ADVISORY BOARD DAVID BALTIMORE SHOUWEI DING PETER C DOHERTY JOHN FAZAKERLY HANS J GROSS BRYAN D HARRISON ROGER HENDRIX KARLA KIRKEGAARD BERNARD MOSS ERLING NORRBY JULIE OVERBAUGH PETER PALUKAITIS FELIX REY JUERGEN RICHT MARILYN ROOSSINCK JOHN J SKEHEL GEOFFREY SMITH MARC H.V VAN REGENMORTEL VERONIKA VON MESSLING Academic Press is an imprint of Elsevier 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK 125 London Wall, London, EC2Y 5AS, UK First edition 2016 © 2016 Elsevier Inc All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein) Notices Knowledge and best practice in this field are constantly changing As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein ISBN: 978-0-12-804821-4 ISSN: 0065-3527 For information on all Academic Press publications visit our website at store.elsevier.com CONTRIBUTORS David C Bloom Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA Abraham L Brass Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA Teresa Hellberg Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany Barbara G Klupp Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany Paul Meraner Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA Thomas C Mettenleiter Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany Lars Paßvogel Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany Jean-Pierre Perreault De´partement de biochimie, Faculte´ de me´decine et des sciences de la sante´, Pavillon de recherche appliquee´ sur le cancer, Universite´ de Sherbrooke, Que´bec, Canada Jill M Perreira Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA Katharina S Schulz Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany Gerhard Steger Institut fuăr Physikalische Biologie, Heinrich-Heine-Universitaăt Duăsseldorf, Duăsseldorf, Germany vii CHAPTER ONE Functional Genomic Strategies for Elucidating Human–Virus Interactions: Will CRISPR Knockout RNAi and Haploid Cells? Jill M Perreira, Paul Meraner, Abraham L Brass1 Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA Corresponding author: e-mail address: abraham.brass@umassmed.edu Contents Introduction Host–Virus Genetic Screens RNAi Genetic Screening Technologies and Approaches 3.1 RNAi Pooled Screening 3.2 Arrayed RNAi Screening 3.3 RNAi Screening Problems and Some Solutions Haploid Cell Genetic Screening Technology and Approach CRISPR/Cas9 Genetic Screening Technologies and Approaches Comparison of HRV-HF Screens: Arrayed MORR RNAi Versus Pooled CRISPR/Cas9 Future Directions Acknowledgments References 2 21 23 28 31 32 34 44 45 45 Abstract Over the last several years a wealth of transformative human–virus interaction discoveries have been produced using loss-of-function functional genomics These insights have greatly expanded our understanding of how human pathogenic viruses exploit our cells to replicate Two technologies have been at the forefront of this genetic revolution, RNA interference (RNAi) and random retroviral insertional mutagenesis using haploid cell lines (haploid cell screening), with the former technology largely predominating Now the cutting edge gene editing of the CRISPR/Cas9 system has also been harnessed for large-scale functional genomics and is poised to possibly displace these earlier methods Here we compare and contrast these three screening approaches for elucidating host–virus interactions, outline their key strengths and weaknesses including a comparison of an arrayed multiple orthologous RNAi reagent screen to a pooled CRISPR/Cas9 human rhinovirus 14–human cell interaction screen, and recount some notable insights made possible by each We conclude with a brief perspective on what might lie ahead for the fast evolving field of human–virus functional genomics Advances in Virus Research, Volume 94 ISSN 0065-3527 http://dx.doi.org/10.1016/bs.aivir.2015.11.001 # 2016 Elsevier Inc All rights reserved Jill M Perreira et al INTRODUCTION The burden imposed upon the health of the world’s population by just three of the major pathogenic viruses is staggering, with nearly 300 million people chronically infected by either HIV-1 (36 million) or HBV (250 million), and another 5–6 million severe infections by influenza A virus (IAV) occurring transiently each year (Ortblad, Lozano, & Murray, 2013; Schweitzer, Horn, Mikolajczyk, Krause, & Ott, 2015) (http://www.who int/immunization/topics/influenza/en/) Collectively these three viruses cause the deaths of over 2.5 million people annually These infections arise because viruses must find and