Journal: Genome Medicine

Article Title: Genome-scale CRISPR screens identify host factors that promote human coronavirus infection

doi: 10.1186/s13073-022-01013-1

Figure Lengend Snippet: Confirmation of host factor involvement by targeted shRNA knockdown and CRISPR knockout. Lentivirus-packaged shRNA clones directed to CTSL , CCZ1 , and EDC4 were transduced into HEK293T-hACE2 cells and selected with puromycin. Lentivirus-packaged sgRNA directed to EDC4 and XRN1 were transduced into SAEC-hACE2 cells and selected with puromycin. A Gene knockdown was assessed using western blotting with antibodies directed to CTSL, CCZ1, and EDC4 in cells transduced with a gene-specific shRNA or empty vector control (EV). Actin expression served as a loading control. B Triplicate wells of knockdown cells were infected with SARS-CoV-2 or OC43 at MOI 0.01. At 2 dpi, viral genome copy numbers were determined by RT-qPCR and normalized to GAPDH levels as a housekeeping control. The data are reported as the relative normalized viral genome copy number in shRNA-expressing cells compared to the EV control ( n = 3 experiments). Error bars denote standard errors of mean and P values were determined using one-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001). C EDC4 and XRN1 knockout in SAEC-hACE2 was assessed using western blotting with antibodies directed to EDC4 and XRN1 in cells transduced with a gene-specific sgRNA or in wild-type cells (WT). Actin expression served as a loading control. D Triplicate wells of SAEChACE2 WT, EDC4ko, and XRN1ko cells were infected with SARS-CoV-2 or OC43 and MOI 0.01. At 0hpi, 1dpi, 2dpi, and 3dpi, cell supernatants were harvested and infectious viral particles were measured by TCID50 ( n = 2 experiments). Error bars denote standard errors of mean and P values were determined using one-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: To confirm gene knockdown and knockout, cell lysates prepared from knockdown/knockout and control cells were tested by western blotting with antibodies directed to CTSL (Invitrogen, BMS1032), CCZ1 (Santa Cruz Biotechnology, sc-514290), EDC4 (Cell Signaling Technology, 2548S), XRN1 (Cell Signaling Technology, 70205S), and actin as a loading control (Sigma-Aldrich, MAB1501R).

Techniques: shRNA, CRISPR, Knock-Out, Clone Assay, Western Blot, Transduction, Plasmid Preparation, Expressing, Infection, Quantitative RT-PCR

Journal: Genome Medicine

Article Title: Genome-scale CRISPR screens identify host factors that promote human coronavirus infection

