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59 Daniel R Perez ed Reverse Genetics of RNA Viruses Methods and Protocols Methods in Molecular Biology vol 1602 DOI 10 1007 978 1 4939 6964 7 5 Springer Science Business Media LLC 2017 Chapter 5 Efficient Reverse Genetic Systems for Rapid Genetic Manipulation of Emergent and Preemergent Infectious Coronaviruses Adam S Cockrell Anne Beall Boyd Yount and Ralph Baric Abstract Emergent and preemergent coronaviruses CoVs pose a global threat that requires immediate intervention Rapid intervention necessitates the capacity to generate grow and genetically manipulate infectious CoVs in order to rapidly evaluate pathogenic mechanisms host and tissue permissibility and candidate antiviral therapeutic efficacy CoVs encode the largest viral RNA genomes at about 28 32 000 nucleotides in length and thereby complicate efficient engineering of the genome Deconstructing the genome into manageable fragments affords the plasticity necessary to rapidly introduce targeted genetic changes in parallel and assort mutated fragments while maximizing genome stability over time In this protocol we describe a well developed reverse genetic platform strategy for CoVs that is comprised of partitioning the viral genome into 5 7 independent DNA fragments depending on the CoV genome each subcloned into a plasmid for increased stability and ease of genetic manipulation and amplification Coronavirus genomes are conveniently partitioned by introducing type IIS or IIG restriction enzyme recognition sites that confer directional cloning Since each restriction site leaves a unique overhang between adjoining fragments reconstruction of the full length genome can be achieved through a standard DNA ligation comprised of equal molar ratios of each fragment Using this method recombinant CoVs can be rapidly generated and used to investigate host range gene function pathogenesis and candidate therapeutics for emerging and preemergent CoVs both in vitro and in vivo Key words Coronavirus CoV Reverse genetics Severe acute respiratory syndrome coronavirus SARS CoV Middle East respiratory syndrome coronavirus MERS CoV Emerging Preemergent Bat coronavirus Porcine epidemic diarrhea virus PEDV 1 Introduction Human coronaviruses HCoVs were first identified in the 1960s HCoV 229E and HCoV OC43 and were primarily associated with mild upper respiratory tract infections with the potential to progress to a severe respiratory disease in young children the elderly and immunocompromised individuals 1 Although addi tional HCoVs were known to circulate at this time these strains were not culturable therefore HCoV 229E and HCoV OC43 60 infections modeled our understanding of CoV disease severity until 2003 1 In Southeast Asia severe acute respiratory syndrome coronavirus SARS CoV emerged in late 2002 and caused acute respiratory distress syndrome and an age dependent mortality rate of 10 50 clearly demonstrating that HCoVs were emerging pathogens with pandemic potential 2 The portent of a worldwide pandemic mobilized the scientific community leading to robust public health intervention strategies that controlled the epidemic Moreover the outbreak spurred academic interest into CoV gene function and pathogenic mechanisms associated with SARS CoV induced disease leading to the development of therapeutic coun termeasures Because of the availability of reverse genetics robust in vitro replication and in vivo animal models of human disease SARS CoV has become the most intensively studied prototype for HCoV research 3 SARS CoV research prompted the generation of novel animal models that have provided insight into 1 genetic changes in the SARS CoV genome that modulate respiratory pathogenesis 4 2 the impact of SARS CoV on host innate and adaptive immune responses 5 6 3 the role of host genes in regulating SARS CoV pathogenesis in mice 4 7 and 4 novel strategies for the development of vaccines and therapeutic counter measures 3 Two novel HCoVs NL63 and HKU1 were identified shortly after the emergence of SARS CoV 8 10 and nearly a decade later in 2012 the world saw the emergence of Middle East respira tory syndrome coronavirus MERS CoV Fig 1 MERS CoV causes acute respiratory distress syndrome ARDS severe pneumonia like symptoms and multi organ failure with a case fatality rate of 36 11 Human cases of MERS CoV have been predominantly observed in Saudi Arabia and the Middle East MERS CoV infected individuals have also traveled internationally illustrating the potential for global spread For example a South Korean native returning home from the Middle East in May 2015 initiated an outbreak that infected 186 people resulting in 20 mortality and a nationwide economic crisis 12 Transmission of MERS CoV has been mostly observed among health care workers in the hospital setting Accumulating evidence indicates that Middle Eastern individuals working in close contact with drome dary camels are at increased risk of acquiring MERS CoV 13 Camels