exploit the host’s cellular resources and machinery to produce their progeny Elucidating human pathogenic viral dependencies has been a longstanding pursuit of health science researchers whose goal is to use this knowledge to treat and cure infections For decades, mammalian in vitro tissue culture systems have proved tremendously useful for studying host–virus interactions Over this same period, loss-of-function genetic screening produced an impressive number of discoveries and illuminated gene and pathway function in multiple model systems While loss-offunction genetic screening proved extremely valuable in model systems, such technologies did not exist for mammalian cells until the discovery and implementation of RNA interference (RNAi) (Fire et al., 1998) The initial technologic revolution of RNAi, and later the development of haploid cell screening, resulted in a wave of discoveries that shed new light on many vital human viral requirements (Brass et al., 2008; Hao et al., 2008; Krishnan et al., 2008; Randall et al., 2007; Sessions et al., 2009) The ascendance of CRISPR/Cas9 technologies, which can dramatically alter gene expression, has heralded a new era in mammalian in vitro genetic screening (Shalem, Sanjana, & Zhang, 2015) This review will discuss the available functional genomics strategies, highlight their strengths and weaknesses including a comparison of matched MORR RNAi and CRISRP/Cas9 screens, and provide some future perspectives on the use of mammalian in vitro genetics to elucidate human host–virus interactions HOST–VIRUS GENETIC SCREENS The numbers of host–virus functional genomic screens using these technologies, particularly RNAi, have been increasing rapidly attesting to Functional Genomic Strategies for Elucidating Human–Virus Interactions their innovative discovery power, generalizability and remarkable ease of use (Table 1) Drosophila cell in vitro RNAi screens were the first to detect novel host factor interactions for several human pathogens with the practical focus being on arboviruses, although an elegant approach using a recombinant virus also made it possible to screen for IAV dependency factors in this system (Arkov, Rosenbaum, Christiansen, Jonsson, & Munchow, 2008; Cherry et al., 2005; Hao et al., 2008) RNAi screens using human cells have now been done for the majority of major human pathogenic viruses (Table 1); these efforts have largely used arrayed siRNA libraries combined with high-throughput imaging or plate reader-based assays as readouts for viral replication Collectively these works have identified multiple previously unappreciated dependencies for each virus, as well as host cell defense mechanisms Recent publications covering viruses that have been functionally interrogated by multiple independent groups including HIV-1, IAV, and HCV have been discussed elsewhere in detail (Bushman et al., 2009; Hao et al., 2013; Stertz & Shaw, 2011; Zhu et al., 2014) In this work, we focus on the functional genomic screening technologies and provide a resource noting many of the published host–virus screens along with some of their key attributes RNAi GENETIC SCREENING TECHNOLOGIES AND APPROACHES Nearing a decade ago the Nobel Prize winning discovery of RNAi in C elegans and its mercurial extension into mammalian systems provided virologists and geneticists alike with a powerful new tool for detecting viral dependencies (Elbashir et al., 2001; Fire et al., 1998; Grishok & Mello, 2002) Academia and industry both quickly embraced RNAi and paired it with the contemporaneous completion of the genetic annotation of the entire human genome to create multiple large-scale libraries for functional genomic screening (Paddison et al., 2004; Root, Hacohen, Hahn, Lander, & Sabatini, 2006; Silva et al., 2005) Because the RNA-induced silencing complex (RISC) machinery’s expression is ubiquitous, virtually all mammalian cell lines can carry out RNAi, permitting host–virus screens to be carried out with any tropic cell line and virus pairing (Elbashir et al., 2001) Two major types of RNAi libraries, pooled and arrayed, have been constructed and dictate the two methods of screening discussed below Table Functional Genomic Screens for Elucidating Host–Viral Interactions Haploid cells Knockdown/ Challenge Out Time Time Viral Dependency Factors Viral Dependency Factor Selection Criteria Viral Competitive or Restriction Factors Viral Competitive or Restriction factors Selection Main Criteria Candidates Stage of