Journal: Cell

Article Title: Therapeutic alphavirus cross-reactive E1 human antibodies inhibit viral egress.

doi: 10.1016/j.cell.2021.07.033

Figure Lengend Snippet: Figure 7. Model for mechanism of action of human E1-specific mAbs (A) Human E1-specific mAbs that target cryptic or partially exposed epitopes do not inhibit SINV/EEEV egress. Exposure for cryptic epitopes depends on pre- treatment conditions, such as acidic pH or addition of the nonionic detergent Tween 20. Treatment efficacy (EEEV-138 and EEEV-346) of EEEV infection following s.c. challenge is minimally significant due to low survival efficacy and presence of viral RNA levels in the serum of treated mice. (B) Human E1-specific mAbs that target exposed, pH-independent epitopes can inhibit SINV/EEEV egress. Quaternary epitopes between two adjacent E1 proteins of neighboring trimeric spikes may aid in binding to infected cells for inhibition of virus egress and enable broad alphavirus cross-reactivity (EEEV-179 [New World]). Treatment efficacy (EEEV-109 and -179) of EEEV infection following s.c. challenge corresponds with SINV/EEEV egress inhibition potency due to

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies EEEV-76 (hybridoma-produced IgG1) This paper N/A EEEV-104 (hybridoma-produced IgG1) This paper N/A EEEV-109 (hybridoma-produced IgG1) This paper N/A EEEV-126 (hybridoma-produced IgG1) This paper N/A EEEV-127 (hybridoma-produced IgG1) This paper N/A EEEV-138 (hybridoma-produced IgG1) This paper N/A EEEV-157 (hybridoma-produced IgG1) This paper N/A EEEV-179 (hybridoma-produced IgG1) This paper N/A EEEV-307 (hybridoma-produced IgG1) This paper N/A EEEV-312 (hybridoma-produced IgG1) This paper N/A EEEV-320 (hybridoma-produced IgG1) This paper N/A EEEV-342 (hybridoma-produced IgG1) This paper N/A EEEV-346 (hybridoma-produced IgG1) This paper N/A EEEV-354 (hybridoma-produced IgG1) This paper N/A EEEV-368 (hybridoma-produced IgG1) This paper N/A EEEV-377 (hybridoma-produced IgG1) This paper N/A EEEV-379 (hybridoma-produced IgG1) This paper N/A EEEV-387 (hybridoma-produced IgG1) This paper N/A EEEV-398 (hybridoma-produced IgG1) This paper N/A EEEV-400 (hybridoma-produced IgG1) This paper N/A rEEEV-97 IgG (recombinant Expi293F-produced IgG1) Williamson et al., 2020 N/A rEEEV-109 IgG (recombinant Expi293F-produced IgG1) This paper N/A rEEEV-109 LALA-PG (recombinant Expi293F-produced IgG1) This paper N/A rEEEV-126 IgG (recombinant Expi293F-produced IgG1) This paper N/A rEEEV-157 IgG (recombinant Expi293F-produced IgG1) This paper N/A rEEEV-346 IgG (recombinant Expi293F-produced IgG1) This paper N/A rDENV-2D22 IgG (recombinant ExpiCHO-produced IgG1) Fibriansah et al., 2015 N/A Murine mAb: EEEV-66 Michael S. Diamond Kim et al., 2019 Eastern equine encephalomyelitis immune ascites fluid ATCC Cat# VR-1242AF Venezuelan equine encephalitis virus hyperimmune ascitic fluid ATCC Cat# VR-1249AF Western equine encephalomyelitis virus immune ascitic fluid ATCC Cat# VR-1251AF Chikungunya virus immune ascitic fluid ATCC Cat# VR-1241AF Mayaro virus immune ascitic fluid ATCC Cat# VR-1277AF 1A4B-6 Millipore Cat# MAB8754; RRID:AB_11211668 1A3B-7 Millipore Cat# MAB8755; RRID:AB_95409 2A3D-5 Millipore Cat#MAB8746; RRID:AB_95399 (Continued on next page) Cell 184, 4430–4446.e1–e12, August 19, 2021 e1

