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    Synthetic Recombinant Bat SARS Like Coronavirus Is Infectious in PDF

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    mariavillares

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    Michelle , Becker*, QUENSINGEESID, Eric F Dana Ay Sieh Tiny Shesnan, Raymond J. Pickles, Davide Cort!, Robert E. Johnston’ 3, and Mark R. Denison*5? lrestabuimonary Rearend Treatment Canter and Depart Departments of “Pediatrics and #Microbiology and immunology, Vanderbit University, Neshile, TN eee pel Hil, NC27809; and institute for Research In Blomadicne,cH-€500 Bellnzon pathways of animal-to-human movement has been hindered by challenges in Identifying reservoir species, cultivating zoonotic or bat and human recovery of hy: lestigate: mecha. ‘isms of transspecies movement of zoonoses a 1 aid in rapid publichealth responses to known or me Ing microbial threats. emerging pathogers | syntheticbiology | vaccine development | zoonoses, Eom: of zoonotic human pathogens is increasingly recog nized asa threat to public health (1). Human popalation growth and eavironmental changes have created new opportunities for contact between humans and zoonotic organisms that may result in cross-species transmission and human disease (1,2). Recent exam- plesinclude RNA viruses such a severe acute respiratory syndrome coronavirus (SARS-CoV), Ebola, Hanta, Nipah, and Chikungunya Viruses 2). However, the determinants regulating successful trans- species movement remain poorly understood due to challenges in identifying viral precursors and animal reservoirs, thereby comy cating vaecine and therapeutic design (3). SARS-CoV, which ‘exemplifies the challenges inherent in studying emerging. patho- gens, was the frst 2st century emerging virus to exhibit efficient human-to-human transmission and rapid slobal spread (4-6), Although zoonotic SARS-CoV strains were isolated from civets ‘and raccoon dozs (7-9), the epidemic likely originated from strains circulating in bats (Bat-SCoVs) (10-12). Bat CoVs cluster in both ‘major mammalian CoV taxonomic groups, raising the possibility that Bat CoVs may be progenitorsto ll 4 known pathogenic human CoVs (1, 13). Batsare also predicted to function as reservoirs for other important emerging human and animal viruses (14, 13), “Although several complete Bat CoV genome sequences are aval able, no Bat CoV has been successfully cultivated in cell culture or in animals (11, 13), severely limiting the identification of determi- nants of zoonotic CoV transspecies movement “The SARS-CoV Spike isa 180-KDa type I membrane glycopro- {cin that contains a well-defined receptor-binding domain (RBD), 19844-19849 | PNAS | December 16,2008, | vol 105. | ne. 50 and approves October 14, 2008 eceved for review August 1, 2008) ‘The SARS-CoV genome is likely mosaic of sequences derived from moliple recombination events, although this hypothesis is somewhat controversial (16). However, recombination within Spike has been described often (17), suggesting thatthe RBDs may be interchangeable between stains (1820). During the SARS-CoV ‘outbreak, evolution in the Spike RBD allowed for more efficient use of human angiotensia-converting enzyme 2 (hACE2) 35 receptor for entry (21,22) Because future zoonoses are likely, its ‘teal to identity srategics used by viruses to adapt ia human populations In this studs, we have combined phylogenetic and bioinformatics analyses, large-scale CDNA synthesis chimerie gene desien, and reverse genetics to generate a consensus Bal-SCOV. Successul recovery of the infectious chimeric virus, Bat SRBD, ‘which includes the RBD within Spike from human SARS-CoV, ‘demonstrates the plasticity of the CoV type 1 ahcoprotein. The synthetic reconstriction and recovery of this novel chimeric virus identifies a necessary genetic element for CoV cross-species tans- mission, establishes 4 model system for testing experimental evo- lution of zoonotic CoVs, and