【病毒外文文獻】2003 In Vitro and In Ovo Expression of Chicken Gamma Interferon by a Defective RNA of Avian Coronavirus Infectious Bronc

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JOURNAL OF VIROLOGY May 2003 p 5694 5702 Vol 77 No 10 0022 538X 03 08 00H110010 DOI 10 1128 JVI 77 10 5694 5702 2003 Copyright 2003 American Society for Microbiology All Rights Reserved In Vitro and In Ovo Expression of Chicken Gamma Interferon by a Defective RNA of Avian Coronavirus Infectious Bronchitis Virus Karen Hackney Dave Cavanagh Pete Kaiser and Paul Britton Institute for Animal Health Compton Laboratory Compton Newbury Berkshire RG20 7NN United Kingdom Received 25 November 2002 Accepted 28 February 2003 Coronavirus defective RNAs D RNAs have been used for site directed mutagenesis of coronavirus genomes and for expression of heterologous genes D RNA CD 61 derived from the avian coronavirus infectious bronchitis virus IBV was used as an RNA vector for the expression of chicken gamma interferon chIFN H9253 D RNAs expressing chIFN H9253 were shown to be capable of rescue replication and packaging into virions in a helper virus dependent system following electroporation of in vitro derived T7 RNA transcripts into IBV infected cells Secreted chIFN H9253 under the control of an IBV transcription associated sequence derived from gene 5 of the Beaudette strain was expressed from two different positions within CD 61 and shown to be biologically active In addition following infection of 10 day old chicken embryos with IBV containing D RNAs expressing chIFN H9253 the allantoic fluid was shown to contain biologically active chIFN H9253 demonstrating that IBV D RNAs can express heterologous genes in vivo Infectious bronchitis virus IBV is a highly infectious and economically important pathogen of chickens that causes re spiratory disease diminished growth rate and substantial de cline in egg production Although infectious bronchitis is con sidered primarily a disease of the respiratory system strains of IBV have wide and variable tropisms and the clinical manifes tations of the disease can be diverse 9 Genetically very similar viruses cause disease in turkeys 6 and pheasants 7 IBV is a group 3 member of the genus Coronavirus of the family Coronaviridae in the order Nidovirales 12 being an enveloped RNA virus with an unsegmented 5H11032 end capped 3H11032 end polyadenylated single stranded positive sense RNA genome of 27 608 nucleotides nt 4 Coronaviruses produce a3H11032 coterminal nested set of subgenomic mRNAs sg mRNAs that are polycistronic These are produced by a discontinuous transcription process during synthesis of the negative strand and contain identical 5H11032 ends due to the addition of a leader sequence derived from the 5H11032 end of the genomic RNA gRNA 35 36 Preceding the body sequence of each sg mRNA is a consensus sequence the transcription associated sequence TAS 15 involved in the acquisition of the leader sequence All coronavirus envelopes contain at least three membrane proteins the spike glycoprotein a small membrane protein and an integral membrane protein In addition the coronavirus virion also contains a nucleocapsid protein that interacts with the gRNA Coronavirus defective RNAs D RNAs which lack large parts of the genome are produced following virus passage at a high multiplicity of infection While all D RNAs contain cis acting sequences necessary for replication only a subset of D RNAs contain sequences necessary for packaging into viri ons in the presence of a helper virus Coronavirus D RNAs have been used for site directed mutagenesis of the virus ge nome 25 and for expression of heterologous genes 1 2 13 17 20 22 39 42 43 Cytokines are regulatory proteins that act as a communica tion network between cells throughout immunological devel opment Avian homologues of several mammalian cytokines have been isolated including chicken gamma interferon chIFN H9253 10 Mammalian IFN H9253 is a pleiotropic cytokine initially produced by natural killer cells during