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Infectious Enveloped RNA Virus Antigenic Chimeras (Alphaviruses/Random Mutagenesis/Virus Vectors/Protein Engineering/Glycoproteins) STEVEN D

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Infectious Enveloped RNA Virus Antigenic Chimeras (Alphaviruses/Random Mutagenesis/Virus Vectors/Protein Engineering/Glycoproteins) STEVEN D Proc. Natl. Acad. Sci. USA Vol. 89, pp. 207-211, January 1992 Biochemistry Infectious enveloped RNA virus antigenic chimeras (alphaviruses/random mutagenesis/virus vectors/protein engineering/glycoproteins) STEVEN D. LONDON*, ALAN L. SCHMAUOHNt, JOEL M. DALRYMPLEt, AND CHARLES M. RICE*t *Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8230, St. Louis, MO 63110-1093; and tUnited States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011 Communicated by Edwin D. Kilbourne, October 7, 1991 (receivedfor review August 26, 1991) ABSTRACT Random insertion mutagenesis has been used would allow recovery of infectious chimeric viruses. The to construct infectious Sindbis virus structural protein chime- insertional mutagen was an oligonucleotide encoding an ras containing a neutralization epitope from a heterologous 11-amino acid neutralization epitope (4D4) derived from the virus, Rift Valley fever virus. Insertion sites, permissive for external G2 glycoprotein of Rift Valley fever virus (RVFV). recovery of chimeric viruses with growth properties similar to Both a polyclonal anti-peptide antiserum and a monoclonal the parental virus, were found in the virion E2 glycoprotein and antibody (mAb), mAb 4D4, that react with this epitope on the secreted E3 glycoprotein. For the E2 chimeras, the epitope RVFV virions and also with denatured and reduced RVFV was expressed on the virion surface and stimulated a partially G2 (13, 14) are available. Thus, chimeric viruses expressing protective immune response to Rift Valley fever virus infection this epitope can be identified by Western analysis. Further- in vivo. Besides providing a possible approach for developing more, the antigenic and immunogenic properties of Sindbis- live attenuated vaccine viruses, insertion ofpeptide ligands into 4D4 chimeras are of interest since 4D4 epitope-specific virion surface proteins may ultimately allow targeting of virus antibodies inhibit RVFV plaque formation in cell culture and infection to specific cell types. protect mice against lethal challenge with RVFV (13, 14). Sindbis virus, a relatively benign alphavirus, has been used MATERIALS AND METHODS as a model enveloped RNA virus for inserting heterologous epitopes into virion structural glycoproteins. Alphaviruses Plasmid Constructs and Random Insertion Mutagenesis. are a diverse group of enveloped animal RNA viruses trans- Standard recombinant DNA techniques were used in all mitted in experiments (15). The plasmid used for random insertion nature to their vertebrate hosts by mosquitoes mutagenesis, pMTE2, consists of the Stu I (nt 8572)-BssHII (reviewed in ref. 1). Besides providing models for studying (nt 9804) Sindbis cDNA fragment (Sindbis nucleotide posi- virus entry and membrane protein biogenesis and function (1, tions refer to the full-length cDNA sequence reported in ref. 2), recent studies suggest that engineered alphaviruses may 5) from pTR2000 (16) cloned into pMT21 (obtained from also be used as live immunogens (3) and gene expression H. V. Huang, Washington University). The plasmid DNA vectors (ref. 4; C. S. Hahn, A. Diehl, C. Xiong, H. V. was linearized by treatment with DNase I in the presence of Huang, and C.M.R., unpublished results). Sindbis virus 1.5 mM MnCl2 (12) and repaired with T4 DNA polymerase contains a single-stranded, positive-sense RNA genome of and Escherichia coli DNA ligase (15). The resultant linear 11,703 nucleotides (nt) (5). Icosahedral nucleocapsids, com- molecules were isolated on a 0.8% low melting temperature posed of genomic RNA complexed with basic capsid protein agarose gel and ligated to the nonphosphorylated double- molecules, bud from the surface of infected vertebrate cells stranded 45-mer encoding the RVFV 4D4 epitope (see Fig. and acquire a host-derived lipid bilayer containing two trans- 2A). Further details of the library construction are given in membrane viral glycoproteins, El and E2. The characteristic Results and Fig. 2B. The transcription vector used to regen- virion spikes visualized by electron microscopy (6, 7) are erate full-length Sindbis virus cDNA, pTR2002, has a 604-nt believed to be composed of trimers of an E1-E2 heterodimer lethal deletion [Ban I (8862)-Dra 1 (9470)] in E2. The parental (Fig. 1A; ref. 9). Although this heterodimer is likely a virus for these experiments, TR2001, was derived from functional unit, some properties have tentatively been as- transcripts of pTR2001. pTR2001 is a derivative of pTR2000 signed to individual glycoproteins. E2 is believed to be (16) in which the Sac I recognition site upstream from the SP6 involved in receptor binding (10), and E2-specific monoclonal promoter has been eliminated. antibodies often neutralize virus infectivity (11). El, the viral Generation of Chimeric Virus cDNA Clones. Double- hemagglutinin, probably mediates fusion of the virion enve- stranded cDNA was prepared