allows for testing of vaccine and therapeutics against posible future zoonotic strains Results Consensus Bat-SCoV Sequence Design and Construction, When this study was initiated, 4 Ba-SCoVs had been identified (HKU3-1, HKU3-2, HKU3-3, and RP3) as the virus reservoir populations from which SARS-CoV emerged (10-12), Because none hacl been recovered in culture, the infectivity of the reported viral genomic RNA sequences was hypothetical, having been derived from RT- PCR sequencing of bat fecal or rectal swab samples. Sequence databases have error frequencies from 1/500 to 1/10,000, making viable genome reconstruction problematic with inereasing size (23) ‘Therefore, we used the 4 reported Bat-SCoV sequences to establish «putative consensus Bat-SCoV sequence (GenBank accession no, FI211859) and designed eDNA fragments with junetions precisely aligned to the existing SARS-CoV reverse genetics system [Fig. 4, ‘supporting information (SI) Fig. St] (24). The defined and func: tional SARS-CoV 5! UTR and transcriptional regulatory sequences Author contin MMB, RLG, EF, RS. and MRD dese retenen: RLM (ontrnaedneweagentfansistock MM, RLG, EF 0,58, ACS, RIP, andRS taleed dts and MIM RLG.RSB, andUARD. ote the paper cont ot eres sateen: RE. convertor a he Venezunan Eq Encephale NEE expesion vector teensy an hls an ey Iter n Wma ne 8 eompany that hs bene ths ecology om the Unverty o Norh Carona. “Th arte PHAS Dic Submis Fey aetabe online veugh the PAS open cs open Dat depekion-Thesquances opera inthipaperhasbendepotodinth GenBank MARE ard... conte uted equly to tha work 2to whom conepondence may be adsreed, Ema barceeralunc ed or mark “sare centanssvportng Information cnine a wpa orenontnta ice er0sCsuplement © 2008 by The National Academy of Scere fhe USA arpa orga doi/101073/pma 08081 6105 hh ve oe c D = AP Tipe mRNA ee ors = manne BE «ws Fig. 1 Schematic roprestaton of SARSCOV and Bat SOV vrs. Schematic roprosentation of SARS CoV and Bat SCaV (GenBank sczesion no. 1211855) genomes and reverse genes syste (Tap) Arowheads inate rsp Processing sites within the ORF ab polyprotein open arrowheads, papain Proteinase mediate fled aroweads rsp5 [SC lke protelrase] medltx). Innmediatly belowarethe fragments wedin te reverse genetissystem, beled ‘Rttwovgh F The fragments synthesized o generate Bat SCOV exactly recap late the fragment junctions of SARS-CoV with the seception tha the Bat SCoV thas 2 agents, Bat Et and Bac £2, whichcerespondto MheSARS-E ragmant. (8 Schematic ropresntation showing organation ofthe SARS CaV ana Bat CoV Ske proteins. The engineered Spike protein are pictured below with the Wis ame to th aft st SH Include al ofthe EatSe=V Spo saquence except that the Bat SCoV RED (Bat SCoV amino acd 323-505) replaced withthe SARS CoV RED (amino aid 319-518) (GenBank accesion no. 211860), Bat SRBD-MA cludes he MAS Spike RED change at SARS-COV a YAQ6H, Ba SRBM ‘cludes the minimal 13 SARS-COV residues ctl for ACEZ contact resulting in 23 chimeric RD of Bat SCOV amino acd 32314257 ond SARS COV amino add 426R5180. Bat Hinge is Bat SRBN sequence, with Bat SCoV amino add 382 3OTE replaced with SARS CoV aminoacid 3880-3930. Bat Fncudes re 1-24057 {of SARS CoV (to Spike amino acé 838), withthe remaining 3” sequance from Bat SCeV Tote ightof te echematierpresetationsabeenationaftranript actvityand approximate stock titers atpasage 1 (P1) areindieated.NDinccates to infectious tected by plaque ney. (Cand D) Pretenceof genomic ad Subgenomic tansaipis after electroporation of n vito warscribed vial RNA, and coresponding © mRNA nccates the presence of genomic RNA, ether ‘lecroporated genomic RNA or progeny genomic RNA, and the presance ofa band conesponding to mANAG nccates to presence of leader containing sub- ‘genomic RNA, consistent with mRNA Wanscrbtion. were used because the 5’ UTRsof the Bat-SCoVs were incomplete. ‘The genomic cDNA fragments were commercially synthesized, inserted into vectors, assembled into a full-length