immune induc tion and then by committed T helper 1 Th1 cells as a regu lator and effector molecule for driving inflammatory responses for a review see reference 8 IFN H9253 has a major role in activating antiviral immune responses through augmentation of major histocompatibility complex expression on antigen presenting cells for interaction with T cells stimulation of antibody Ab formation promotion of Ab isotype class switching and development of cytotoxic T cells Several studies have been undertaken to investigate the in vivo potential of IFN H9253 as a vaccine adjuvant Coadministration of bovine IFN H9253 with vesicular stomatitis virus G glycoprotein to cattle resulted in an increased formation of protective Ab against vesicular stomatitis virus 41 In a mouse model fusion of IFN H9253 to human immunodeficiency virus gp120 resulted in enhanced primary Ab responses against gp120 enhanced an tigen specific T cell proliferation and IFN H9253 production 26 The use of recombinant feline IFN H9253 as a vaccine adjuvant increased Ab responses against rabies virus and calicivirus an tigens 37 The role of recombinant chIFN H9253 as an adjuvant and ther apeutic agent has been investigated Coadministration of chIFN H9253 with sheep red blood cells SRBCs resulted in an increased secondary Ab response with amounts of SRBCs 10 fold smaller than the dose of SRBCs given alone 23 24 Administration of recombinant chIFN H9253 prior to challenge with avian coccidia resulted in decreased intracellular sporo zoite development and oocyst production with an enhanced level of body weight gain 21 chIFN H9253 had an adjuvant effect reducing parasite replication when used in a DNA vaccine regimen against Eimeria acervulina 27 Coexpression of New Corresponding author Mailing address Division of Molecular Biology Institute for Animal Health Compton Laboratory Compton Newbury Berkshire RG20 7NN United Kingdom Phone 44 1635 578411 Fax 44 1635 577263 E mail paul britton bbsrc ac uk 5694 on April 4 2015 by UNIV OF CALIF SANTA CRUZ http jvi asm org Downloaded from castle disease virus antigens with chIFN H9253 by using fowlpox virus resulted in an earlier Ab response with the best protec tive immune response of the recombinant fowlpox virus vac cines 32 In this study we describe both the in vitro and in ovo expres sion of biologically active chIFN H9253 from an IBV D RNA We demonstrate for the first time the expression of a chicken cytokine from an IBV D RNA and the in ovo expression of a biologically active heterologous gene from an IBV D RNA MATERIALS AND METHODS Cells and viruses IBV Beaudette was grown in 11 day old embryonated do mestic fowl eggs harvested from allantoic fluid 24 h postinfection and used as helper virus for the rescue of IBV D RNAs 31 IBV was passaged and titrated on primary chick kidney CK cells 30 HD11 cells an avian leukosis virus MC29 transformed cell line 3 were cultured in RPMI 1640 medium Life Technologies containing 2 5 fetal calf serum 2 5 chick serum 10 tryptose phosphate broth 20 mM L glutamine 0 225 NaHCO 3 1Uofpenicillin ml and 1 H9262g of streptomycin ml Oligonucleotides The oligonucleotides used in this work were obtained from Invitrogen and are listed in Table 1 Recombinant DNA techniques Standard procedures were used to produce recombinant DNA 33 or methods were according to the manufacturers in structions Construction of TAS chIFN H9253 gene cassette A TAS chIFN H9253 cassette was produced by PCR for insertion into the IBV D RNA CD 61 cDNA sequence in pIBV Vec 13 Oligonucleotides IBV5IFN H9253START and SmaIIFN H9253 END corresponding to the 5H11032 end and complementary to the 3H11032 end respectively of the chIFN H9253 sequence were used to generate the TAS chIFN H9253 cassette Oligonu cleotide IBV5IFN H9253START contained the restriction endonuclease SmaI and PmaCI sites the Beaudette derived gene 5 TAS 39 and the first 15 nt of the chIFN H9253 sequence Oligonucleotide SmaIIFN H9253 END consisted of the last 15 nt complementary of the chIFN H9253 gene