Viral Lifecycle Impacted Candidate Validation and Follow up Assays RT-PCR; immunofluorescence; complementation with cDNAs Citation Virus Cell Line Pooled/ Arrayed Library Carette et al (2009) Influenza virus (PR/8/34; H1N1) Haploid human suspension cells KBM-7 N/A Pooled Haploid cell Insertional mutagenesis with lentiviral exon trap 2–3 weeks Survival Yes Multiple independent integrations No N/A CMAS; SLC35A2 Entry Carette et al (2011) Pooled Haploid cell N/A rVSV-GP-Ebola Haploid human Insertional virus adherent cells mutagenesis (HAP1) with lentiviral exon trap Unknown Survival Yes Multiple independent integrations No N/A NPC1, HOPS complex Entry, viral fusion Complementation with in lysosomal cDNAs; test against compartment related viruses; smallmolecule U1866A and imipramine; immunofluorescence/ electron microscopy viral entry assays; primary cell lines Jae et al (2013) rVSV-GP-Lassa virus HAP1 Pooled Haploid cell Gene-Trap Unknown Survival Insertional mutagenesis with lentiviral exon trap Yes Multiple independent integrations No N/A TMEM5; B3GALNT2; B3GNT1; SLC35A1; SGK196 Entry, presentation of laminin-binding carbohydrate Null alleles TALENs; rescue cDNAs; analysis of know polymorphisms; flow cytometry; RT-PCR; clinical comparison Kleinfelter et al (2015) rVSV-Andes virus-GP HAP1 Pooled Haploid cell N/A Insertional mutagenesis with lentiviral exon trap Yes Multiple independent integrations No N/A S1P; S2P; SREBF2; SCAP; LSS; SQLE; ACAT2 Entry S1P CRISPR/Cas9 gene editing in U2OS; complementation with cDNA; small-molecule inhibitor days Readout Survival siRNA Haploid Petersen et al cell and (2014) siRNA Brass et al (2008) Hao et al (2008) HAP1 rVSV-Andes virus, either recombinant or pseudoparticles expressing Renilla luciferase HIV-1-IIIB Pooled Haploid cell N/A Insertional mutagenesis with lentiviral exon trap weeks Survival HEK29 72 h Arrayed Ambion druggable genome library (9102 genes) (4 siRNAs/ gene) (2 siRNAs/ well) 24 h Renilla luciferase Yes expression 210 dsRNAs; 112 genes reconfirmed In both pools: Z score for infection 2.4 SDs; Viability reduction Z score >-3 Yes Influenza A virus DL1 Flu-VSVG-GFP Arrayed Ambion 48 h Drosophila RNAi library (13,071 genes) 24 h Yes Renilla luciferase Yes activity 176 candidate genes—110 confirmed Multiple independent integrations No 123 candidate genes—11 genes confirmed N/A N/A Increase >3 SDs; viability reduction Z score >À3 SCAP; S1P; Entry S2P; SREBF2 Functionally deficient cells S1P, S2P, or SCAP null CHO and SREBP2 KD HEK293T; TALEN-mediated gene disruption; smallmolecule PF-429242 and mevastatin SREBF2 Entry additional unique siRNAs screened with ANDV and VSV-G pseudoparticles; validated by siRNA repeating finding two times 105 candidate genes—33 validated—9 specific for ANDV Subcellular localization; gene ontology (GO) biological processes analysis; Expression Genomic Institute of the Novartis Research Fund (GNF); individual shRNAs; individual siRNAs; infection with VSV-g; other cell lines Jurkat; qPCR RAB6A Fusion TNPO3 Cytosolic postRT–pre integration MED28 Transcription COX6A1 PB2/ RT-PCR; reagent PB1-F2-mediated redundancy; test human functions homologues, knockdown in HEK293 Fusion cells; individual siRNAs; small-molecule RNA export inhibitors; related pathway viruses: WSN, H5N1 Influenza A/Indonesia/ 7/05, VSV, VACV ATP6V0D1 NXF1 Continued Table Functional Genomic Screens for Elucidating Host–Viral Interactions—cont'd Citation Virus Cell Line Krishnan et al (2008) West Nile virus WNV strain 2471 HeLa Dengue virus DENV New Guinea C strain Tai et al (2009) Hepatitis C virus Huh7/RepSubgenomic Feo genotype 1b replicon Pooled/ Arrayed Library Knockdown/ Challenge Out Time Time Arrayed Dharmacon 72 h siARRAY siRNA library (21,121 siRNA pools) 24 h Arrayed Dharmacon 72 h siARRAY human genome siRNA library (21,094 genes) N/A Readout Viral Dependency Factors % Infectivity Yes (viral E-proteins) 30 h Hepatitis C virus Huh 7.5.1 JFH-1 Arrayed Dharmacon 72 h siARRAY siRNA library; human genome (19,470 genes) 48 h Viral Competitive or Restriction Factors Viral Competitive or Restriction factors Selection Main Criteria Candidates Stage of Viral Lifecycle Impacted Infection reduction of >twofold No NA Entry 283 candidates Viral replication (luciferase) Yes 236 pools— 186 replicated—96 confirmed Li et al (2009) Viral Dependency Factor Selection Criteria % Infectivity (HCV Core Antibody 6G7) Yes 407 candidate pools CBLL1 MCT4 Replicon expression decreases by >2 SDs Yes 