Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs

Voraphoj Nilaratanakul,Debra A Hauer,Diane E Griffin

doi:10.1038/s41598-020-61573-3

Abstract

Sindbis virus (SINV) can infect neurons and cause encephalomyelitis in mice. Nonstructural proteins are translated from genomic RNA and structural proteins from subgenomic RNA. While visualization of viral proteins in living cells is well developed, imaging of viral RNAs has been challenging. RNA aptamers that bind and activate conditional fluorophores provide a tool for RNA visualization. We incorporated cassettes of two F30-scaffolded dimers of the Broccoli aptamer into a SINV cDNA clone using sites in nsP3 (genomic RNA), the 3′UTR (genomic and subgenomic RNAs) and after a second subgenomic promoter resulting in 4–28 Broccoli copies. After addition of the cell-permeable 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI-1T) conditional fluorophore and laser excitation, infected cells emitted green fluorescence that correlated with Broccoli copy numbers. All recombinant viruses replicated well in BHK and undifferentiated neural cells but viruses with 14 or more Broccoli copies were attenuated in differentiated neurons and mice. The signal survived fixation and allowed visualization of viral RNAs in differentiated neurons and mouse brain, as well as BHK cells. Subgenomic RNA was diffusely distributed in the cytoplasm with genomic RNA also in perinuclear vesicle-like structures near envelope glycoproteins or mitochondria. Broccoli aptamer-tagging provides a valuable tool for live cell imaging of viral RNA.

Highlights

Sindbis virus (SINV), an alphavirus in the Togaviridae family, is an enveloped plus-strand RNA virus that can cause seasonal outbreaks of rash and arthritis in humans and encephalomyelitis in experimentally infected mice[1,2,3]
The RNA aptamer Spinach[2] within a tRNA scaffold was incorporated into a cDNA clone of the TE strain of SINV to produce recombinant viruses that expressed viral RNA tagged with the aptamer[17]
To construct TE strain SINV cDNAs with Broccoli insertions, plasmids with either full-length TE22 or TE engineered with a second subgenomic promoter (TEds)[23] were used

Summary

Introduction

Sindbis virus (SINV), an alphavirus in the Togaviridae family, is an enveloped plus-strand RNA virus that can cause seasonal outbreaks of rash and arthritis in humans and encephalomyelitis in experimentally infected mice[1,2,3]. Spinach[2] tagging allowed visualization of SINV RNA in live cells as green fluorescence after addition of the non-toxic cell-permeable conditional fluorophore DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one) and excitation at 488 nm[17]. To determine the applicability of this system to analysis of viral RNA dynamics, we constructed and characterized SINVs with different F30-2xdBroccoli numbers and insertion sites These recombinant viruses were used for analysis of RNA synthesis and distribution after infection of neural and non-neural cells

Methods

Results

Conclusion