including the termination codon fol lowed by a SmaI site The chIFN H9253 sequence was amplified by PCR by using Taq Pwo DNA polymerase Hybaid from plasmid pGEM T IFN H9253 19 con taining a cDNA derived from chIFN H9253 mRNA The TAS chIFN H9253 cassette was digested with XmaI and ligated into XmaI digested pBluescript II SK H11001 Strat agene resulting in pBS IFN H9253H11001ENDS in which the TAS chIFN H9253 sequence was verified by sequence analysis The TAS chIFN H9253 cassette was removed from pBS IFN H9253H11001ENDS by using PmaCI and SmaI and inserted into either the PmaCI site or the SnaBI site in the CD 61 sequence of pIBV Vec In vitro rescue of chIFN H9253 containing D RNAs by IBV Beaudette In vitro T7 derived D RNA transcripts were synthesized from 1 H9262g of plasmid DNA and electroporated into IBV infected CK cells passage 0 P 0 39 Virus V 1 in1ml of cell medium was used to infect CK cells and viruses V 2 to V 6 were serially passaged every 24 h for six passages P 1 to P 6 In ovo rescue of chIFN H9253 containing D RNAs Cell medium 100 H9262l from P 3 CK cells corresponding to peak D RNA levels was used to infect 10 day old Rhode Island Red specific pathogen free embryos The infected embryos were incubated at 37 C for 16 h and cooled to 4 C overnight Allantoic fluid was collected from the infected eggs centrifuged at 1 500 H11003 g for 10 min and stored at H1100270 C Allantoic fluid 100 H9262l containing IBV and any potential D RNA was used to infect CK cells Identification of IBV derived RNAs Total cytoplasmic RNA was extracted from infected CK cells by using RNeasy Qiagen and electrophoresed in dena turing 1 agarose 2 2 M formaldehyde gels The RNAs were Northern blotted onto Hybond XL nylon membranes Amersham and IBV derived RNAs were detected with probes covalently labeled with psoralen biotin BrightStar Am bion 13 The probes were hybridized to the RNAs at 42 C for 16 h detected with streptavidin alkaline phosphatase conjugate in the presence of an alkaline phosphatase 1 2 dioxetane chemiluminescent substrate CDPStar and BrightStar Biodetect Ambion and exposed to film at room temperature for 2 h An IBV specific 309 nt 3H11032 untranslated region UTR probe corresponding to the last 309 nt of the 3H11032 end of the IBV genome was used to detect all IBV derived RNA species including IBV derived D RNAs 13 A 209 nt 5H11032 IBV specific probe produced by PCR with oligonucleotides BG 67 and BG 2 Table 1 corresponding to nt 265 to 474 within the 5H11032 UTR of the Beaudette genome was used to detect IBV gRNA and D RNAs A 360 nt chIFN H9253 specific probe produced by PCR with primers IFN 3 and IFN 7 Table 1 was used to detect RNAs containing the chIFN H9253 sequence chIFN H9253 bioassays and neutralization assays The chIFN H9253 bioassays and neutralization assays were carried out as described by Lawson et al 19 Essen tially triplicate samples 200 H9262l of serially diluted CK cell medium or allantoic fluid either from mock infected embryos or embryos infected with IBV were added to 4 H11003 10 4 HD11 cells in 96 well flat bottomed plates Recombinant chIFN H9253 prepared as described by Lawson et al 19 was serially diluted two fold and used as a positive control chIFN H9253 neutralization assays were carried out by using an anti chIFN H9253 neutralizing monoclonal Ab MAb 1E 12 18 and an anti bovine granulocyte macrophage colony stimulating factor polyclonal Ab CC305 kindly provided by Paul Sopp Institute for Animal Health Comp ton United Kingdom Both antibodies were diluted 1 1 000 and incubated with CK cell medium and allantoic fluid samples for2hatroom temperature before being added to HD11 cells For controls all samples were also preincubated with the isotype control Ab CC305 and media alone All HD11 cells were incubated at 41 Cin5 CO 2 for 48 h for the chIFN H9253 bioassays Nitric oxide NO produced from HD11 cells resulting from induction with chIFN H9253 was measured in HD11 cell medium 100 H9262l as nitrite NO 2 H11002 by using a modification of the Griess