are suspected to be intermediate hosts between bats and humans that can repeatedly allow for reemergence of MERS CoV in the human population Though camels show only mild sympto mology during MERS CoV infection zoonotic CoV infections can be highly pathogenic in animals Demonstrated recently by the emergence of a porcine CoV in the United States porcine epidemic diarrhea virus PEDV has caused severe disruption to the pork industry with the deaths of tens of millions of animals in the first 2 years and a 90 mortality rate 14 Fig 1 PEDV Adam S Cockrell et al 61 Fig 1 T imeline of emerging corona virus events and infectious c lones genera ted using reverse genetics systems RGS RGS timeline spans from the first RGS c lones TGEV in 2000 through the most recently genera ted RGS c lone preemergent ba t corona virus infectious c lone WIV1 CoV in 2016 Red indica tes outbreak and viral identifica tion events Blue indica tes the publica tion of RGS c lones T ransmissible gastroenteritis virus TGEV mouse hepa titis virus MHV severe acute respira tor y syndrome corona virus SARS CoV Hong K ong University HKU Middle East respira tor y syndrome corona virus MERS CoV porcine epidemic diarrhea virus PEDV Revere Genetics of Emerging Coronaviruses 62 MERS CoV SARS CoV HCoV NL63 HCoV HKU1 and 229E HCoV are thought to have emerged from a bat reservoir while OC43 HCoV is thought to have originated among bovine coronaviruses In 2013 preemergent SARS like CoVs were identi fied in horseshoe bats and found to be poised for entry into the human population 15 Fig 1 Importantly much of the HCoV research over the last 15 years has been possible because of the capacity to generate infectious clones using highly efficient reverse genetics platforms 16 coupled with robust small animal models of human disease 17 18 Reverse genetic systems for coronaviruses were difficult to achieve because of the large size of the viral RNA genome genome stability in bacterial vectors difficulty in driving full length 30 kb RNA transcripts in vitro poor transfection efficiencies and low infectivity of the viral genome In 2000 the first molecular clone was developed for transmissible gastroenteritis virus TGEV using targeted splice junctions to increase genome stability in low copy baculovirus vectors 19 Fig 1 A few months later our group published an alternative TGEV reverse genetic strategy 20 and then again applied this technique for the group 2 murine coronavirus 21 A final innovation in CoV molecular clone design was the insertion of full length HCoV 229E molecular clone into vaccinia virus in 2001 22 Each approach has proven to be a potentially powerful system to probe the role of CoV genes in rep lication and pathogenesis 3 23 24 This review primarily focuses on the reverse genetic strategy developed in the Baric laboratory a technology that partitions the CoV genome into discrete frag ments and uses class IIS and IIG restriction endonucleases to sys tematically and seamlessly assemble full length cDNA genomes of CoVs After in vitro transcription and transfection of full length genomes into permissive cells recombinant viruses are recovered which contain the genetic content of the molecular clone Although the reverse genetic system RGS described here achieved prominence shortly after the emergence of SARS CoV 16 this platform has been used to generate CoV infectious clones that span nearly the entire breadth of the Coronaviridae family including pathogenic viruses from groups 1a and 1b of the alphacoronaviruses and groups 2a 2b and 2c of the betacoronavi ruses Fig 1 The entire CoV fragments are joined by type IIS or IIG restriction sites e g BglI SapI and BsaI that support direc tional seamless ligation into full length genome Fig 2 For type IIS e g SapI restriction enzymes the recognition sequences are separated from its cleavage site by one or more variable nucleotides leaving three to four nucleotide unique overhangs Fig 2 Thus these enzymes leave 64 256 unique ends providing directionality during multi segment assembly Moreover the recognition site is not palindromic allowing for seamless assembly of component cDNA clones into full length genes and genomes By orienting the Adam S Cockrell et al 63 Fig 2 Organiza tion of corona virus genomes and infections c lones used to genera te recombinant corona virus a Left Genome organiza tion of MERS CoV PEDV and SHC014 Right Organiza tion of corona virus cDNA fra gments used in subc loning in order to genera te a genome length cDNA templa te prior to transcription Color coded restriction sites denote distinct type IIS Sa pI or type IIG BglI restriction sites An example of each is sho wn for the first junction encoded in the MERS CoV and PEDV genomes b A benefit of the RGS infectious c lone system is the ease of directed genome muta tion By s wa pping fra gments between wild type and mutant MERS CoV or even between various corona virus species useful CoV variants such as wild type spike or open reading frame mutants