from poly(A)+ RNA obtained lope and the endosomal membrane upon acidification, re- from chicken embryo fibroblasts (CEF) cells infected with sulting in delivery ofthe nucleocapsid to the cytoplasm (1, 2, twice plaque-purified virus isolates from the full-length ran- 11). dom insertion library (15, 17). The Stu I-BssHII cDNA Sindbis structural proteins are produced from a polypro- fragment was cloned into pTR2002 to generate cDNA clones tein by a complex series of processing events (Fig. 1B; ref. for each chimeric virus. The sequence of the Stu I-BssHII 2). PE2, the E2 precursor, was initially selected for insertion region was determined for each of the three clones used to of heterologous peptides since this precursor is cleaved, late generate virus stocks for further characterization. in virus maturation, to yield both secreted (E3) and virion- associated (E2) polypeptides. In addition, E2 is the less conserved of the two alphavirus virion glycoproteins (11). RESULTS Since high-resolution structures of the Sindbis virion glyco- Construction of the RVFV 4D4 Epitope-Sindbis Virus PE2 proteins are not yet available, random insertion mutagenesis Random Insertion Library. The scheme for PE2 mutagenesis, (12) was used to identify permissive insertion sites, which Abbreviations: mAb, monoclonal antibody; nt, nucleotide(s); pfu, The publication costs of this article were defrayed in part by page charge plaque-forming units; RVFV, Rift Valley fever virus; CEF, chicken payment. This article must therefore be hereby marked "advertisement" embryo fibroblasts. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 207 Downloaded by guest on October 1, 2021 208 Biochemistry: London et al. Proc. Natl. Acad. Sci. USA 89 (1992) A. [vIn 45-mer oligonucleotide (Fig. 2A), encoding the 11-amino acid Ii 4D4 epitope and flanking restriction sites (5' Xba I and 3' Sac I) not present in Sindbis virus cDNA (or cloning vectors), was ligated to repaired linear molecules of a PE2 subclone (pMTE2), which had been produced by limited DNase I digestion (Fig. 2B). In the initial random insertion library, -7% of the colonies contained the RVFV 4D4 oligonucleo- tide, which corresponded to -8000 independent insertions in the Stu I-BssHII region (which spans from amino acid residue 46 of E3 to residue 391 of E2). To enrich for insertion mutations, plasmid DNA was digested with Sac I and the linear molecules were isolated, recircularized by ligation at 13 low DNA concentration, and used to transform E. coli. After the first Sac I selection, the Stu I-BssHII region from the 26)4 ;Lit 6-11 t 423 a >> iii 4t11.) diie random insertion library was subcloned into pMTE2 to I... ( aiipsal I S. 2 -_ ................- eliminate insertions in the plasmid sequences, and two fur- -T--L-- - ther Sac I selections were performed. Finally, a library of A t insertion mutations in the context of a transcription vector FIG. 1. Organization and processing of the Sindbis structural containing the full-length Sindbis cDNA was constructed by proteins. (A) Schematic representation of the spike heterodimer. subcloning the mutagenized Stu I-BssHII fragment into Oligosaccharides (o-o), covalently attached fatty acids (-), and the pTR2002, followed by two additional Sac I selections. uncleaved signal sequence (stippled box) present in the N-terminal Recovery and Characterization of Infectious Chimeras. SP6 region of E3 are indicated. El and E2 each contain an N-terminal RNA transcripts obtained from the full-length random inser- ectodomain, a membrane anchor near the C terminus, and a short tion library were used to transfect CEF or BHK-21 cells (17, C-terminal cytoplasmic tail. Permissive insertion sites for the 4D4 19). Transcripts obtained from the insertion library were 70- peptide are indicated (A). The figure is not drawn to scale. (B) The Sindbis virus structural polyprotein and its cleavage sites. After to 77-fold less efficient at generating plaques at 37TC than autocatalytic cleavage of the capsid protein (at A), the N-terminal parental (pTR2001) RNA transcripts, suggesting that, as portion of PE2 (the E2 precursor) acts as an uncleaved signal expected, all sites in the mutagenized region were not per- sequence to initiate cotranslational translocation into the endoplas- missive for insertion of the 4D4 oligonucleotide. mic reticulum. A series of stop transfer and internal signal sequences Individual plaques, isolated from monolayers transfected result in the formation of a PE2-E1 oligomer in the infected cell with RNA obtained from the random insertion library and membrane. Cellular signalase catalyzes the cleavages flanking the incubated at 37°C, were used to generate first passage virus 6-kDa (6K) protein (*), and the delayed cleavage of PE2 into E3 and E2 ( t ) occurs in a post-Golgi compartment, possibly mediated by a stocks. Plaques morphologically similar to those of the pa- host protease (1, 2). Stippled areas represent hydrophobic regions rental strain of Sindbis virus were selected with the hope that (8), and the facing arrows indicate the mutagenized region ofthe PE2 they would have growth characteristics similar to that of the gene. The sizes of the individual proteins are indicated.
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