13 pools Increased PI1KA replicon expression with threshold of q < 0.10 Individual siRNAs, small-molecule: MG132, cyclohexamide; colocalization; Replication phase enrichment analysis using Panther; gene expression—microarray; protein interaction network Replication complex formation, generation of HCV nonstructural protein-associated membranes COPICoatomer Early Hepcidin Cellular translation Infectivity Yes Infectivity RAB9p40 150% pf plate 114 candidate mean; cell mean; cell number >50% pools number >50% of plate mean plate mean Candidate Validation and Follow up Assays Needed for both HCV and HIV Gene ontology; clustered; literature review; other cell line: OR6 replicon cell line, UHCVcon57.3; protein expression; Western blot; small-molecule Wortmannin, brefeldin A; reagent redundancy; shRNAs; localization studies; virus: HCVJFH1 Individual siRNAs, enrichment analyses for molecular function and biological process according to Panther classification; network analyses interactome screens + HPRD; RT-PCR Sessions et al (2009) Dengue virus DENV-S2 Brass et al (2009) Influenza A virus U2OS A/Puerto Rico/ 8/34 Influenza A virus HBECs IAV PR8 Shapira et al (2009) Dipteran cells Arrayed Genome-wide 48 h RNAi library DRSC 2.0 (22,632 dsRNAs) RNA accumulation Gene ontology; in vivo mosquito Ae aegypti; validation of human homologue siRNAs in Huh-7 cells; other viruses: YFV 17D vaccine strain, Coxsackie B3 (strain 20; CB3); RT-qPCR >200% IFITM3 infectivity; viability >40% Early Rescreened candidates; (GO) enrichment analysis; other cell lines primary lung fibroblasts, HeLa, A549, ChEFs, MDCKs; other viruses: HIV, PR8, H3N2 A/ Udorn/72, A/Brisbane/ 59/07 H1N1, A/ Uruguay/716/07 H3N2, A/Aichi/2/68 H3N2, MLV, VSV-G; pseudoparticles MLV with the following envelopes: H1, H3, H5, H7, MACH, MLVRescue construct; overexpression; Western blot; immunofluorescence Change >twofold more replication compared to median NS1 related Pathway analysis; clustering of expression data; functional annotations; yeast hybrid 72 h Expression of Yes envelope protein 218 candidate dsRNAs— rescreen 179 dsRNA— identified 118 dsRNA ¼ 116 genes—111 novel Inhibited No infection !1.5-fold with p < 0.05 N/A Arrayed Dharmacon 72 h siARRAY siRNA library; human genome (17,877 genes) 12 h % Infectivity (anti-HA antibody) 40% 22 pools Arrayed Dharmacon SMARTpool 48 h Change >twofold less replication compared to median 72 h Viral particle production (reinfection); IFN production Yes 312 pools Yes Yes FLJ20254; TAZ; EXDL2; CNOT2 WNT/p53 pathway Continued Viroid Structure 163 mutants did replicate but were restricted to specific tissues such as galls and roots, whereas the corresponding inoculation with wild-type PSTVd led to systemic infection Obviously Agrobacterium-mediated inoculation could result in local replication but cell-to-cell or long distance movement was prevented It was concluded that the structure or stability of the right terminal stem-loop is essential for cell-to-cell and/or long distance movement Possibly an essential RNA movement protein interaction was disrupted (Hammond, 1994) VirP1, a bromodomain-containing protein with an atypical RNA-binding domain and a nuclear localization signal, binds to the RY motifs located in the TR domain of (+) PSTVd (Fig 2) N tabacum and N benthamiana plants with a suppressed VirP1 were not infected by PSTVd or CEVd through mechanical inoculation, and VirP1-suppressed protoplasts were unable to sustain viroid replication (Gozmanova et al., 2003; Kalantidis et al., 2007) 5.1.4 Pathogenicity (or Virulence) Modulating Domain All attempts to correlate the thermodynamic stability of the P region with virulence have failed (Owens et al., 1996) A hypothesis derived from model building that postulated a higher degree of bending for more virulent strains does not accord with the extraordinary pathogenicity of PSTVd AS1 (Matousˇek et al., 2007) However, viroid infection is associated with the appearance of viroid-specific small RNA, similar in size to endogenous small interfering RNA and microRNA (Papaefthimiou et al., 2001) Thus, they might alter normal gene expression in the host plant by pathways similar to transcriptional and/or posttranscriptional silencing thereby inducing viroid-typical symptoms that vary dramatically, depending on both the plant cultivar and the viroid variant This hypothesis was verified for PLMVd, a member of the Avsunviroidae (see Section 5.2.3; for an overview on this topic, see for example, Hammann & Steger, 2012; Navarro et al., 2012b) 5.2 Important Structural Motifs for the Replication of PLMVd 5.2.1 The Initiation Site and Its Vicinity The rolling circle replication of PLMVd is initiated by a polymerization step (Fig 1) Two different strategies have led to the identification of the same initiation site for both PLMVd polarities (Delgado, Martı´nez de Alba, Herna´ndez, & Flores, 2005; Motard, Bolduc, Thompson, & Perreault, 2008) For the strand of (+) polarity, positions A50 and C51, with a prevalence for C51, were identified, with U284 identified for the (À) strand 164 Gerhard Steger and Jean-Pierre Perreault (Fig 10, circled nucleotides) These initiation sites are located in the last helix of the P11 stem, adjacent to the P1 and P10 stems Hence, these initiation sites are in the vicinity of the self-cleavage and ligation sites It has been suggested that these are the universal initiation sites for each polarity However, there is no evidence to discard the possibility that more than one initiation site is used Subsequently, an in vitro selection procedure based on a model rolling circle replication assay was developed This model system as well as previous experiments revealed that the viroid template is the double-stranded P11 stem (Motard et al., 2008; Pelchat, Cote, & Perreault, 2001; Pelchat, Grenier, & Perreault, 2002) This selection experiment led to the identification of a conserved CAGACG box which is reminiscent of the sequence found in the vicinity of the PLMVd initiation sites; specifically, the sequences 47CAGACU52 and 287CAGACA292 are retrieved for the (+) and the (À) strands, respectively (Fig 10, nucleotides in the green (dark gray in the print version) box; Motard et al., 2008) Experiments performed to identify proteins that bind PLMVd led to the recovery of the peach elongation factor 1-alpha (eEF1A), i.e., a protein of 447 amino acids (Dube, Bisaillon, & Perreault, 2009) eEF1A has been frequently reported to be involved in the replication and translation of RNA viruses The PLMVd and eEF1A complex was confirmed as occurring in leaf and further characterized This protein appears to bind close to the universal initiation site, indirectly supporting a potential contribution for PLMVd replication (i.e., the initiation of polymerization) However, additional physical support for the involvement of eEF1A remains to be obtained It is tempting to speculate that together, these features in addition to the nuclear-encoded chloroplastic DNA-dependent RNA polymerase (Rodio et al., 2007) may be essential components for the initiation of polymerization 5.2.2 The Hammerhead Self-Cleaving Sequences In the second step of rolling-circle replication, the generated longer-thanunit conformers self-cleave into monomers (Fig 1) The P11 stem contains the hammerhead self-cleaving RNA sequences of both the (+) and (À) polarities (i.e., the lower and the upper strand, respectively; Figs and 10) Folded into its active structure, the hammerhead motif catalyzes the selfcleavage reaction in the presence of MgCl2 The hammerhead motif is composed of three stems flanking an apparently unpaired core region that includes highly conserved nucleotides (Fig 10) This is an alternative Viroid Structure 165 structure of higher energy than the native structure Previous reports have shown that changes in the catalytic core composition of the hammerhead self-cleaving motif significantly affect cleavage ability This received important support from a recent high-throughput sequencing analysis showing that within almost 4000 new sequence variants the nucleotides composing the catalytic core were perfectly conserved (Glouzon, Bolduc, Wang, Najmanovich, & Perreault, 2014) Conversely, the nucleotides composing all three stems have shown significant substitutions maintaining basepairing by covariation This supports the existence of hammerhead motifs in all PLMVd sequence variants and is in agreement with the existence of a selective pressure in favor of the self-cleaving activity, which is essential for the release of the monomer from the novel multimeric nascent strands of both polarities during replication The left-handed domain appears to contain most, if not all, features required for replication When adopting the P11 stem–loop structure (i e., the most stable one), it is responsible for the polymerization step as well as the ligation step required for circularization to occur Conversely, when adopting the alternative, less stable