Journal: International Journal of Molecular Sciences

Article Title: tRNA Modifications: A Tale of Two Viruses—SARS-CoV-2 and ZIKV

doi: 10.3390/ijms26157479

Figure Lengend Snippet: Elongator complex loss reduces SARS-CoV-2 and ZIKV infection. ( A ) Schematic representation of U34 tRNA modification pathway in FD (ELP1-deficient) cells. The Western blot shows ACE2 receptor and actin levels in cells transduced by ACE2-expressing lentivector (VLP ACE2 ). ( B ) Quantification of U34 modification levels (ncm 5 U, mcm 5 U, and mcm 5 s 2 U) in wild-type ( wt ) versus FD cells determined by mass spectrometry on tRNA-enriched RNA fractionations. ( C ) SARS-CoV-2 infection levels measured by RT-qPCR in wt and FD cells at different multiplicities of infection (MOI) 24 h post-infection. ( D ) ZIKV infection levels measured by RT-qPCR in wt and FD cells at different MOIs 48 h post-infection.

Article Snippet: Seventy-two hours after transduction, accurate ACE2 expression was controlled by Western blots using anti-ACE2 antibody (Human ACE-2 Antibody, AF933, R&D systems (a Bio-Techne brand (Minneapolis, MN, USA)).

Techniques: Infection, Modification, Western Blot, Expressing, Mass Spectrometry, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: tRNA Modifications: A Tale of Two Viruses—SARS-CoV-2 and ZIKV

doi: 10.3390/ijms26157479

Figure Lengend Snippet: ALKBH8 and CTU1 knockdown individually impairs SARS-CoV-2 and ZIKV infection. ( A ) Schematic showing shRNA-mediated knockdown approach targeting U34 ALKBH8 and CTU1 components of U34 modification pathway. The Western blot analysis of ACE2 expression in A549 cells with and without lentiviral vector expressing ACE2 (VLP ACE2 ). ( B ) Western blot validation of ALKBH8 and CTU1 knockdown efficiency with corresponding actin loading controls. Numbers indicate relative protein levels. ( C ) SARS-CoV-2 infection levels measured by RT-qPCR following knockdown of the indicated genes 24 h post-infection. ( D ) ZIKV infection levels measured by RT-qPCR following knockdown of the indicated genes 48 h post-infection.

Article Snippet: Seventy-two hours after transduction, accurate ACE2 expression was controlled by Western blots using anti-ACE2 antibody (Human ACE-2 Antibody, AF933, R&D systems (a Bio-Techne brand (Minneapolis, MN, USA)).

Techniques: Knockdown, Infection, shRNA, Modification, Western Blot, Expressing, Plasmid Preparation, Biomarker Discovery, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: Tuning VLP viral tropism by altering viral glycoprotein. ( A ) Different types of VLPs were produced using the 3P system by varying the viral glycoprotein (VSV-G, SARS2 spike, SARS spike, or MERS spike) and reporter genes (luciferase or EGFP). ( B ) One microgram of the viral glycoprotein was used to create various 3P Luc-PS9 VLPs and these were used to infect five cell types: WT 293T (293T), 293T-hACE2, A549-hACE2-TMPRSS2, Calu-3, and 293T-DPP4. SARS2 and SARS spike VLPs displayed similar tropism and entered only hACE2-expressing cells (293T-hACE2, A549-hACE2-TMPRSS2, and Calu-3), with SARS exhibiting higher luminescence intensity compared with SARS2. MERS VLPs infected Calu-3 cell at low level and efficiently entered 293T-DPP4 cells. VSV-G VLPs entered all cell types. ( C ) 3P EGFP-PS9 VLPs were produced with 4 μg VSV-G, 1 μg SARS2 spike, or 1 μg MERS spike plasmid. VSV-G VLPs entered all cell types. SARS2 VLPs only entered ACE2 cells. MERS VLPs only entered DPP4 cells. Methods provide detailed steps for VLP production. Data are mean ± STD. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: These include anti-SARS2 N protein mAb 1035111 (IgG2b, Catalog #: MAB10474), anti-SARS2 spike subunit 2 (S2) mAb 1034617 (IgG2a, Catalog #: MAB10557), Alexa Fluor 647-conjugated anti-human ACE2 mAb 535919 (IgG2a, Catalog #: FAB9332R), and rabbit anti-mouse ACE2 mAb 2818I (IgG, Catalog #: FAB34372G), all from R&D Systems (Minneapolis, MN).

