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Synthetic Virology: Building Viruses to Better Understand Them

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Synthetic Virology: Building Viruses to Better Understand Them Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Additional Perspectives articles for Influenza: The Cutting Edge book collection are available at http://perspectivesinmedicine.cshlp.org/cgi/collection/influenza_the_cutting_edge. Synthetic Virology: Building Viruses to Better Understand Them Benjamin R. tenOever Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA Correspondence: [email protected] Generally comprised of less than a dozen components, RNA viruses can be viewed as well-designed genetic circuits optimized to replicate and spread within a given host. Understanding the molecular design that enables this activity not only allows one to disrupt these circuits to study their biology, but it provides a reprogramming framework to achieve novel outputs. Recent advances have enabled a “learning by building” approach to better understand virus biology and create valuable tools. Below is a summary of how modifying the preexisting genetic framework of influenza A virus has been used to track viral movement, understand virus replication, and identify host factors that engage this viral circuitry. nfluenza virus biology demands cellular entry exposes NP and enables nuclear import in Iand successfully launching of an RNA-based which each RNP is “launched” by a bound viral circuit to generate the necessary components for RNA-dependent RNA polymerase (RdRp). The nuclear import, transcription, replication, nu- RdRp, composed of three viral gene products clear export, and ultimate egress. Influenza virus (PA, PB1, and PB2), is responsible for generat- circuitry is maintained as a ribonucleoprotein ing 10 major viral products shared by all influ- (RNP) complex, comprised of the nucleoprotein enza A virus (IAV) subtypes. Apart from HA, (NP) and viral polymerases bound to eight in- M1, M2, NP, PA, PB1, and PB2, the RdRp also dependent RNA segments of negative polarity generates transcripts that encode proteins re- and packaged into a host-derived lipid mem- sponsible for RNP export (nuclear export pro- brane decorated by viral glycoproteins (Bouvier tein [NEP]) and egress (neuraminidase [NA]). www.perspectivesinmedicine.org and Palese 2008). Cellular entry of the eight The 10th transcript generated by IAV encodes RNPs is mediated by a trimeric glycoprotein the nonstructural 1 protein (NS1), which is not (hemagglutinin or HA), which triggers endocy- thought to contribute to the virus circuit, but tosis and fusion within the endosome (Bouvier rather is a product dedicated to protecting it and Palese 2008). During this process, a small from unwanted host interference (Ayllon and ion channel (formed by the Matrix-2 or M2 García-Sastre 2015). In addition to these 10 ma- protein) enables acidification of the virion and jor products shared among all influenza Avirus- the dissociation of the RNPs from Matrix-1 es, additional strain-specific gene products have (M1), a viral protein required for RNP encapsi- also been described that, like NS1, generally dation and nuclear export. Dissociation of M1 function to shut down antiviral host defenses Editors: Gabriele Neumann and Yoshihiro Kawaoka Additional Perspectives on Influenza: The Cutting Edge available at www.perspectivesinmedicine.org Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a038703 Cite this article as Cold Spring Harb Perspect Med 2020;10:a038703 1 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press B.R. tenOever 1. Entry (HA,M2) 3. Transcription (NP, RdRp) 2. Nuclear import (NP) 4. Switch (NEP) 6. Nuclear export (NEP, M1) 7. Egress (HA, NA) 5. Replication (NP, RdRp) Figure 1. The biological circuitry of influenza A virus. This conceptual schematic depicts each major event in the life cycle of IAV and denotes the proteins responsible. (Vasin et al. 2014). Collectively these proteins virus biology. For IAV, it was the minireplicon are responsible for multiple outputs that func- systems that enabled one to deduce the neces- tion in concert to achieve a biological circuit sary RNA sequences required for RdRp recog- designed for amplification and spread (Fig. 1). nition, amplification, and packaging (Hsu et al. This understanding of IAV biology has been 1987; Luytjes et al. 1989; Enami et al. 1990). made possible largely through modification of Informed by this work, reverse genetic systems this circuit and its individual modules through soon followed ushering in a new era of virus a process of genetic engineering. engineering (Neumann et al. 2002). The capac- The ability to reprogram viruses began with ity to generate IAV entirely from DNA has en- the discovery that one could launch a replica- abled us to probe the virus at single-nucleotide tion-competent virus from a DNA-based plas- resolution to deduce the circuitry involved at mid—a method now commonly referred to as both the RNA and protein level. Moreover, our reverse genetics. The first reverse genetic