Detection of human immunodeficiency virus RNAs in living cells using Spinach RNA aptamers

• Published in: Virus research Vol. 228; pp. 141 - 146
• Main Authors: Burch, Brandon D., Garrido, Carolina, Margolis, David M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.01.2017
• Subjects: Aptamers; Aptamers, Nucleotide; Cell Line; Cells, Cultured; HIV - genetics; HIV Infections - diagnosis; HIV Infections - virology; HIV-RNA; Humans; Lentivirus; Microscopy, Fluorescence - methods; Retroviridae; RNA, Plant; RNA, Viral - genetics; SELEX Aptamer Technique; Spectrometry, Fluorescence - methods; Spinach; Spinacia oleracea; Spinacia oleracea - genetics; Virion; Virus Replication; HIV-RNA; Aptamers; Spinach
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2016.11.031

•Limited techniques allow in vivo RNA expression to be imaged in real time.•Better understanding the kinetics of transcriptional regulation and expression in vivo is an unmet need.•We present a novel technique that allows the real-time, single cell, visualization of HIV RNA expression in vivo in unmanipulated, living cells. Many techniques currently used to measure HIV RNA production in cells suffer from limitations that include high background signal or the potential to destroy cellular context. Fluorophore-binding RNA aptamers offer the potential for visualizing RNAs directly in living cells with minimal cellular perturbation. We inserted a sequence encoding a fluorophore-binding RNA aptamer, known as Spinach, into the HIV genome such that predicted RNA secondary structures in both Spinach and HIV were preserved. Chimeric HIV-Spinach RNAs were functionally validated in vitro by testing their ability to enhance the fluorescence of a conditional fluorophore (DFHBI), which specifically binds Spinach. Fluorescence microscopy and PCR were used to verify expression of HIV-Spinach RNAs in human cells. HIV-1 gag RNA production and fluorescence were measured by qPCR and fluorometry, respectively. HIV-Spinach RNAs were fluorometrically detectable in vitro and were transcribed in human cell lines and primary cells, with both spliced and unspliced species detected by PCR. HIV-Spinach RNAs were visible by fluorescence microscopy in living cells, although signal was reproducibly weak. Cells expressing HIV-Spinach RNAs were capable of producing fluorometrically detectable virions, although detection of single viral particles was not possible. In summary, we have investigated a novel method for detecting HIV RNAs in living cells using the Spinach RNA aptamer. Despite the limitations of the present aptamer/fluorophore combination, this is the first application of this technology to an infectious disease and provides a foundation for future research into improved methods for studying HIV expression.