Techniques: Infection, Binding Assay, Inhibition, Virus

Journal: RNA

Article Title: Noncanonical cytoplasmic processing of viral microRNAs

doi: 10.1261/rna.2303610

Figure Lengend Snippet: (A) Schematic representation of Sindbis viral products. 5′ and 3′ ends of mRNA and negative-strand genome products are depicted as are NH2 (N) and COOH (C) terminals of polyproteins. The noncoding region (NCR) represents the extra subgenomic insertion site where the pri-miRNA transcript was incorporated. The nonstructural genes (nsP1-4) are translated into a large polyprotein that forms four unique nonstructural proteins. The complementary minus strand [(−) Genome] is used as a template for the genomic RNA along with both the subgenomic mRNA and the extra subgenomic NCR depicted. The endogenous subgenomic message is translated into a second polyprotein that is processed into the C, E3, E2, 6K, and E1 proteins. (B) Human fibroblasts mock-treated, transfected with miR-124 producing plasmid (p124), or infected with SV or SV124 (MOI of 5) and harvested at the indicated hours post-infection (hpi). (Upper two frames) Northern blots probed for miR-124 (top) and U6 (bottom). (Lower two frames) Immunoblots depicting Sindbis virus core protein and actin. (C) Confocal microscopy of cells mock-treated or infected with SV or SV124 (MOI of 2). Cells stained for Sindbis virus core protein (green) and cell nuclei (blue). Scale bar, 10 μm.

Article Snippet: The polyclonal Sindbis antibody was purchased from ATCC as Sindbis Ascitic Fluid (VR-1248AF).

Techniques: Transfection, Plasmid Preparation, Infection, Northern Blot, Western Blot, Virus, Confocal Microscopy, Staining

Journal: RNA

Article Title: Noncanonical cytoplasmic processing of viral microRNAs

doi: 10.1261/rna.2303610

Figure Lengend Snippet: (A) Murine embryonic fibroblasts derived from wild-type (WT) or Dicer-deficient (Dcr1−/−) mice mock-treated or infected with SV or SV124 for 24 h (MOI of 1). (Top three panels) Northern blots probed for miR-124 (top), miR-93 (middle), and U6 (bottom). (Bottom two panels) Western blots for Sindbis virus core protein and actin. (B) Human fibroblasts transfected with scrambled short interfering RNAs (Scbl siRNA) or siRNAs directed against Exportin-5 (Xpo5 siRNA). Forty-eight hours post-transfection, cells were mock-treated or infected with SV or SV124 for 24 h (MOI of 1). (Top two panels) Northern blot probed for miR-124 (top) and U6 (below). (Bottom three panels) Immunoblots for Exportin-5, Sindbis core, and actin. (C) Sequence analysis of Sindbis-derived miR-124. The pre-miR-124 sequence is depicted at the top, with the mature miR-124 sequence in red and the predicted secondary structure below. The number of reads corresponding to each RNA species is indicated.

Article Snippet: The polyclonal Sindbis antibody was purchased from ATCC as Sindbis Ascitic Fluid (VR-1248AF).

Techniques: Derivative Assay, Infection, Northern Blot, Western Blot, Virus, Transfection, Sequencing

Journal: RNA

Article Title: Noncanonical cytoplasmic processing of viral microRNAs

doi: 10.1261/rna.2303610

Figure Lengend Snippet: (A) Murine embryonic fibroblasts derived from wild-type (WT), Dicer-deficient (Dcr1−/−), DGCR8-deficient (Dgcr8−/−), or IFN-I-deficient (Ifnar1−/−) mice were mock-treated or infected with SV or SV124 for 24 h (MOI of 2). (Top three panels) Northern blots probed for miR-124, miR-93, and U6. (Bottom two panels) Western blots for Sindbis virus core protein and actin. (B) Human fibroblasts transfected with a miR-124-targeted GFP plasmid (GFP_miR-124t) were additionally transfected with an miR-124-producing plasmid (p124) or infected with SV or SV124 for 24 h (MOI of 2). (Top three panels) Western blots for green fluorescent protein (GFP), Sindbis virus core protein, and actin. (Bottom three panels) Northern blots probed for miR-124, miR-93, and U6.

Article Snippet: The polyclonal Sindbis antibody was purchased from ATCC as Sindbis Ascitic Fluid (VR-1248AF).

Techniques: Derivative Assay, Infection, Northern Blot, Western Blot, Virus, Transfection, Plasmid Preparation

Journal: RNA

Article Title: Noncanonical cytoplasmic processing of viral microRNAs

doi: 10.1261/rna.2303610

Figure Lengend Snippet: (A) Multicycle growth curve of SV and SV124 performed in wild-type murine fibroblasts (WT), or fibroblasts lacking either Dicer (Dcr1−/−) or a functional IFN-I receptor (Ifnar1−/−). Cells were infected at an MOI of 0.1 and plaqued at the indicated time points. P-values of the difference between SV and SV124 replication levels in WT, Dcr1−/−, and Ifnar1−/− at 48 hpi are 0.008, 0.164, and 0.015, respectively. (B) Human fibroblasts were mock-treated or transfected with vector or miR-124-producing plasmid (p124). Twenty-four hours post-transfection, cells were infected with SV or SV124 (MOI of 2) and harvested 24 hpi. (Top two panels) Western blots for Sindbis virus core protein and actin. (Bottom three panels) Northern blots probed for miR-124, miR-93, and U6. (C) Schematic of miR-124 targeting of the SV124 genome (top) or the SV124 negative-strand genome.

