small RNA methods
Recorded terms: luciferase 3-prime UTR reporter assay; luciferase reporter silencing assay; reporter silencing assay; reporter-based post-transcriptional silencing assay
SARS-CoV generates three discrete small viral RNAs during infection of mouse lung, and blocking the one derived from the nucleocapsid gene with a locked nucleic acid antagomir reduces pulmonary inflammation, tissue damage and proinflammatory cytokine expression without measurably lowering lung virus titres.
Poxviruses degrade host microRNAs through the catalytic subunit of their own poly(A) polymerase, VP55, which adds short nontemplated adenosine tails to argonaute-loaded guide strands and thereby marks them for cellular decay, while small RNAs carrying a 3 prime terminal 2 prime O-methyl group are spared.
Primary microRNA transcripts generated in the cytoplasm by a recombinant Sindbis virus are cleaved without any nuclear involvement yet still require Drosha, which relocalises from nucleus to cytoplasm on infection while the endogenous microRNA profile of the cell remains largely unchanged.
Insertion of a primary microRNA locus into the exclusively cytoplasmic Sindbis virus genome yields mature, functional miR-124 through a Dicer-dependent but microprocessor- and Exportin-5-independent route, defining a cytoplasmic hairpin-processing activity in vertebrate cells that the authors term a virtron.