Techniques: Produced, Luciferase, Expressing, Infection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: VLPs deliver functional Cas9 mRNA into target cells to achieve gene editing. ( A ) 3P Cas9-P2A-dTo-T20 VLPs (dTo: dTomato) were used for gene editing studies, with surface glycoprotein encoding for either VSV-G or SARS2 spike. ( B ) General workflow of gene editing study performed in panels (C)–(E). sgRNAs were transfected into cells on day −1 using plasmids carrying BFP reporter. VLP-carrying spCas9 mRNA was introduced into cells on day 0. Tropism of the VLP depends on surface glycoprotein. Gene editing efficiency was quantified on day 6. Editing efficiency quantified percentage of BFP(+) population that either turned EGFP(−) (panels C and E) or lost ACE2 expression based on anti-hACE2 binding (panel D). ( C ) sgRNAs targeting EGFP were introduced into 293T-hACE2-EGFP and spCas9 mRNA was delivered using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. ( D ) sgRNAs against hACE2 were introduced to knockout the receptor in 293T-hACE2 cells using 3P SARS2 Cas9-P2A-dTo-T20 VLPs. ( E ) sgRNAs targeting EGFP were introduced in 293T-EGFP cells, with genome editing being performed using 3P Cas9-P2A-dTo-T20 VLPs bearing either VSV-G or SARS2 spike. In all panels, the target gene ( EGFP or hACE2 ) was knocked out in 20%–35% of cells expressing sgRNA. Higher VLP amount resulted in greater editing. ( F ) 293T-hACE2 stably expressed sgRNAs against SLC35A1 were infected with 3P SARS2 Cas9-P2A-dTo-T20 VLPs (1.885 μg/μl N protein equivalent) or without SARS2 spike (1.385 μg/μl N protein equivalent). Gene editing efficiency was evaluated based on increase in fluorescent peanut agglutinin lectin (PNA) binding to cells. More than 70% gene editing was observed upon using 3P VLPs to edit endogenous genes. Volume of VLP used in each assay is specified in individual panels. Data are mean ± STD. *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: These include anti-SARS2 N protein mAb 1035111 (IgG2b, Catalog #: MAB10474), anti-SARS2 spike subunit 2 (S2) mAb 1034617 (IgG2a, Catalog #: MAB10557), Alexa Fluor 647-conjugated anti-human ACE2 mAb 535919 (IgG2a, Catalog #: FAB9332R), and rabbit anti-mouse ACE2 mAb 2818I (IgG, Catalog #: FAB34372G), all from R&D Systems (Minneapolis, MN).

Techniques: Functional Assay, Transfection, Expressing, Binding Assay, Knock-Out, Stable Transfection, Infection

Journal: Pathogens and Immunity

Article Title: Characteristics and Functions of Infection-enhancing Antibodies to the N-terminal Domain of SARS-CoV-2

doi: 10.20411/pai.v9i2.679

Figure Lengend Snippet: Mechanism of infection-enhancement of SARS-CoV-2. (A) Infection-enhancing activity of IgG and purified Fab generated from a panel of NTD-binding, infection-enhancing mAbs. Enhancement was measured in VSV-SARS-CoV-2 S pseudovirus (PV) assays using PV bearing S from Wuhan, Delta, or Omicron variants. Mean values for percentage enhancement by IgG and Fab against each PV were compared using a 2-tailed Student's t test, *** P <0.0001, NS=not significant. (B) Flow cytometry of cell-surface ACE2 expression on 293T-ACE2, intestinal (Caco-2), and respiratory (Calu-3) epithelial cell lines as compared to an isotype matched IgG2A control antibody. MFI=Mean Fluorescent Intensity. (C) Infection of 293T-ACE2, Caco-2 and Calu-3 epithelial cell lines with a Wuhan-PV. Luciferase activity was measured in cell lysates 24 hours after infection and expressed as the median of relative light units (RLU) for 5 replicate wells. (D) Percentage enhancement of Wuhan-PV infection by NTD-binding mAbs in 293T-ACE2, Calu-3, and Caco-2 cells. Mean values for percentage enhancement of infection between 2 cell types were compared using a 2-tailed Student's t test, *** P <0.0001.