Article Snippet: The polyclonal Sindbis antibody was purchased from ATCC as Sindbis Ascitic Fluid (VR-1248AF).

Techniques: Functional Assay, Infection, Transfection, Plasmid Preparation, Western Blot, Virus, Northern Blot

Journal:

Article Title: Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

doi: 10.1128/JVI.77.11.6450-6465.2003

Figure Lengend Snippet: Comparison of large-plaque-forming JEV isolate CNU/LP2 and original K87P39 strain. (A) Plaque morphology. BHK-21 cells were mock infected or infected with the original JEV K87P39 strain, which formed a heterogeneous mixture of viral plaque sizes. The CNU/LP2 isolate purified in this study formed a homogeneous population of large plaques (CNU/LP2-infected). (B) Levels and patterns of JEV protein expression. Naïve BHK-21 cells were mock infected or infected with K87P39, CNU/LP2, or the yellow fever virus strain YF17D; 18 h later they were fixed and stained with JEV-specific mouse hyperimmune ascites followed by fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (green fluorescence) and confocal microscopy. Nuclei were visualized by staining with propidium iodide (red fluorescence) in the presence of RNase A.

Article Snippet: After three washes with washing solution, membranes were incubated at room temperature for 2 h with either a monoclonal antiactin antibody (A4700) that recognizes the epitope conserved in the C terminus of all actin isoforms (Sigma, St. Louis, Mo.) or mouse hyperimmune ascites fluid specific for JEV (ATCC VR-1259AF; American Type Culture Collection).

Techniques: Infection, Purification, Expressing, Staining, Fluorescence, Confocal Microscopy

Journal:

Article Title: Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

doi: 10.1128/JVI.77.11.6450-6465.2003

Figure Lengend Snippet: Comparison of synthetic JEVs with parental virus CNU/LP2. (A) Representative plaque assays of synthetic JEVs and parent CNU/LP2. BHK-21 cells were infected with parent or synthetic viruses, overlaid with agarose, and stained 3 days later with crystal violet. (B) Growth kinetics in BHK-21 cells of synthetic JEVs and parent CNU/LP2 infected at multiplicities of infection of 0.01, 1, and 10. Viruses were harvested at the hour postinfection (h.p.i.) indicated, and titers were determined by plaque assays. The data shown represent one of two independent experiments yielding similar results. (C and D) Viral protein and RNA levels were analyzed by immunoblotting (C) and Northern blotting (D), respectively. BHK-21 cells were infected at a multiplicity of infection of 1 with synthetic JEVs (lanes 1 to 4) or CNU/LP2 (lane 5) or mock-infected (lane 6) and cultured for 18 h. (C) Protein extracts were prepared from approximately 3 × 104 cells and separated on 10% SDS-polyacrylamide gels. Viral proteins were visualized by immunoblotting with JEV-specific mouse hyperimmune ascites (top panel). In parallel, actin protein was detected as a loading and transfer control (bottom panel). The positions of viral protein-related cleavage intermediates and actin are indicated with arrowheads on the left. Molecular mass markers (in kilodaltons) are indicated on the right. (D) Total RNA from approximately 105 cells was extracted and analyzed by Northern blotting with a 32P-labeled antisense riboprobe hybridizing to the sequence in the NS5 gene encompassing nt 9143 to 9351 (top panel). Ethidium bromide-stained 18S rRNA bands are shown as a loading control (bottom panel). The positions of full-length genomic viral RNA (11 kb) and 18S rRNA are indicated on the left.

Article Snippet: After three washes with washing solution, membranes were incubated at room temperature for 2 h with either a monoclonal antiactin antibody (A4700) that recognizes the epitope conserved in the C terminus of all actin isoforms (Sigma, St. Louis, Mo.) or mouse hyperimmune ascites fluid specific for JEV (ATCC VR-1259AF; American Type Culture Collection).

Techniques: Infection, Staining, Western Blot, Northern Blot, Cell Culture, Labeling, Sequencing