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