hammerhead structure, it catalyzes selfcleavage 5.2.3 An Extra Stem–Loop Domain Associated with Severe Symptoms An additional stem–loop of 12 or 13 nts was reported to occur within the left terminus of the P11 stem–loop (Fig 10; Malfitano et al., 2003) The presence of this stem–loop capped by a UUUU loop was demonstrated to be responsible for extensive chlorosis of peach leaves known as peach calico after PLMVd infection The PLMVd variants responsible for this phenotype interfere with the maturation of plastid rRNA impairing chloroplast biogenesis (Rodio et al., 2007) Recently, it has been demonstrated that viroid small RNA derived from the peach calico insertion direct RNA-induced silencing complex cleavage of a specific host mRNA (Navarro et al., 2012a) Importantly, the peach calico insertion has not yet been probed in solution; however, its stem–loop structure is supported by sequence covariation of some of the basepairs forming the helical region (Rodio, Delgado, Flores, & Di Serio, 2006) PERSPECTIVE The determination of secondary structure of viroids in solution has flourished immensely over the last few years If previously the classical 166 Gerhard Steger and Jean-Pierre Perreault methods for structure determination were technically complex to perform, time-consuming and the resulting data not always easy to interpret, the adaptation of the high-throughput SHAPE has completely changed the situation This method is precise, fast, and can be performed by any laboratory It has already permitted the elucidation of a sequence variant of most of the viroid species, and we can expect that the structure of one species of each viroid will be determined in solution very soon Such structures of single sequences in equilibrium state will also support the prediction of consensus structures, which then might help to gain insight on the impact of mutations in the variants and on the delineation of functional elements The important achievement by high-throughput SHAPE has set the path for future work on the unaddressed and difficult questions on the viroid’s structure The current available structures were determined using RNA alone (i.e., in the absence of host proteins) and in conditions favoring structural homogeneity, ignoring any structure equilibrium Thus, it will be important to study the structure of the viroids in planta because the physiological conditions as well as the presence of proteins may favor the folding of distinct structures The latest issue becomes even more important when the viroid is considered as a quasispecies in terms of multiple sequences present in the host, which may favor the folding into various RNA structures Elucidation of viroid structure in vivo would be an important step forward; however, it remains highly challenging The knowledge of the three-dimensional structure of viroid remains a relatively unexplored issue The rod-like structure of viroid was reported a long time ago based on electron microscope imaging (Saănger et al., 1976; Sogo, Koller, & Diener, 1973) Production of viroid in a large quantity should permit to resolve the detailed RNA structure using present-day 3D biophysical approaches By including host proteins, such a structure may be considered as a ribonucleoprotein complex, thus opening up new avenues 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transcriptional activity during latency, 68–70 Alphaherpesvirus latency, establishment of, 59–66 initiation of transcriptional activation/ suppression, 60–61 role of cell type in, 60 Alphaherpesvirus latency model, 55 cell culture models, 58–59 explant models, 57–58 implant tissue models, 57 natural host animal models, 56 nonnatural host models, 56 in vitro models, 57–59 Ambion Silencer Select library, 27–28 Apoptosis, 93 Apscaviroids, UCCR of, 152f Arabidopsis thaliana, 162 Arena virus (ANDV) host factors, 41 Arrayed RNAi screening, 23–28 ASBVd, 159 Avsunviroidae, 142–145 structure, 154–160, 158f symmetrical replication of, 145f Capsid vertex specific component (CVSC), 98 C-capsid specific complex (CCSC), 98 CCCVd, 148–149 partial sequence duplications in, 153f CChMVd, 159 Cell culture models, 58–59 Cell-to-cell, and long distance movement motif, 162–163 Cellular nuclear export mechanism, nuclear egress as, 113–115 Cellular vesicular nucleocytoplasmic transport, vs herpesvirus nuclear egress, 114t Central conserved region (CCR), 142 PSTVd, 161f Chronic myelogenous leukemia (CML), 31–32 CPE See Cytopathic effect (CPE) CRISPR/CAS9 