assay described by Lawson et al 19 and a Spectra Max 250 enzyme linked immunosorbent assay reader Molecular Devices Wok ingham United Kingdom The amount of NO present in the HD11 cell media was expressed as the concentration of NO 2 H11002 millimolar for the chIFN H9253 bio assays or as the optical density at 543 nm for the neutralizing assays RESULTS D RNAs containing TAS chIFN H9253 Expression of heterolo gous genes from coronavirus D RNAs requires the genes to be under the control of a TAS for synthesis of an sg mRNA The chIFN H9253 sequence was placed under the control of the IBV Beaudette gene 5 TAS 39 by using PCR and a chIFN H9253 mRNA derived cDNA 19 generating a TAS chIFN H9253 cas sette for IBV controlled expression The gene 5 TAS was orig inally chosen for expression of heterologous genes because it has the shortest sequence between the 3H11032 end of the TAS and the AUG of open reading frame ORF 5a and also because the Beaudette sg mRNA 5 is one of the most abundantly TABLE 1 Oligonucleotides used for generation of TAS chIFN H9253 cassette and hybridization probes Oligonucleotide Sequence a Position b Polarity IBV5IFN H9253START TCC CCC GGG CAC GTG TTT TAC TTA ACA AAA ACT TAA CAA ATA CGG ACG ATG ACT TGC CAG ACT NA H11001 SmallFN H9253 END ACC CCC GGG GGT TAG CAA TTG CAT CT NA H11002 BG 67 GGC TGG TTC GAG TGC GAG Beaudette 265 282 H11001 BG 2 TCA GGG GTT GTT TGG CAC T Beaudette 455 473 H11002 IFN 3 ATG ACT TGC CAG ACT TAC AA chIFN H9253 1 20 H11001 IFN 7 CAG GTC CAT GAT ATC TTT CAC chIFN H9253 340 360 H11002 a Underlined sequences correspond to the IBV sequence Nucleotides marked in bold correspond to the IBV canonical TAS and those in italics correspond to restriction endonuclease sites used for cloning b The positions of the nucleotides refer to the IBV Beaudette sequence 4 or the chIFN H9253 sequence 10 NA not applicable VOL 77 2003 EXPRESSION OF chIFN H9253 BY AN IBV D RNA 5695 on April 4 2015 by UNIV OF CALIF SANTA CRUZ http jvi asm org Downloaded from expressed sg mRNAs 39 The TAS chIFN H9253 cassette was initially inserted into pBluescript II SK H11001 under the control of the T7 promoter A protein with a size corresponding to that of chIFN H9253 was produced in vitro by using the TNT T7 coupled wheat germ extract system Promega data not shown indi cating that a product of the expected size could be produced from the TAS chIFN H9253 cassette Two restriction endonuclease sites PmaCI and SnaBI within the IBV D RNA CD 61 have been used for the expres sion of heterologous genes 13 39 The PmaCI site is within domain III of CD 61 and not within the D RNA specific ORF 31 whereas the SnaBI site within domain II of CD 61 in terrupts the D RNA specific ORF Fig 1B The TAS chIFN H9253 cassette excised from pBS IFN H9253H11001ENDS was in serted into the PmaCI site or the SnaBI site of the CD 61 sequence in pIBV Vec Four plasmids pIBV Vec IFN H9253PmaCI and pIBV Vec IFN H9253SnaBI with the TAS chIFN H9253 gene cassette in the correct orientation and pIBV Vec H9253 NFIPmaCI and pIBV Vec H9253 NFISnaBI with the TAS chIFN H9253 gene cassette in the opposite incorrect orientation were identified for generating D RNAs Fig 1 In vitro rescue of chIFN H9253 containing D RNAs In vitro T7 derived transcripts of D RNAs IBV Vec IFN H9253PmaCI IBV Vec IFN H9253SnaBI IBV Vec H9253 NFIPmaCI and IBV Vec H9253 NFISnaBI were electroporated into IBV infected CK cells and progeny virus with D RNAs was serially passaged on CK cells P 0 to P 6 Northern blot analyses using the IBV 3H11032 UTR and chIFN H9253 probes on RNA isolated from P 3 cells identified an RNA species of 6 8 kb Fig 2 This RNA was not present in cells infected with IBV only and corresponded in size to the in vitro T7 derived D RNA transcript The chIFN H9253 specific probe did not hybridize to IBV gRNA or sg mRNAs indicat ing that the 6 8 kb RNA represented D RNAs containing the TAS chIFN H9253 sequence As expected the chIFN H9253 probe de tected D RNAs IBV Vec H9253 NFIPmaCI and IBV Vec H9253 NFISnaBI with the TAS