Article Snippet: Cells were then stained with either anti-ACE2-Alexa Fluor® 647-conjugated antibody (R&D Systems) or an isotope-matched control mouse IgG2A antibody (R&D systems) for 30 minutes at room temperature.

Techniques: Infection, Activity Assay, Purification, Generated, Binding Assay, Flow Cytometry, Expressing, Control, Luciferase

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: Phylogenetic tree of bat ACE2 protein sequences with mammalian or avian ACE2 sequences. Phylogenetic trees were constructed by aligning CVB and PB ACE2 protein sequence with previously studied mammalian or bat ACE2 sequences. Alignments were performed using the Jukes–Cantor genetic distance model, tree was built using neighbor-joining algorithm using Chicken-ACE2 as an outgroup since it does not bind to SC2 S protein. Consensus tree was generated by resampling with 500 bootstraps. Branch labels indicate substitutions per site. Scale bar is shown.

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Construct, Sequencing, Generated

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: CVB-ACE2 and PB-ACE2 cells support infection of SC2 variants. Line graphs show log 10 TCID 50 titers/mL of WA1, Delta, Lambda, and Omicron lineage SC2 viruses in CVB-ACE2/hTMPRSS2 ( A ), PB-ACE2/hTMPRSS2 expressing DF-1 cells ( B ) and DF-1 cells expressing human ACE2 and TMPRSS2 ( C ). Data represent mean ± SD from three independent experiments for each time point. Statistical comparisons were conducted with 2-way ANOVA using repeated measures with Geisser–Greenhouse correction and Tukey multiple comparisons test with individual variances computed for each comparison. Lines with different lowercase letters indicate statistically significant differences ( p < 0.05). Titers are indicated on Y-axis and time points of infection are indicated on the X-axis. CVB = Common vampire bat (Desmodus rotundus ), PB = Pallid bat ( Antrozous pallidus ), Hs = Homo sapiens .

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Infection, Expressing, Comparison

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: Immunofluorescence microscopy of SC2 infected CVB-ACE2 cells. Confluent (75%) monolayers of CVB-ACE2 expressing DF-1 cells on iBID chamber slides were infected with WA1 ( A ), Delta ( B ), Lambda ( C ) or Omicron ( D ) variant of SC2 for 48 h and then stained for SC2 S protein and counterstained for nuclei using DAPI as stated in materials and methods. Scale bars at the bottom right represent 10× magnification.

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Immunofluorescence, Microscopy, Infection, Expressing, Variant Assay, Staining

Journal: Viruses

Article Title: The ACE2 Receptor from Common Vampire Bat ( Desmodus rotundus ) and Pallid Bat ( Antrozous pallidus ) Support Attachment and Limited Infection of SARS-CoV-2 Viruses in Cell Culture

doi: 10.3390/v17040507

Figure Lengend Snippet: Immunofluorescence microscopy of SC2 infected PB-ACE2 cells. Confluent (75%) monolayers of PB-ACE2 expressing DF-1 cells on iBID chamber slides were infected with WA1 ( A ), Delta ( B ), Lambda ( C ) or Omicron ( D ) variant of SC2 for 48 h and then stained for SC2 S protein and counterstained for nuclei using DAPI as stated in materials and methods. Scale bars at the bottom right represent 10× magnification.

Article Snippet: Primary antibodies included mouse anti-human ACE2 (1:1500 dilution) (Catalog # TA803844, Origene, Rockville, MD, USA), rabbit anti-human TMPRSS2 (1:1000), (Catalog # ab10913, AbCam, Cambridge, UK) and mouse anti-beta actin (1:2000) (Invitrogen, San Jose, CA, USA).

Techniques: Immunofluorescence, Microscopy, Infection, Expressing, Variant Assay, Staining

Journal: medRxiv

Article Title: The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera

doi: 10.1101/2021.01.19.21249592

Figure Lengend Snippet: (A) Strategy: Vero-E6 cells were transduced with a lentiviral vector expressing mACE-2 and a puromycin resistance gene. Cells were selected for mACE-2 expression by puromycin selection. (B) Expression in the selected polyclonal population was confirmed by Western blot.

Article Snippet: Anti-Tubulin and anti-mACE-2 (R&D Systems, Cat# MAB3437) primary antibodies were used at dilution of 1:1000 while secondary HRP-conjugated antibodies were used at dilutions of 1:10000 in 3% BSA-containing TBST.

Techniques: Transduction, Plasmid Preparation, Expressing, Selection, Western Blot