genetic screening technology and approach, 32–34 MORR RNAi vs pooled, 34–44 pooled, 25t screen for HRV host factors, 37f Cyclin-dependent kinase (CDK), 94 Cyclin-dependent kinase (CDK1), 87 Cytopathic effect (CPE), 36–38 Cytorhabdovirus, 118 B Barrier-to-autointegration factor (BAF), 88 Biological function, structural motifs and, 160–165 Bovine herpesvirus-1 (BHV-1), 65 Budded viruses (BV), 118 D Dendritic cells (DCs), 43 Dephosphorylation, 87 Drosophila cell, in vitro RNAi screens, 2–3 E Elongation factor 1-alpha (eEF1A), 164 ELVd, 160 Emerin, 88 Endonuclease processed siRNA (esiRNA) pools, 28 173 174 Endosomal sorting complex required for transport (ESCRT), 109–110 esiRNA pools See Endonuclease processed siRNA (esiRNA) pools Explant models, in vitro, 57–58 G Gene expression, 21–23 Genetic screening technology CRISPR/CAS9, 32–34 haploid cell, 31–32 RNAi, 31 Genetic screens, host–virus, 2–3 Genome-wide enrichment of seed sequence matches (GESS) method, 30–31 Genomic screen, for elucidating host–viral interactions, 4t GESS method See Genome-wide enrichment of seed sequence matches (GESS) method GFP See Green fluorescent protein (GFP) Giant unilamellar vesicles (GUVs), 109–110 Green fluorescent protein (GFP), 36–38 GUVs See Giant unilamellar vesicles (GUVs) H Hairpin I (HPI), 149 Hairpin II (HPII), 149 pospiviroidae, 153–154, 154f Hammerhead self-cleaving sequence, 164–165 Hannon–Elledge Open Biosystems shRNA library, 21 Haploid cells genetic screening technology and approach, 31–32 pooled RNAi, 25t Herpes simplex virus (HSV), 54–55 Herpes simplex virus-1 (HSV-1), 61–64 genome and noncoding RNAs, 62f in situ hybridization, 63f Herpes simplex virus-2 (HSV-2), 64 Herpesvirus, 82–83 replication cycle, 83f Herpesvirus nuclear egress vs cellular vesicular nucleocytoplasmic transport, 114t Index deenvelopment, 112–113 INM environment to facilitate nucleocapsid access, 106–108 intranuclear movement of nucleocapsids to INM, 106 nuclear egress complex, 99–106 nucleocapsid docking at NEC, 108 nucleocapsid formation, 97–98, 99f primary virion, 110–112 vesicle formation and scission, 109–110 Herpesvirus nuclear escape pathways, 115–117 High-throughput SHAPE (hSHAPE), 154–155, 158–160 Host models natural, 56 nonnatural, 56 Host–viral interactions genomic screens for elucidating, 4t genomic strategies for elucidating, 22f Host–virus genetic screens, 2–3 HRV-HF screens, 34–44 HRV host factors CRISPR/Cas9 screen for, 37f MORR/RIGER screen for, 35f hSHAPE See High-throughput SHAPE (hSHAPE) HSV See Herpes simplex virus (HSV) Human–virus interaction, genomic screening strategies for, 25t Human–virus loss-of-function genetic screens, 34–36 I Immunohistochemistry (IHC), 54–55 Implant tissue models, 57 Influenza A virus (IAV), Inner nuclear membrane (INM), 84–85 environment to facilitate nucleocapsid access, 106–108 intranuclear movement of nucleocapsids to, 106 proteins of, 87–89 In situ hybridization (ISH), 54–55 In vitro models, 57–59 Ion Torrent sequencer, 38–39 ISH See In situ hybridization (ISH) 175 Index K Kaposi’s sarcoma-associated herpesvirus (KSHV), 100 KASH domain, 89–90 KBM-7 cells, 31–32 KSHV See Kaposi’s sarcoma-associated herpesvirus (KSHV) L Lamina-associated polypeptides (LAP), 88 Lamin B receptor (LBR), 87–88 Lamins, 85–87 Laser capture microdissection (LCM) analysis, 69 Latency See also specific types of latency definition, 54–55 Latent genomes, 54 LBR See Lamin B receptor (LBR) Linker of nucleoskeleton and cytoskeleton (LINC), 84–85 between cytoskeleton and nucleus, 89–90 Lipopolysaccharide (LPS), 43 LNAs See Locked nucleic acids (LNAs) lncRNAs See Long noncoding RNAs (lncRNAs) Locked nucleic acids (LNAs), 27–28 Long noncoding RNAs (lncRNAs), 56 expression, 61–66 M Marek’s Disease Virus (MDV), 54, 65–66 MicroRNAs (miRNAs), 56 expression, 61–66 Moloney Leukemia virus (MLV)-GFP, 37f MORR See Multiple orthologous RNAi reagents (MORR) Motif and biological function, 160–165 cell-to-cell and long distance movement, 162–163 pathogenicity/virulence modulating domain, 163 in processing oligomeric intermediates to circles, 161–162 in strand synthesis, 160–161 Multiple orthologous RNAi reagents (MORR), 29–30 and CRISPR/Cas9 HRV-HF screen, 40f and RIGER screen for HRV host factors, 35f RNAi vs pooled CRISPR/CAS9, 34–44 N Natural host animal models, 56 NE breakdown (NEBD), 92–93 NEC See Nuclear egress complex (NEC) Nerve growth factor (NGF), 58 NES See Nuclear export signal (NES) NGF See Nerve growth factor (NGF) NLS See Nuclear