chIFN H9253 sequence in the opposite orientation The chIFN H9253 probe also detected two other RNAs of 1 7 kb Fig 2B lane 3 and 5 kb Fig 2B lane 4 following the rescue of D RNAs IBV Vec IFN H9253PmaCI and IBV Vec IFN H9253SnaBI respectively in addition to the 6 8 kb D RNA These RNAs corresponded to the expected sizes of chIFN H9253 mRNAs expressed from the TAS chIFN H9253 sequence in the D RNAs The IBV Vec IFN H9253PmaCI 1 7 kb chIFN H9253 mRNA was routinely observed in smaller amounts than the IBV Vec IFN H9253SnaBI 5 kb chIFN H9253 mRNA The IBV 3H11032 UTR probe detected RNAs larger than the 6 8 kb D RNA and the 7 3 kb IBV sg mRNA 2 Fig 2A which were not detected by the chIFN H9253 probe Fig 2B These RNAs were shown by using Northern blot analysis and an IBV 5H11032 UTR probe Fig 3 to be new IBV derived D RNAs not containing the TAS chIFN H9253 sequence To investigate the rescue profile of the D RNAs Northern blot analyses were carried out on RNA isolated from P 0 to P 6 CK cells containing D RNAs IBV Vec IFN H9253PmaCI and IBV Vec IFN H9253SnaBI by using the IBV 5H11032 UTR 3H11032 UTR and chIFN H9253 probes Fig 3 The 6 8 kb D RNAs were detected by the two IBV probes in RNA isolated from P 1 to P 6 CK cells Fig 3A B D and E and analyses using the chIFN H9253 probe confirmed that they contained the chIFN H9253 sequence Fig 3C and F Both D RNAs irrespective of whether the chIFN H9253 sequence was inserted into the PmaCI or SnaBI site were initially detected at P 1 and increased in amount upon serial passage with the largest amount in P 4 CK cells after which the amount of D RNA decreased RNAs larger than the 6 8 kb chIFN H9253 containing D RNA and the IBV sg mRNA 2 were detected from P 3 to P 6 by using both IBV probes The use of the IBV 5H11032 probe indicated that the RNAs corresponded to new IBV derived D RNAs Fig 3A B D and E The new D RNAs did not contain the chIFN H9253 sequence Fig 3C and F indicating that they were derived from IBV gRNA and were observed in increasing amounts in RNA isolated from P 3 to P 6 cells with concomitant gradual loss of the 6 8 kb chIFN H9253 containing D RNAs Fig 3 Analysis of P 3 derived RNA following the rescue of D RNA FIG 1 Schematic diagrams of the IBV D RNAs containing the TAS chIFN H9253 sequence A pIBV Vec showing the PmaCI and SnaBI sites within CD 61 for insertion of heterologous genes HH9254R hepatitis delta virus antigenomic ribozyme B IBV D RNA CD 61 showing the positions of the restriction sites The BamHI site was used for determining the orientation of inserts The TAS chIFN H9253 gene cassette was used for insertion into CD 61 The 998 amino acid CD 61 specific ORF 30 31 is indicated as a thick black line C The D RNAs resulting from insertion of the TAS chIFN H9253 cassette into the two restriction endonuclease sites in both orientations within CD 61 The T7 promoter HH9254R and T7 termination sequences of pIBV Vec 13 and the TAS chIFN H9253 sequences are as indicated in panel A The chIFN H9253 mRNAs of 1 7 and 5 0 kb derived from D RNAs IBV Vec IFN H9253PmaCI and IBV Vec IFN H9253SnaBI respectively are indicated as black arrows 5696 HACKNEY ET AL J VIROL on April 4 2015 by UNIV OF CALIF SANTA CRUZ http jvi asm org Downloaded from IBV Vec IFN H9253SnaBI identified a 5 kb D RNA derived chIFN H9253 mRNA This mRNA was detected in RNA from P 1 to P 6 CK cells containing IBV Vec IFN H9253SnaBI with the amounts detected varying in accordance with the amounts of D RNA present the largest amount being detected at P 4 Fig 3F An RNA corresponding to the IBV Vec IFN H9253PmaCI 1 7 kb chIFN H9253 mRNA was not detected following serial pas sage of the D RNA Fig 3C However from the amounts of the RNA detected the most likely explanation for this result was that the amount of the 1 7 kb chIFN H9253 mRNA was below the detection level of the analysis The Northern blot analyses showed that the two chIFN H9253 containing D RNAs with the TAS chIFN H9253 insert in the cor rect orientation were rescued in IBV infected CK cells upon ser