can be ra pidly genera ted in order to understand the role of specific muta tions or viral genes Multiple infectious c lones with different genetic muta tions can be genera ted in parallel provided tha t the muta tions are on different fra gments Example Three different viruses are genera ted from muta tions in the nsP3 gene fra gment A blue and the spike gene fra gment E purple Revere Genetics of Emerging Coronaviruses 64 recognition sequence external to the cleavage site upon digestion and ligation the restriction site is lost seamlessly joining the cDNAs while preserving ORF integrity and sequence authenticity A second approach uses type IIG e g BglI restriction endonucle ases which has a palindrome restriction site bisected by a variable domain of five nucleotides and also leaves 64 different overhangs for directional assembly of large genome molecules Fig 2 In this instance the restriction site is retained in the assembled product As coronavirus genomes are unstable in bacteria junctions are ori ented within toxic sequence domains thereby bisecting region tox icity and increasing component clone stability Plasmids are digested with type IIS or IIG restriction enzymes to isolate each fragment of the CoV genome Fig 2 Fragments are then resolved on an agarose gel purified and ligated Fig 2 Following ligation the coronavirus genome length mRNA is in vitro transcribed from a T7 promoter added at the 5uni2032 end of the 5uni2032 UTR In some instances strong T7 stop sites are mutated to promote full length transcript synthesis in in vitro transcription reactions 20 Resulting genome length mRNA is electroporated into a permissive mammalian cell line Fig 2 Cloning success and viral fitness can be measured by plaque assay and growth curves Fig 3 During viral replication Fig 2 continued Adam S Cockrell et al 65 CoVs have the unique capacity to generate a nested set of sub genomic viral RNAs sgRNAs harboring similar 5uni2032 and 3uni2032 untrans lated regions UTRs Fig 4 Northern blot analysis of CoV RNA and or PCR of viral cDNA affords visualization of sgRNA and confirmation of replication competent virus Fig 4 The efficiency of RGS is exemplified by the fact that an infectious clone of SARS CoV icSARS was generated and published 16 within weeks of the initial publication of the SARS CoV Urbani strain sequence 26 Fig 1 The use of DNA synthesis companies to synthesize a full length coronavirus genome was achieved in 2008 using HCoV NL63 as a model 27 and then applied to MERS CoV and various bat SARS like CoVs in 2008 2013 2015 and 2016 25 28 30 Fig 3 Confirmation of MERS CoV production and growth characteristics a A comparison of growth curves in wild type filled square MERS CoV recombinant MERS CoV open square rMERS CoV and a recombinant MERS CoV containing a tissue culture adapted mutation in the spike gene filled triangle rMERS CoV T1015N b A comparison of plaque formation in wild type MERS CoV rMERS CoV and rMERS CoV T1015N The recom binant MERS CoV with the tissue culture adaptation cloned back in using RGS exhibits plaque sizes similar to wild type MERS CoV All images reprinted from 25 Revere Genetics of Emerging Coronaviruses 66 Finally our group has applied this approach to generate stable molecular clones for flaviviruses that include dengue and the newly emerged Zika virus 31 32 Importantly fragmenting HCoV genomes confers additional biological safety benefits over reverse genetics systems that would otherwise maintain a CoV genome as a cDNA molecule compris ing greater than 2 3 of the full length genome As sanctioned by NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules NIH Guidelines April 2016 Section III E 1 indicates that Recombinant or synthetic nucleic acid molecules containing no more than two thirds of the genome of any eukaryotic virus may be propagated and maintained in cells in tissue culture using BL1 containment Moreover full length CoV genomes that encode select agents such as SARS CoV are further regulated under the Federal Select Agent Program www selecta gents gov and must be handled according to specific guidelines Wild Type Mouse Lung Transgenic Mouse Lung ORF1a ORF1b S 3 4a 4b 5 E M N 8b 5 UTR 3 UTR 3 UTR 5 3 UTR 3 UTR 3 UTR 3 UTR 3 UTR 3 UTR 5 5 5 5 5 5 1 2 3 4 5 6 7 8 Fig 4 MERS CoV subgenomic RNA sgRNA sgRNA is generated during active coronavirus replication and can be visualized by northern blot left and PCR right Using a biotinylated or radioactive probe against the CoV N gene all sgRNA species can be visualized by running RNA isolated from CoV infected cells on a northern gel left Northern blot image is reprinted from 25 Alternately PCR primers contained within the leader sequence and N gene can be used to generate PCR products from viral cDNA in order to visualize sgRNA signifying productive CoV replication right Here PCR products confirm productive replication of MERS CoV in the lungs of a mouse permissive to MERS CoV infection transgenic mouse lung but not in a nonpermissive wild type mouse lung Both of these methods can be used to confirm productive CoV replication following RGS clone generation Adam S Cockrell et al 67 Partitioning the SARS CoV genome allows for efficient handling of genomic fragments under standard BSL1 2 containment conditions Fig 2 For reconstruction of full length genomes encoding CoVs restricted to BSL3 containment SARS CoV MERS CoV preemergent bat CoVs fragment ligation and all subsequent steps are executed under BSL3 conditions including recovery of recombinant viruses Fig 2 One advantage of a segmented molecular clone design is that muta genesis can occur in parallel on multiple fragments and that the indi vidual fragments can be reassorted to make larger panels of derivative mutants encoding mutation subsets Fig 2 For example an early application included the introduction of over 27 mutations into the SARS CoV genome at 9 different genome transcription regulatory sequences thereby demonstrating for the first time that the transcription regulatory circuit of a virus could be rewired 33 The applicability of the RGS was ratified in ensuing reports describ ing the cause and effect relationship between site directed mutations introduced into the viral genome which altered viral fitness and in vivo pathogenesis phenotypes 17 18 33 36 In a seminal pathogenesis study RGS was used to validate that six mutations acquired during mouse adaptation of the SARS CoV Urbani MA15 strain indeed caused lung pathology associated with severe acute respiratory distress syndrome 18 Since these mutations were dispersed across the entire genome RGS proved to be an efficient method to introduce all six mutations simultaneously in order to generate a robust mouse adapted SARS CoV strain Notably because MA15 SARS CoV is a novel virus strain RGS is an effective method to validate Koch s postulates by demonstrating a cause and effect relationship After nearly a decade of research the MA15 strain continues to play a dominant role in SARS CoV mouse pathogenesis studies including vaccine and therapeutic evaluations 7 17 37 39 In today s research environment these studies would be considered gain of function GOF studies and thereby would have been subject to increased government oversight which would have inevitably stymied progress into understanding the molecular mechanisms governing emerging coronavirus pathogenesis host range receptor usage virus replication and vaccine and therapeutic efficacy 3 Notably SARS CoV gain of function GOF studies have yielded invaluable information regarding the role of viral proteins in pathogenesis in animal models and in tissue culture studies 17 36 Applying the combined technologies of GOF studies with the RGS will be essential to future research on emergent and preemergent coronaviruses More recently RGS applications were extended to MERS CoV wherein a replication competent MERS CoV expressing the tomato red fluorescent protein tRFP was rapidly generated reproduced from 25 Fig 5 The infectious clone icMERS CoV tRFP has 1 1 Applications of the Reverse Genetics Platform Revere Genetics of Emerging Coronaviruses 68 Fig 5 A pplica tion for an infectious c lone of MERS CoV expressing tRFP a Genomic representa tion of an infectious c lone of MERS CoV containing the toma to red fluoresc e n t protein tRFP substituted for the orf 5 gene icMERS CoV tRFP b Protein sequence alignment of human mouse and chimeric dipeptid yl peptidase 4 DPP4 MERS CoV host receptor sequence with five specific amino acid changes indica ted by red arro ws F igure reprinted from 40 c The indica ted chimeric DPP4 receptor constructs were overexpressed in 293T cells and subsequently infected with the icMERS CoV tRFP virus to ma p the minimal number of amino acids required to facilita te MERS CoV infection T wo amino acid changes A288L and T330R were sufficient to support infection and replica tion of MERS CoV F igure reprinted from 40 Adam S Cockrell et al 69 since been applied to understanding host factors that restrict MERS CoV infection 40 41 Figure 5 demonstrates how the icMERS CoV tRFP was used to facilitate mapping of specific amino acids in the host dipeptidyl peptidase 4 DPP4 receptor that are necessary to permit MERS CoV infection and replication 40 Humanizing the mouse DPP4 by changing a minimum of two amino acids A288L and T330R on the mouse DPP4 protein conferred effi cient infection replication of MERS CoV as determined by expres sion of tRFP from the icMERS CoV tRFP virus Fig 5 40 The following protocol outlines the RGS used to generate infectious clones of MERS CoV and provides detailed methods for building recombinant coronaviruses using this technique 2 Materials 1 Seven plasmids containing MERS fragments A F cloned into pCR XL TOPO Invitrogen Carlsbad CA or pUC57 vectors 2 Appropriate restriction enzymes and buffers for screening each MERS fragment see Table 1 3 Chemically competent bacterial cells Top 10 Invitrogen 4 LB broth supplemented with carbenicillin 100 uni03