight a new host-targeting strategy for potentiating the antiviral activity of RdRp inhibitors.

    View details for DOI 10.1016/j.chembiol.2020.05.002

    View details for PubMedID 32442424

    View details for PubMedCentralID PMC7241336