doi: 10.1186/s13073-022-01013-1

Figure Lengend Snippet: Summary of genes found in this and other studies and their potential roles in the SARS-CoV-2 life cycle. The host factors identified in CRISPR screens are presented adjacent to the putative stage of viral replication where they function. The genes are color-coded based on their identification in our and other published studies, as indicated in the legend. Candidate pan-HCoV host factors are indicated with red asterisks. The virus replicates through a series of well-defined molecular steps. 1 – 2 After virion binding to ACE2, SARS-CoV-2 can fuse at the plasma membrane or following endocytosis. Heparan sulfate proteoglycans enhance viral attachment to cells so host factors involved in heparan sulfate biosynthesis (B3GAT3, EXT1, EXTL3, SLC35B2) and glycosylation (A4GALT, ALG5, ALG9) may play a role in viral entry. The IFITM proteins are proposed to promote fusion at the cell surface but inhibit fusion in endosomes. Host factors involved in endocytosis (C18orf8, CCZ1, CCZ1B, CLTC, EPN1, WDR81, WDR91), vesicular transport (DNM2, PIK3C3, RAB7A, TMEM106B, SNX27, VAC14, VPS35), and amphisome maturation/lysosome fusion (ATP6VIE1, ATPCV1G1, ATP6V1A, CTSL, GDI2, TMEM41B) likely facilitate virion uncoating. 3 The positive-sense RNA genome is then translated to produce the nonstructural polyproteins which are co-translationally cleaved to form the mature nsps. Certain host factors like RNH1 and DAZ3 may serve to protect the viral genome from degradation by host enzymes. 4 The nsps form the viral replicase which assembles on organellar membranes to form the replication and transcription complexes (RTCs) where progeny genomes and structural/accessory protein transcripts are produced, respectively. P-body components EDC4 and XRN1, identified in this study, may play a role in maintaining viral RNA stability or assembly of the RTC. 5 Structural and accessory proteins are translated, and structural proteins insert into the ER membrane. ER-localized SLC39A1 may play a role in this process. 6 Nucleocapsids bud into the ERGIC, potentially aided by host factors ERGIC3, SEC63, SLC33A1, and SCAP. 7 Progeny virions form as they traverse through the Golgi and structural proteins are glycosylated. 8 Virions exit the cell through either typical exocytosis (DNM2, EXOC2, EXT1, EXTL3, MYH13, SNX27, VPS35) or nonclassical lysosomal egress (GNPTAB, GNPTG, NAGPA, NPC1, TMEM106B, PIP4P1). Numerous host factors with less obvious direct roles in promoting steps in the viral life cycle have also been identified in CRISPR screens. For example, numerous factors regulating the cell cycle (BAX, CDK4, CDKN1A, DYRK1A, HRK, MPLKIP, PTCH1, STRADA, TP53) were identified in our screens in AGM and human cells. Furthermore, multiple nuclear-localized host factors including diverse transcriptional regulators and two components of the integrator complex (INTS6, INTS12) were identified. Overall, the large number of diverse host factors that promote SARS-CoV-2 replication illustrates the large-scale exploitation of cellular processes required for successful viral propagation. Adapted from BioRender template titled Life Cycle of Coronavirus generated by the Britt Glaunsinger laboratory. Created with BioRender.com

Article Snippet: To confirm gene knockdown and knockout, cell lysates prepared from knockdown/knockout and control cells were tested by western blotting with antibodies directed to CTSL (Invitrogen, BMS1032), CCZ1 (Santa Cruz Biotechnology, sc-514290), EDC4 (Cell Signaling Technology, 2548S), XRN1 (Cell Signaling Technology, 70205S), and actin as a loading control (Sigma-Aldrich, MAB1501R).

Techniques: CRISPR, Binding Assay, Produced, Generated

Journal: mBio

Article Title: Dengue and Zika Virus 5′ Untranslated Regions Harbor Internal Ribosomal Entry Site Functions

doi: 10.1128/mBio.00459-19

Figure Lengend Snippet: Translation of monocistronic reporter RNAs containing the DENV2/ZIKV 5′-UTRs with different 5′ modifications in A549 cells treated with Xrn-1 siRNA. (A) Xrn-1-specific siRNA was transfected onto A549 cells with control irrelevant siRNA (IR). Total protein was harvested, and Xrn-1 levels were analyzed by immunoblotting assay. Actin served as a loading control. (B and C) A549 cells treated with siRNA (-Xrn-1) and without siRNA (+Xrn-1) were transfected with two types of reporter RNAs, D5 G D3 (B) and Z5 G Z3 (C). Gluc activities were measured at different time points posttransfection as indicated. Means ± SEM from three independent experiments are plotted. (D and E) RNAs extracted from A549 cells transfected with D5 G D3 (D) and Z5 G Z3 (E) at different time points posttransfection were subjected to qRT-PCR analysis generating 5′-end fragments. Each reaction product was finally normalized to GAPDH and presented as the fold change relative to input RNAs. The amount of RNAs at 2 hpt was used as time zero posttransfection and artificially set at 1. The means ± SEM from three independent experiments are plotted. Values that are significantly different from the value for control RNA (at 2 hpt) by a two-tailed t test are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01). Values that are not significantly different (ns) are indicated.

Article Snippet: Xrn-1 siRNA was purchased from Santa Cruz Biotechnology, Inc., Dallas, TX (catalog no. sc-61811).

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Two Tailed Test