sys- understanding of IAV biology has also allowed tems established were for positive-strand RNA us to reprogram the virus to run additional mod- www.perspectivesinmedicine.org viruses, as these generally launch independently ules from the central IAV program concurrent of any other viral component (Taniguchi et al. with the infection. This latter technology has 1978; Racaniello and Baltimore 1981). In con- enabled significant advancements toward our trast, because negative-strand RNA viruses understanding of virus replication, transmis- require an RNA with precise termini bound sion, and pathogenesis. These findings and the properly to NP in the presence of the cognate designs responsible for their discovery are sum- RdRp, reverse genetic systems for this order of marized below. viruses were significantly more complicated. As a result, early attempts at recapitulating aspects DEFINING IAV CIRCUITRY of negative-strand RNA virus replication began as minireplicon systems, DNA-based platforms The successful generation of IAV-based rescue that use small genomic mimics with NP to study systems was essential in elucidating the relative RdRp biology (Neumann et al. 2002). The gen- importance of each base or residue with very eration of these early systems to build virus-like high resolution. In fact, the laboratories respon- circuits laid the groundwork for all of our sible for pioneering the minireplicon systems, understanding regarding negative-strand RNA and then later the reverse genetic systems, were 2 Cite this article as Cold Spring Harb Perspect Med 2020;10:a038703 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Synthetic Virology also the ones who initially applied this new tool orated through more current methodologies to better understand the biology of IAV RNA (Dadonaite et al. 2019; Majarian et al. 2019). (Fodor et al. 1999; Neumann et al. 1999). This knowledge of packaging requirements en- Some of the first studies that applied this new abled one to build viral recombinants expressing methodology focused on the capacity to gener- a foreign segment and complement the missing ate foreign RNAs that would be packaged by the viral protein using stably expressing cell lines virus (Luytjes et al. 1989; Fujii et al. 2003). These (Shinya et al. 2004; Martínez-Sobrido et al. studies were the first to show that the packaging 2010; Ozawa et al. 2011). Furthermore, the fact signals for each segment extended beyond the thateachsegmentcan bedefinedbyitspackaging promoter on the termini of each segment. The sequence allows one to swap this material be- capacity to introduce modified segments using tween open reading frames and build a virus reverse genetics was quickly used to define the that is incapable of reassortment with any IAV minimal packaging material required for each of found in nature (Gao and Palese 2009). the eight segments (Duhaut and Dimmock Use of reverse genetics to probe for RNA 2000; Fujii et al. 2005; Liang et al. 2005; Marsh packaging signals was quickly followed by stud- et al. 2007, 2008; Hutchinson et al. 2008; Essere ies aimed at defining the tolerance for modifica- et al. 2013; Gavazzi et al. 2013). This work re- tion in IAV proteins. Through some form of vealed that each viral RNA consists of one or mutagenesis on a segment of interest, one can more open reading frames that are flanked by perform DNA-based rescue en masse using the segment-specific untranslated regions and con- heterogeneous population of DNA to ascertain served, partially complementary 50 and 30 pro- which mutants result in replication-competent moterelements.Much ofthenoncodingmaterial virus. The first study to perform genome-wide folds to generate a promoter element necessary insertional mutagenesis of IAV used the bac- for RdRp recognition or generation of a poly(A) teriophage Mu transposase to determine the tail, but it is insufficient for packaging. Instead, relative tolerance of each of the IAV proteins packaging sequences have been found to extend (Heaton et al. 2013b). In brief, a mouse-adapted into the open reading frame and the length of laboratory strain of IAV was subjected to a trans- material required appears to be unique for each posable element which, when removed, left a segment (Fig.2).Thisworkhasledtotheconcept 15-nt insertion at the site of integration. Virus that IAV packaging is mediated by RNA:RNA rescue was performed using seven wild-type interactions, a hypothesis that has been corrob- genomes alongside the mutant gene pool of the www.perspectivesinmedicine.org A IAV -ve strand RNA segment B Transgenic segments 1–3 Open reading frame GOI C Segment 8 RdRp promoter elements/noncoding NS1 NEP Packaging sequence lntergenic segment 8 Open reading frame NS1 NEP Figure 2. Engineering framework for influenza A virus. (A) Schematic of the viral RNA (vRNA) segments depicting the RdRp promoter elements flanking the open reading frame with overlapping packaging sequences. (B) Schematic of transgenic designs in which segments 1–3 are the most tolerable for expression of a gene of interest (GOI).
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