Screening for small molecule inhibitors of HIV-1 Gag expression Ahalya Balachandran, Alan Cochrane ⇑ Dept. of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada article info Article history: Received 17 March 2017 Received in revised form 8 May 2017 Accepted 5 June 2017 Available online xxxx Keywords: RNA splicing Small molecule modulators abstract The control of RNA processing plays an important role in the nature and quantity of protein generated from mammalian genes. Consequently, efforts to manipulate RNA processing have the capacity to signif- icantly impact gene function. Although multiple strategies have been developed to alter splice site selec- tion using oligonucleotide occlusion of splice sites or splicing regulatory elements, systemic delivery of such agents remains problematic. Outlined in this chapter is a protocol to screen for small molecule inhi- bitors of HIV-1 Gag expression that have been subsequently determined to modulate viral RNA process- ing. Identification and characterization of such RNA processing modulators offers the potential for the development of therapeutic lead compounds or probes for investigating the mechanism underlying the regulation of select RNA processing events. Ó 2017 Elsevier Inc. All rights reserved. 1. Introduction Sequencing of multiple genomes has revealed that gene number alone cannot account for the complexity of an organism. While transcription can determine when during development and in what tissue a gene is expressed, the segmented nature of genes means that RNA splicing can affect the nature of the protein gener- ated. Comparison of the transcriptomes of humans and worms has revealed that, although they have similar numbers of protein cod- ing genes (20,000), they vary greatly in how they manipulate the transcript. While alternative RNA splicing is used in only a minor- ity (25%) of instances in worms, >90% of human transcripts undergo alternative splicing with the potential to expand the num- ber and nature of the proteins generated [1] (however, whether this actually occurs has been contested [2]). Control of when and where the various spliced mRNA isoforms are produced required the evolution of additional layers of regulation that can be manip- ulated independent of transcription. While the general mechanism of RNA splicing is conserved from yeast to humans, the regulation of exon recognition and inclusion is not. Modulating the frequency of exon inclusion can involve multiple sequence components including inefficient splice sites (either splice donor (5 0 ss) or acceptor (3 0 ss)) sequences as well as sequences that inhibit or enhance splice site recognition [1]. Con- sequently, modulation of exon inclusion within a mRNA is depen- dent upon the abundance and/or activity of factors that recognize these elements [3–5]. In addition to expression level, there are multiple examples of splicing factor activity being regulated by covalent modification (i.e. phosphorylation) or localization that in turn is dependent upon various signaling networks [6–8]. This complexity of transcript processing is not limited to higher eukary- otes but has also been exploited by multiple viruses (influenza, adenovirus, HIV-1, human papilloma virus, Herpes) to expand their coding capacity [9–17]. As an example, an integrated HIV-1 pro- virus generates a single 9 kb transcript that is subsequently trans- formed through alternative splicing into >40 mRNAs [18–21]. Regulating the extent of HIV-1 RNA processing is essential to virus replication as mutation of select splicing regulatory sequences severely impairs viral protein expression and ultimately the quan- tity and quality of the virions generated [22–24]. Consequently, strategies to modulate HIV-1 RNA processing offer alternatives to existing drugs for the control of this infection. Multiple studies have demonstrated the feasibility of such an approach with the characterization of several small molecules that modulate RNA splicing or alter HIV-1 RNA accumulation and inhibit virus replica- tion (see Table 1) [25–32]. Current efforts are directed at under- standing how these compounds elicit their response in an effort to enhance their antiviral activity while minimizing their effect on host cell gene expression. Outlined in this report are the reagents and methods to screen for inhibitors of HIV-1 Gag expres- sion, several of which operate by altering various steps of HIV-1 RNA processing (splicing, polyadenylation, transport, translation), as well as the subsequent characterization of their effects on viral protein and RNA accumulation. While the methods outlined here http://dx.doi.org/10.1016/j.ymeth.2017.06.007 1046-2023/Ó 2017 Elsevier Inc. All rights reserved. ⇑ Corresponding author at: Dept. of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S1A8, Canada. E-mail address: alan.cochrane@utoronto.ca (A. Cochrane). Methods xxx (2017) xxx–xxx Contents lists available at ScienceDirect Methods journal homepage: www.elsevier.com/locate/ymeth Please cite this article in press as: A. Balachandran, A. Cochrane, Methods (2017), http://dx.doi.org/10.1016/j.ymeth.2017.06.007