localization signal (NLS) Noncoding RNAs, 61, 62f long, 56, 61–66 Nonnatural host models, 56 Non-Watson–Crick basepairs, 152f NPC See Nuclear pore complex (NPC) Nuclear egress as cellular nuclear export mechanism, 113–115 in virus families, 117–118 Nuclear egress complex (NEC), 99–106 nucleocapsid docking at, 108 pUL31 component of, 101f pUL34 component of, 103f Nuclear envelope (NE), 83–85, 86f dynamics of, 92–93 trafficking across, 91–92 Nuclear escape pathways, herpesvirus, 115–117 Nuclear export mechanism, nuclear egress as cellular, 113–115 Nuclear export signal (NES), 91–92, 101f Nuclear localization signal (NLS), 91–92, 101f Nuclear membrane, fusion mechanisms comprising, 94–96 Nuclear pore complex (NPC), 29, 83–85, 91 Nucleocapsid, 82–83 access, 106–108 docking at NEC, 108 formation, 97–98 intranuclear movement of, 106 Nucleocytoplasmic transport, 83–84, 114t, 115 Nucleophagy, 93 176 Nucleorhabdovirus, 118 Nucleus, 84–96 cyclin-dependent kinases, 94 dynamics of NE, 92–93 fusion mechanisms comprising nuclear membranes, 94–96 lamins, 85–87 LINC between cytoskeleton and nucleus, 89–90 nuclear envelope, 83–84, 86f nuclear pore complex, 91 proteins of INM, 87–89 torsin, 90–91 trafficking across NE, 91–92 O Occlusion-derived viruses (ODV), 118 On-target-plus (OTP), 27 Orthomyxovirus, 117–118 OTEs, 28–33 Outer nuclear membrane (ONM), 84–85 P Pathogenicity (P) domain, pospiviroidae, 150 PLMVd initiation site and vicinity, 163–164 as model viroid, 154–158 secondary structure, 155f, 157f Pooled screening, RNAi, 21–23 Pooled sgRNA retroviral vectors, 33 Pooled shRNA approach, 24–27 screen, 21–23 Pospiviroidae (PSTVd), 142, 147–149, 162 asymmetrical replication of, 145f biological function of structural motifs in, 160–163 CCR, 161f central (C) domain, 150–152 consensus structure, 146f, 147–148 hairpin II, 153–154 pathogenicity (P) domain, 150 secondary structure schemes of, 146f structure, 147–154 terminal left (TL) domain, 149–150, 150f terminal right (TR) domain, 153 variable (V) domain, 153 Premelting (PM) region, pospiviroidae, 150 Index Protein kinase C (PKC), 87 Proteins of INM, 87–89 Prototypic herpesvirus replication cycle, 83f PRV See Pseudorabies virus (PRV) Pseudorabies virus (PRV), 54, 65 PSTVd See Pospiviroidae (PSTVd) R Ran GTPase activating protein (RanGAP), 92 Recombinant vesicular stomatitis viruses (rVSVs), 39–41 Relative light units (RLUs), 41–42 Rhabdovirus, 118 Ribonucleoprotein particles (RNPs), 113–114 RIGER See RNAi gene enrichment ranking (RIGER) RISC See RNA-induced silencing complex (RISC) RNAi See RNA interference (RNAi) RNAi gene enrichment ranking (RIGER), 29–30 RNA-induced silencing complex (RISC), 3, 21 RNA interference (RNAi), arrayed (siRNA), 25t arrayed RNAi screening, 23–28 Drosophila cell in vitro, 2–3 genetic screening technologies and approaches, 31 haploid cells pooled, 25t pooled (shRNA), 25t pooled screening, 21–23 screening problems and some solutions, 28–31 RNPs See Ribonucleoprotein particles (RNPs) Rough endoplasmic reticulum (RER), 85 rVSVs See Recombinant vesicular stomatitis viruses (rVSVs) S sgRNAs See Short guide RNAs (sgRNAs) SHAPE, 146f, 147–148, 154–159, 165–166 Short guide RNAs (sgRNAs), 32–33 177 Index Short hairpin RNAs (shRNAs), 21–23 detection, 23 pooled, 24–27 screen, 21–23 Siberian C, 157f siGENOME, 27 siRNA, 21–28, 22f, 30–31, 41–42 SMARTpool library, 27–28 siGENOME library, 27 Stem–loop domain, with severe symptoms, 165 T T cell regulation, of latency, 70–71 Terminal left (TL) domain, pospiviroidae, 149–150, 150f Terminal right (TR) domain, pospiviroidae, 153 Tissue models, implant, 57 Torsin, 90–91 U UCCR of apscaviroids, 152f structural alignment, 151f V Vacuolar ATPase (V-ATPase), 29 Variable (V) domain, pospiviroidae, 153 Varicella zoster virus (VZV), 54, 65 Viroids, 142 biophysical properties as basis for functional activity, 147 compilation of, 143t consensus structures of, 147–148 PLMVd as model, 154–158 VirP1, 163 VZV See Varicella zoster virus (VZV) W West Nile virus (WNV), 33–34 ... Elucidating Host–Viral Interactions—cont'd Citation Virus Cell Line Krishnan et al (2008) West Nile virus WNV strain 2471 HeLa Dengue virus DENV New Guinea C strain Tai et al (2009) Hepatitis C virus. .. human virus functional genomics Advances in Virus Research, Volume 94 ISSN 0065-3527 http://dx.doi.org/10.1016/bs.aivir.2015.11.001 # 2016 Elsevier Inc All rights reserved Jill M Perreira et al INTRODUCTION... experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety