【病毒外文文献】2005 [Current Topics in Microbiology and Immunology] Coronavirus Replication and Reverse Genetics Volume 287 __ Reverse

CTMI 2005 287 199 227 C23 Springer Verlag 2005 Reverse Genetics of Coronaviruses Using Vaccinia Virus Vectors V Thiel 1 S G Siddell 2 1 Research Department Cantonal Hospital St Gallen St Gallen Switzerland volker thiel kssg ch 2 Department of Pathology and Microbiology School of Medical and Veterinary Sciences University of Bristol Bristol UK 1 Introduction 200 2 The Use of Vaccinia Virus as a Vector for Coronavirus cDNA 202 2 1 Cloning of Full Length Coronavirus cDNA into the Vaccinia Virus Genome 202 2 2 Mutagenesis of Cloned Coronavirus cDNA 204 2 3 Rescue of Recombinant Coronaviruses 206 2 3 1 Rescue of Recombinant Coronaviruses Using Full Length In Vitro Transcripts 207 2 3 2 Rescue of Recombinant Coronavirus Using Full Length cDNA 209 2 3 3 Expression of the Nucleocapsid Protein Facilitates the Rescue of Recombinant Coronaviruses 210 3 Recombinant Coronaviruses 211 3 1 Analysis of HCoV 229E Replicase Polyprotein Processing 212 3 2 Analysis of IBV Spike Chimeras 215 3 3 Recombinant MHV Is Fully Pathogenic in Mice 215 4 Generation of Replicon RNAs 217 4 1 Replicase Gene Products Suffice for Discontinuous Transcription 217 4 2 Generation of Autonomously Replicating RNAs 219 5 Development of Coronavirus Based Multigene Vectors 221 5 1 Multigene Expression Using Coronavirus Based Vectors 222 5 2 Coronavirus Based Vectors as Potential Vaccines 223 6 Discussion 225 References 226 Abstract In this article we describe the reverse genetic system that is based on the use of vaccinia virus cloning vectors This system represents a generic approach to coro navirus reverse genetics and was first described for the generation of recombinant hu man coronavirus 229E representing a group I coronavirus Subsequently the same ap proach has been used to generate recombinant avian infectious bronchitis coronavirus and recently recombinant mouse hepatitis virus representing group III and group II coronaviruses respectively We describe how vaccinia virus mediated homologous re combination can be used to introduce specific mutations into the coronavirus genom ic cDNA during its propagation in vaccinia virus and how recombinant coronaviruses can be isolated Finally we describe how the coronavirus reverse genetic system has now been extended to the generation of coronavirus replicon RNAs 1 Introduction The development of systems for manipulating the coronavirus genome using traditional reverse genetic approaches has presented a consider able technological challenge because of both the genome size and the in stability of specific coronavirus cDNA sequences in bacterial systems However recently reverse genetic systems for a number of coronavirus es have been established using non traditional approaches which are based on the use of bacterial artificial chromosomes Almazan et al 2000 the in vitro ligation of coronavirus cDNA fragments Yount et al 2000 and the use of vaccinia virus as a vector for the propagation of co ronavirus genomic cDNAs Thiel et al 2001a With the systems now available it is possible to genetically modify coronavirus genomes at will Recombinant viruses with gene inactivations deletions or attenuat ing modifications can be generated and used to study the role of specific gene products in viral replication or pathogenesis Genetically attenuat ed viruses can be produced which are potential vaccine candidates and modified coronavirus genomes have been developed as eukaryotic mul tigene expression vectors Thiel et al 2003 In this article we shall de scribe the reverse genetic system that is based on the use of vaccinia vi rus cloning vectors This system represents a generic approach to coro navirus reverse genetics and was first described for the generation of re combinant human coronavirus 229E HCoV 229E Thiel et al 2001a representing a group I coronavirus Subsequently we have used the same approach to generate recombinant avian infectious bronchitis coronavi rus IBV Casais et al 2001 and recently recombinant mouse hepatitis virus MHV A59 Coley et al manuscript in preparation representing group III and group II coronaviruses respectively The basic strategy for the generation of recombinant coronaviruses can be divided into three phases The Assembly of a Full Length Coronavirus Genomic cDNA This nor mally involves the generation of numerous subgenomic cDNA fragments 200 V Thiel S G Siddell that are either amplified as bacterial plasmid DNA or prepared in large amounts by preparative reverse transcriptase polymerase chain reaction RT PCR The cDNAs are then ligated sequentially in vitro to produce a small number of cDNAs which encompass the entire genome The spe cific ligation strategy is dictated by the sequence of the coronavirus in question but a common feature is the use of convenient naturally oc curring or engineered restriction sites especially if they cleave for ex ample interrupted palindromic sequences It is also necessary to modify the cDNAs which represent the 5 0 and 3 0 ends of the coronavirus ge nome Normally a transcription promoter sequence for the bacterio phage T7 RNA polymerase is positioned upstream the coronavirus ge nome and a unique restriction site followed by the hepatitis d ri bozyme is placed downstream of the poly A tail of the coronavirus ge nome The terminal cDNA constructs must also have appropriate EagI or Bsp120I restriction sites to facilitate cloning into a unique NotI restriction site present in the genomic DNA of vaccinia virus strain vNotI tk The Cloning and Propagation of the Coronavirus Genomic cDNA in Vac cinia Virus Vectors The next stage is to ligate in vitro the coronavirus cDNA fragments and the long and short arms of NotI cleaved vNotI tk genomic DNA Merchlinsky and Moss 1992 This ligation is done in the presence of NotI to prevent religation of the vaccinia virus DNA Subse quently the ligation reaction is transfected into mammalian cells which have been previously infected with fowlpox virus Recombinant vaccinia virus the genome of which includes a full length copy of the coronavi rus genome is rescued Rescue of Recombinant Coronaviruses Essentially recombinant coron aviruses are rescued by generating genomic length RNA transcripts from the coronavirus component of the recombinant vaccinia virus DNA template These transcripts are then transfected into permissive cells The transcription reaction is normally done in vitro but it is also possible to rescue recombinant coronaviruses via the transcription of template DNA in the permissive cell itself This requires the introduction of non infectious i e restriction enzyme digested recombinant vaccin ia virus DNA and a source of bacteriophage T7 RNA polymerase nor mally a recombinant fowlpox virus into the permissive cell Also as will be described below we have found that the ability to rescue recombinant coronaviruses is significantly enhanced by but not dependent on the Reverse Genetics of Coronaviruses Using Vaccinia Virus Vectors 201 directed expression of the coronavirus nucleocapsid protein in the transfected cells In addition to these basic concepts this article will describe how vac cinia virus mediated homologous recombination can be used to intro duce specific mutations into the coronavirus genomic cDNA during its propagation in vaccinia virus Once an infectious coronavirus cDNA has been obtained this element of the reverse genetic approach is actually the rate limiting step It is therefore imperative that a rapid and easy procedure is available Finally we shall describe how the coronavirus re verse genetic system has now been extended to the generation of corona virus replicon RNAs 2 The Use of Vaccinia Virus as a Vector for Coronavirus cDNA The first use of vaccinia virus as a cloning vector for full length corona virus cDNA was described for the human coronavirus 229E HCoV 229E system Vaccinia virus vectors were chosen for several reasons First poxvirus vectors are suitable for the cloning of large cDNA It has been shown that they have the capacity to accept at least 26 kb of foreign sequence Smith and Moss 1983 and recombinant vaccinia virus gen omes of this size are stable infectious and replicate in tissue culture to the same titre as non recombinant virus Second vaccinia virus vectors have been developed which are designed for the insertion of foreign DNA by in vitro ligation Merchlinsky and Moss 1992 This obviates the need for plasmid intermediates carrying the entire cDNA insert Third the cloned cDNA insert should be accessible to mutagenesis by vaccinia virus mediated homologous recombination Finally conventional clon ing strategies based on procaryotic cloning systems e g plasmid vec tors bacterial artificial chromosomes or bacteriophage lambda vectors were not applicable to the stable propagation of full length HCoV 229E cDNA 2 1 Cloning of Full Length Coronavirus cDNA into the Vaccinia Virus Genome As outlined above the overall strategy to insert full length coronavirus cDNA fragments into the vaccinia virus genome involves two steps First the full length coronavirus cDNA is assembled by in vitro ligation using 202 V Thiel S G Siddell multiple cDNAs representing the entire coronavirus genomic RNA Sec ond the vaccinia virus vNotI tk genome is used as a cloning vector to insert the full length cDNA again by in vitro ligation The assembly of full length coronavirus cDNAs for HCoV 229E IBV and recently MHV A59 has involved two three or four cDNA frag ments respectively The DNA fragments corresponding to the 5 0 end of the coronavirus genomes contained the bacteriophage T7 RNA polymer ase promoter sequence and if not encoded at the 5 0 end of the coronavi rus genome one or three additional G nucleotides which are required for efficient initiation of the in vitro transcription reaction The cDNA fragment corresponding to the 3 0 end of the coronavirus genome con tained a synthetic poly A stretch comprised of 20 40 nucleotides nt followed by a hepatitis delta ribozyme element and a convenient restric tion site that can be used to generate so called run off transcripts In or der to insert the full length cDNAs into a single NotI site of the vaccinia virus vNotI tk vector genome the cDNA fragments corresponding to the 5 0 and 3 0 genomic termini contained the restriction sites EagIor Bsp120I After cleavage the resulting DNA ends are compatible with NotI cleaved vaccinia virus vector DNA The insertion of full length coronavirus cDNA fragments into the vac cinia virus vNotI tk genome by in vitro ligation required optimization of the procedure Purified vaccinia virus vNotI tk genomic DNA fragments which had been cleaved with NotI were found to be poor substrates for in vitro ligation most probably because of their large size In contrast in vitro assembled full length coronavirus cDNA fragments which had been cleaved with EagI were found to ligate efficiently Thus in ligation reac tions containing NotI cleaved vaccinia virus vector DNA and coronavirus cDNA inserts the ligation products were predominantly comprised of multiple insert fragments Ligation products comprised of vector arms and insert cDNA fragments were not readily detectable To resolve this problem we therefore included the NotI enzyme in the ligation reaction and using alkaline phosphatase dephosphorylated the coronavirus in sert DNA fragments As illustrated in Fig 1 this strategy resulted in the production of detectable amounts of ligation products comprised of two vaccinia virus vector arms and the coronavirus cDNA insert To rescue recombinant vaccinia virus clones containing the full length coronavirus cDNA the ligation reaction was transfected into CV 1 cells Because vaccinia virus genomic DNA is not infectious fowlpox vi rus has been used as a helper virus Scheiflinger et al 1992 Thus CV 1 cells were infected with fowlpox virus before transfection At 2 3 h after infection transfection the cells were collected and transferred with a Reverse Genetics of Coronaviruses Using Vaccinia Virus Vectors 203 fivefold excess of fresh CV 1 cells into 96 well plates During a period of 14 days virus stocks were collected from 96 well plates displaying cyto pathic effect Because fowlpox virus infection of mammalian cells is abortive the resulting virus stocks contained exclusively vaccinia virus es Furthermore the analysis of genomic DNA of rescued vaccinia virus es by Southern blotting showed that a high percentage of the viruses contained the coronavirus cDNA insert Thiel et al 2001a b 2 2 Mutagenesis of Cloned Coronavirus cDNA One major advantage of using vaccinia virus as a cloning vector is that the cloned coronavirus cDNA is amenable to site directed mutagenesis using vaccinia virus mediated homologous recombination Ball 1987 We will show one example to demonstrate the ease of using vaccinia vi rus mediated recombination to genetically modify coronavirus cDNA inserts Fig 1 Cloning of coronavirus cDNA into the vaccinia virus genome A schematic overview of the optimized ligation reaction using EagI cleaved and dephosphorylat ed coronavirus cDNA 1 and NotI cleaved vaccinia virus genomic DNA is illustrat ed The ligation reactions are carried out at 25C30C in the presence of NotI enzyme Also shown is a pulse field gel electrophoresis analysis of the ligation reaction prod ucts Fragments corresponding to the insert cDNA and ligation products comprised of insert cDNA and vaccinia virus DNA 2 3 4 5 and 6 are indicated 204 V Thiel S G Siddell Fig 2 Mutagenesis of cloned coronavirus cDNA by vaccinia virus mediated homol ogous recombination The generation of the recombinant vaccinia virus vVec GN containing a modified HCoV 229E genome is illustrated see text for details Also shown is a PCR analysis of the region in which the homologous recombination took place within the genomes of the parental vaccinia virus clone vHCoV inf 1 the inter mediate vaccinia virus clone vRec 1 and the recombinant vaccinia virus clone vVec GN Lanes 1 12 show 12 randomly picked recombinant vaccinia virus plaques ob tained after the gpt negative selection indicating the 100 recovery of desired geno types Reverse Genetics of Coronaviruses Using Vaccinia Virus Vectors 205 A region corresponding to HCoV nt 20 569 25 653 which lies within the full length HCoV 229E cDNA insert has been replaced by the gene encoding the green fluorescent protein GFP This results in a recombi nant vaccinia virus clone designated vVec GN The overall strategy of vVec GN construction is illustrated in Fig 2 The procedure is based on using the E coli guanine phosphoribosyl transferase gene gpt as both a positive and a negative selection marker First a region corresponding to nt 21 146 24 200 of the HCoV 229E genome was replaced by the E coli gpt gene To do this we transfected vHCoV inf 1 infected CV 1 cells with a plasmid DNA containing the E coli gpt gene located downstream of a vaccinia virus promoter and flanked by HCoV 229E sequences nt 19 601 21 145 and nt 24 201 25 874 that facilitate recombination Two days after infection transfection a vaccinia virus stock was pre pared To isolate gpt containing vaccinia viruses three rounds of plaque purification were done under gpt positive selection on CV 1 cells in the presence of mycophenolic acid 25 mg ml xanthine 250 mg ml and hypoxanthine 15 mg ml A recombinant vaccinia virus designated vRec 1 which contained the E coli gpt gene at the expected position could be easily identified by PCR and Southern blot analysis In a sec ond step vRec 1 was used to replace the E coli gpt gene by the GFP gene CV 1 cells were infected with vRec 1 and transfected with a plas mid DNA encoding the GFP gene flanked by HCoV sequences nt 19 485 20 568 and nt 25 654 27 273 Again after 2 days we prepared a vaccinia virus stock from the infected transfected cells and did three rounds of plaque purification However this time we used HeLa D980R cells and conditions which allow for the selection of vaccinia viruses that have lost the expression of gpt Kerr and Smith 1991 A PCR analysis of 12 vaccinia virus clones Fig 2 demonstrates that in each case vaccinia virus mediated homologous recombination has taken place at the ex pected position within the cloned HCoV 229E insert DNA One of the re combinant vaccinia virus clones was subjected to sequencing analysis of the region where the vaccinia virus mediated recombination had oc curred and the results revealed that vaccinia virus mediated recombina tion is precise at the nucleotide level 2 3 Rescue of Recombinant Coronaviruses Two strategies have been reported for the rescue of recombinant coron aviruses from full length cDNA cloned in vaccinia virus vectors Initially recombinant HCoV 229E was rescued after transfection of full length in 206 V Thiel S G Siddell vitro transcripts of the cloned HCoV 229E cDNA into MRC 5 cells Thiel et al 2001a Alternatively the rescue of recombinant IBV has been re ported by transfecting full length coronavirus IBV cDNA into chick kid ney CK cells that had been infected by a recombinant fowlpox virus rFPV T7 Casais et al 2001 The fowlpox virus mediates the expression of the bacteriophage T7 RNA polymerase In contrast to the transfection of infectious HCoV 229E RNA the rescue of recombinant IBV required the directed expression of the IBV nucleocapsid protein mediated by transfection of a expression plasmid encoding the IBV nucleocapsid N protein This observation led us to develop a line of BHK cells which express the HCoV 229E N protein and indeed we found that the ex pression of this protein also facilitates the rescue of recombinant HCoV 229E coronaviruses after the transfection of cells with full length in vitro transcripts 2 3 1 Rescue of Recombinant Coronaviruses Using Full Length In Vitro Transcripts The overall strategy to recover recombinant human coronavirus from vaccinia virus vHCoV inf 1 genomic DNA is illustrated in Fig 3 The full length HCoV 229E cDNA is cloned downstream of a bacteriophage T7 RNA polymerase promoter and a ClaI restriction endonuclease rec ognition sequence is located downstream of a synthetic poly A se quence representing the 3 0 end of the HCoV genome Genomic vHCoV inf 1 DNAwas prepared from purified recombinant vaccinia virus stocks and cleaved with ClaI enzyme This DNA was then used as template to transcribe in vitro a capped RNA corresponding to the HCoV genome with bacteriophage T7 RNA polymerase When this RNAwas transfected into MRC 5 cells by lipofection cytopathic effects indicative of human coronavirus infection developed throughout the culture after 6 7 days A virus designated HCoV inf 1 was rescued from the tissue culture su pernatant plaque purified and propagated to produce stocks containing approximately 1C14810 7 TCID50 ml Phenotypic analysis revealed that the growth kinetics cytopathic effect and stability of HCoV inf 1 were in distinguishable from those of parental virus Furthermore Northern hy bridization analysis of poly A containing RNA isolated from infected MRC 5 cells demonstrated that the patterns of viral genomic and subge nomic RNAs of HCoV inf 1 and HCoV 229E were identical To confirm that indeed a recombinant virus had been rescued the presence of a marker mutation which was introduced into the recombinant HCoV Reverse Genetics of Coronaviruses Using Vaccinia Virus Vectors 207 Fig 3 Rescue of recombinant HCoV 229E from cloned full length cDNA To recover recombinant HCoV 229E 5 0 capped RNA transcripts are produced in vitro with bac teriophage T7 RNA polymerase and vHCoV inf 1 genomic DNA as template The transcripts are transfected into MRC 5 cells The recombinant human coronavirus HCoV inf 1 contains marker mutations that are evident in the sequence of an RT PCR product of poly A containing RNA from HCoV inf 1 infected cells Also shown is a Northern hybridization analysis of in vitro transcribed HCoV inf 1 RNA lanes 1 and 4 and poly A containing RNA from parental HCoV 229E infected MRC 5 cells lanes 2 and 5 and HCoV inf 1 infected MRC 5 cells lanes 3 and 6 208 V Thiel S G Sid