cDNA, and transcribed in vitro to yield genomic RNA. Initial attempts to backer eral recover and passage infectious Bat-SCoV failed. Electroporated cells contained high levels of genome and leader-containing sub- ‘genomic transcripts on day 2, but not day 5 postelectroporation (pe) (Fig. 10), indicating that the synthetic consensus Bat-SCoV _Benome expressed a functional replicase. We did recover infectious virus consisting of SARS-CoV genome fragments A-E and Bat fragment F (Fig, 1 B and D).’The resulting virus, Bat-F, encoded a chimeric Spike. Thus, the amino-terminal two-thirds of SARS-CoV Spike, including the RBD, and the fusion core contained within the carboxyLterminial third of BatSCoV Spike can suecessully drive productive infection. Also, because Bat-F contained Bat-SCoV accessory and structural genes 3' to the Spike gene, these down- stream ORFS are clearly interchangeable Generation and Recovery of Chimeric Ba-SRBD. The cctodomain of Spike can be exchanged among CoVs, altering host-range specifi ity @5, 26). To test whother the RBDs of Bat-SCoV and SAR: CoV were interchangeable, we replaced the BatSCoV RBD (amino acid 323-505) with the SARS-CoV RBD (amino acid 319-518) (27,28) (GenBank accession no. FI211860), simulating a theoretical recombination eveat that might occur during mixed infection in Vio (Fig. 1B). After electroporation, But SRBD ge nome RNA and leader-containing subgenomic mRNA transcripts were detected (Fig, 1C), and progeny virions were detected by Plague assay. After 2 addtional passages, the population genome sequence was kntical to the Ba-SRBD molecular clone. How ever, 4 nucleotides exhibited dual peaks on the sequencing ee Utopherograms, suggesting quaispecies variation at these postions (Table Si). Recovery and passage of Bat-SRBBD demoastrated the functional interchangeability of human and animal SARS-CoV ike RBDs ‘The crystal structure of SARS-CoV RBD complexed with its receptor, hACE2 (28), implicated 13 residues within the eazboxyl terminus of the RBD (amino ack! 426R-518D) in ACE2 engags ment. Homology modeling indicated that this reeeptor-binding motif (RBM) may be sufficient to allow ACE2 engagement, and further predicts that inclusion of 6 residues amino-terminal tothe RBM (amino acd 388V-893D) may enhance ACE2enzagement by functioning as a distal “hinge” To test this posbiity, chimeric Bat SCoV genomes were constructed containing either the SARS. CoV RBM (Bat-SREM) or the RBM plus the distal hinge residues (Bat-Hinge) (Fig, 1B) Electroporation yielded genome and sub- _Renomic leader-containing transeripts at day 2, but not 5, pe. (Fig. Cand D), and progeny virions could not be successfully passaged in culture ‘Virus Replication in Primate and Murine Cells, We next infected Vero cells, murine delayed brain tumor (DBT) cells, and DBT cells ‘expressing hACE? or civet (c)ACE2 (DBT-hACE2 and DBT- ‘ACE2) (22) with Bat-SRBD or SARS-CoV at a multiplicity of infection (MOI) of 0.01 oF 1 plaque forming unit (PFU) per cal (Fig. 2). Ba SRED and SARS-CoV exhibited productive growth in Vero, DET-hACE2, and DBT-cACE? eels that was remarkably similar in kinetics and peak tcers (Fig. 2), In contrast, DBT cells lacking ACE2 expression did not support growth of either SARS. CoV of Bat-SRBD (data not shown). These data indicate that Bat-SCoV expressing the SARS-CoV RED is capable of entering cells by using ACE? from humans, nonhuman primates, or civetsas receptor, and replicating efficiently Detection of Bat SRBD Replicase Proteins by SARS-CoV Antibo Comparison of SARS-CoV and Bat SRED predicted high, but aot ‘complete, identity of aminoacid sequences across replicase proteins (Tabie $2), Because antibody cross-reactivity ia potential tool for detection and analysis of Bat-SCoVs, we tesied whether antibodies specific for SARS-CoV proteins (30) could also detect Bat-SRBD homologues. Immunoblots were performed by using rabbit poly-

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